Synthesis and biological evaluation of N-substituted benzo[c] phenanthrolines and benzo[c]phenanthrolinones as antiproliferative agents

Article abstract of DOI:10.1016/j.ejmech.2011.02.065

Benzo[c]phenanthrolines and benzo[c]phenanthrolinones substituted by dialkylaminoalkyl side chains at position N5 and C6, respectively, were synthesised and their biological activity evaluated. They displayed interessant cytotoxicity associated with some DNA interactions. However, the low topoisomerase 1 affinity suggests that other cellular targets are responsible for the antiproliferative activity.

Full text of DOI:10.1016/j.ejmech.2011.02.065

European Journal of Medicinal Chemistry 46 (2011) 2117e2131
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of N-substituted benzo[c]phenanthrolines
and benzo[c]phenanthrolinones as antiproliferative agents
,
,
Constance Genès a 1, Gaëlle Lenglet b 1, Sabine Depauw b, Raja Nhili b, Soizic Prado a,
Marie-Hélène David-Cordonnier b, Sylvie Michel a, François Tillequin a, François-Hugues Porée a
,
*
a Université Paris Descartes, Laboratoire de Pharmacognosie, UMR CNRS 8638, 4 Avenue de l’Observatoire, F-75270 Paris Cedex 06, France
b INSERM U837, JPARC (Jean-Pierre Aubert Research Center), Team “Molecular and Cellular Targeting for Cancer Treatment”, Université de Lille 2, IRCL,
Place de Verdun, F-59045 Lille Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Benzo[c]phenanthrolines and benzo[c]phenanthrolinones substituted by dialkylaminoalkyl side chains
at position N5 and C6, respectively, were synthesised and their biological activity evaluated. They dis-
played interessant cytotoxicity associated with some DNA interactions. However, the low topoisomerase
1 affinity suggests that other cellular targets are responsible for the antiproliferative activity.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 13 October 2010
Received in revised form
21 February 2011
Accepted 26 February 2011
Available online 9 March 2011
Keywords:
Benzo[c]phenanthrolines
Benzo[c]phenanthrolinones
Dialkylaminoalkyl side chains
Cytotoxicity
DNA interactions
1. Introduction
program, the influence of the substitution of the basic cores by
dialkylaminoalkyl side chains was envisioned. Compounds
Topoisomerase 1 (Top1), a major enzyme involved in different
cellular processes, is recognised as a clinically validated target for
the research of new anticancer agents [1]. Among the different
promising classes, non camptothecin tetracyclic aromatic struc-
tures derived from natural benzophenanthridine (BZP) alkaloids
nitidine (1) and fagaronine (2) show promising activities, as illus-
trated by the currently developed drugs NSC 725776 3 and Topovale
(ARC 111) 4 (Scheme 1) [2].
Typically, it was shown in these series that the introduction of
an aminoalkyl side chain onto the tetracyclic basic core improves
DNA interactions and solubility in biocompatible solvents, inducing
increased cytotoxicity and topoisomerase affinity [3]. Recently we
focalised on the synthesis of aza-analogues of BZP with a pyridine
cycle A, in the 1,8 and 1,7 benzo[c]phenanthrolinones series
(Scheme 2) [4].
substituted on cycle A in position 1 or 2 exhibited potent cytotoxic
activities, but a moderate Top1 affinity was observed despite good
DNA interactions (Scheme 3) [5].
In the present study, new series of analogues were prepared in
order to examine the influence of the introduction of basic side
chains on cycle
C on the cytotoxicity and Top1 inhibition
(Scheme 4). Two series of compounds were envisaged for this
purpose. The first one involves introduction of the dia-
lkylaminoalkyl side chain at position N5 (A series). In the second
series, the chain is located at position C6 (B series). We describe here
the synthesis and the biological evaluation of these new substituted
tetracyclic derivatives.
2. Chemistry
Unsubstituted compounds in both series were first shown to
display cytotoxic activities within the same order of magnitude as
the parent natural BZP alkaloids [4]. In a second step of this SAR
The different benzo[c]phenanthrolines and benzo[c]phenan-
throlinones were synthetised following the route described in
Schemes 5 and 6. The strategy is based on the construction of the
four benzo[c]phenanthrolinone intermediates 5e8 through an
intermolecular heteroaromatic Suzuki-Miyaura cross coupling,
followed by a nitro reduction-in situ lactamisation sequence from
readily available starting materials (Scheme 5) [6].
* Corresponding author.
E-mail address: francois-hugues.poree@parisdescartes.fr (F.-H. Porée).
Both authors equally contributed to this work.
1
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.065

2118
C. Genès et al. / European Journal of Medicinal Chemistry 46 (2011) 2117e2131
O
R3
N
H
N
R1
R2
N
N
HN
A
N
A
O
N
R3
O
O
O
O
B
N
O
O
B
N
R1
R2
R1
R2
N
N
D
C
N
N
Cl
D
C
O
O
R1, R2: OCH2O Nitidine 1
R1: OH, R2: OCH3 Fagaronine 2
NSC 725776
3
Topovale (ARC 111)
4
benzo[c]-1,7-phenanthroline
benzo[c]-1,8-phenanthroline
R1 = R2 : OCH3 or R1, R2 = OCH2O
R3 = (CH2)nN(CH3)2 or (CH2)nN(C2H5)2
n = 2, 3
Scheme 1. Non-CPT Top1 inhibitors: examples.
The basic side chains were introduced either directly in position
N5 (A series) or, after formation of the chloro derivatives at position
C6 (B series) (Scheme 6). Following these two procedures, 23 new
substituted benzo[c]phenanthrolinones and benzo[c]phenanthro-
lines were prepared (Tables 1 and 2).
Scheme 3. Introduction of alkylaminoalkyl side chains on cycle A.
presented cytotoxicities in the micromolar range, failed to modify
the cell cycle at the tested concentrations, except compound 29
which induced strong G2/M phases cell cycle arrests. Such massive
accumulation of G2/M phase arrests, in correlation with the cyto-
toxic effect, suggests that those compounds induce major pertur-
bation of the integrity of the genomic DNA. It should be noted that
similar observations were previously noted with fagaronine (2) [7].
3. Pharmacology
3.1. Cellular effects
3.1.1. Evaluation of the cytotoxicity
3.2. DNA binding properties and inhibition of topoisomerases 1
and 2
The N5-substituted benzo[c]phenanthrolinones and C6-
substituted benzo[c]phenanthrolines were examined for their
antiproliferative activity against human colon cancer cell line HT-
29. The results (IC50) are reported in Tables 1 and 2 for the N5-
substituted and the C6-substituted series, respectively.
Several chemically related compounds exhibit a good cytotox-
icity in correlation with a good Top1 inhibition and DNA binding
potencies [8]. In order to address the pharmacological profile of the
different substituted tetracycles 17e39, modifications of Top1 and
Top2 activities, and effects on DNA were studied.
The cytotoxicity measurements indicate that methylenedioxy-
substituted compounds in the N5-subtituted series are one order of
magnitude more potent than the corresponding dimethoxy
analogues (Table 1), compounds 23e28 compared with compounds
(17e22). Similar results, but to a much lesser extend, are observed
in the C6-substituted series (serie B, Table 2), unless for the
methylenedioxy compound 36 being 2 fold less potent than its
dimethoxy analogue 30. In this series, the presence of a methyl-
enedioxy or a dimethoxy substituent at position 8,9 on cycle D does
not appear to have a dramatic influence on the biological activity.
The most potent compounds are the N5-substituted 23, 28, and
the C6-substituted 35 compounds, followed by 29, 32, and 37 (one
order of magnitude less than the first cited compounds), whereas
compounds 17, 19 and 20 (methoxy-substituted) display compa-
rable cytotoxic activities comparable to that of the reference niti-
dine (1). Also, these results show that ethylamino side chains
(NCH2CH2N(R2)) are more appropriate than their propylamino
counterparts (NCH2CH2CH2N(R2)) (up to 2 log difference) to
observe potent cytotoxic activities.
3.2.1. DNA binding studies
The ability of the two series of compounds to interact with DNA
was assessed using UV/Vis spectrometry of the compound in
absence or presence of CT-DNA (Fig. 2). The spectra of all tested
compounds were modified in the presence of CT-DNA suggesting
some drug/DNA interaction as evidenced by a strong hypochromic
effect of the peak around 275e285 nm together with a bath-
ochromic effect. In the dimethoxy-C6-substitued series, as well as
with compounds 25 and 27, an additionnal peak was observed
around 320 nm that increased upon addition of CT-DNA (up
arrows). The nature of this binding was then studied using Top1-
induced DNA relaxation studies (Fig. 3) together with CD spec-
troscopy (Fig. 4). The dimethoxy-substituted compounds from the
A or B series presented a strong decrease of the positive CD from the
DNA (270e280 nm) associated with potent DNA intercalation
profile from Top1-DNA relaxation studies. From these dimethoxy-
substitued compounds, the B series (C6-substituted) were more
efficient than the A series (N5-substituted) in relaxing the DNA
upon Top1-DNA cleavage. In the methylenedioxy series,
compounds from the B series were more potent to intercalate in the
DNA helix than their respective counterparts in the A series for
which binding seems to be more complex (Fig. 3).
3.1.2. Cell cycle blockade
The cell cycle effects were studied on the same HT-29 cell line.
As shown in Fig. 1, all six methylenedioxy derivatives in the N5-
substituted series (23e28) induced G2/M phases arrests in corre-
lation with their sub-micromolar cytotoxicities. The two methyl-
enedioxy derivatives in the C6 series 35 and 37 that were cytotoxic
at sub-micromolar concentrations also displayed G2/M phases
arrests at 48 h. By contrast, all other tested compounds that
The CD spectra also evidenced a weak positive peak generated at
w330 or 340 nm upon addition of all C6-substituted compounds,
except for 36 and for the methylenedioxy-N5-substituted
compounds. This positive band is associated with a negative peak at
N
Y
A
N
A
B
N
Y
A
Z
A
B
N
R1
R2
Z
B
B
N
R1
R2
R1
R2
R1
R2
D
Y = CH, Z = N or Y = N, Z = CH,
R1 = R2 : OCH3 or R1, R2 = OCH2O
R3 = (CH2)nN(CH3)2 or (CH2)nN(C2H5)2
n = 2, 3
C
D
D
C
C
D
C
O
N
R3
HN
R3
benzo[c]-1,7-phenanthroline
benzo[c]-1,8-phenanthroline
benzo[c]phenanthroline
benzo[c]phenanthrolinone
R1 = R2 : OCH3 or R1, R2 = OCH2O
serie A
serie B
Scheme 2. Benzo[c]phenanthroline skeletons.
Scheme 4. New series: side chain on cycle C in position N5 (serie A) or C6 (serie B).

C. Genès et al. / European Journal of Medicinal Chemistry 46 (2011) 2117e2131
2119
Table 1
1 ) PdCl2dppf, pinacolborane
Y
Z
Cytotoxicities of N5-substituted Analogues (Serie A).
NaHCO3, CH3CN, Δ
R1
R2
Br
R1
R2
Y
Y = N, Z = CH or Y = CH, Z = N
R1 = R2 = OCH3 or R1, R2
OCH2O
CO2Me
Z
NaH, NaI
DMF,
Y
NO2
CO2Me
2 ) PdCl2dppf, K2CO3,
CH3CN, microwaves
Z
=
R1
R2
R1
R2
9 R1 = R2 : OCH3
10 R1, R2 = OCH2O
N
R
NH
13 R1 = R2 : OCH3, Y = CH, Z = N
14 R1 = R2 : OCH3, Y = N, Z = CH
15 R1, R2 = OCH2O, Y = CH, Z = N
16 R1, R2 = OCH2O, Y = N, Z = CH
Y
Z
R-Cl
Cl
Cl
N
O
Cl
N
R-Cl:
N
O
X
NO2
11 Y = CH, Z = N, X = Br
12 Y = N, Z = CH, X = OTf
Compound
Nitidine 1
R1, R2
Y, Z
Amino chain
IC50 mM (HT-29)
2.75 ꢀ 0.15
1.42 ꢀ 0.60
17-k7755
18-k7773
19-k7758
20-k7775
21-k7792
22-k7774
23-k7896
24-k7953
25-k7952
26-k7893
27-k7969
28-k7895
(OCH3)2
(OCH3)2
(OCH3)2
(OCH3)2
(OCH3)2
(OCH3)2
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
Y ¼ CH,
Z ¼ N
NCH2CH2N(CH3)2
Y
cat Ni Raney, H2 (5 bar)
EtOH, r.t.
Z
5 R1 = R2 : OCH3, Y = CH, Z = N
6 R1 = R2 : OCH3, Y = N, Z = CH
7 R1, R2 = OCH2O, Y = CH, Z = N
8 R1, R2 = OCH2O, Y = N, Z = CH
R1
R2
Y ¼ CH,
Z ¼ N
NCH2CH2CH2N(CH3)2
NCH2CH2N(C2H5)2
NCH2CH2N(CH3)2
6.11 ꢀ 0.50
1.82 ꢀ 0.31
1.66 ꢀ 0.29
4.98 ꢀ 0.78
2.63 ꢀ 0.38
0.078 ꢀ 0.016
0.24 ꢀ 0.125
0.23 ꢀ 0.11
0.12 ꢀ 0.03
0.23 ꢀ 0.08
0.075 ꢀ 0.023
NH
or
Y ¼ CH,
Z ¼ N
SnCl2, MeOH reflux
O
Y ¼ N,
Z ¼ CH
Y ¼ N,
Z ¼ CH
Y ¼ N,
Z ¼ CH
Y ¼ CH,
Z ¼ N
Scheme 5. Preparation of the benzophenanthrolinone intermediates.
NCH2CH2CH2N(CH3)2
NCH2CH2N(C2H5)2
NCH2CH2N(CH3)2
w300 nm (down arrow) in the dimethoxy-C6-substituted series
(29e34), suggesting some differences in the DNA interaction
between the dimethoxy- and methylenedioxy-subtituted series
(Fig. 4).
Neither the Top1-induced DNA relaxation nor the CD spectra
measurements permit to conclude for a typical DNA intercalation or
minor groove binding of the compounds. The DNA binding mode
seems more complex, with some intercalation of part of the
molecule between adjacent base pairs and other mode of binding.
Y ¼ CH,
Z ¼ N
NCH2CH2CH2N(CH3)2
NCH2CH2N(C2H5)2
NCH2CH2N(CH3)2
Y ¼ CH,
Z ¼ N
Y ¼ N,
Z ¼ CH
Y ¼ N,
Z ¼ CH
Y ¼ N,
Z ¼ CH
NCH2CH2CH2N(CH3)2
NCH2CH2N(C2H5)2
3.2.2. Topoisomerases 1 and 2 inhibition assays
The potency of the two series of compounds to inhibit the
activity of human Top1 and Top2 was next envisaged.
DNA cleavage assays were used to examine the effects of the
different compounds on the catalytic activity of topoisomerases 1
and 2 (Figs. 5 and 6). For Top1, the assay was carried out with a 30 end
labelled DNA fragment and camptothecin (CPT) was used as the
reference Top1 inhibitor (Fig. 5) [9]. After resolution on a denaturing
polyacrylamide gel, the induction of Top1-DNA complexes was evi-
denced by the formation of specific DNA cleavage bands. In our case,
no inhibition of the enzyme activity was observed for the different
tetracycles when compared to camptothecin and the structure
related compound nitidine (1). As evidenced by the increased
intensity of the DNA cleavage bands, nitidine (1) presented some
different base sequence preferences, which could ultimately lead to
different pharmacological outcomes. Particularly, nitidine (1)
generated cleavages at CG, CA and TA steps additionally to the typical
CPT-induced cleavage at TG steps.
essential target responsible for the cytotoxicity measured and
introduction ofanaminoalkyl sidechaininpositionN5orC6does not
influence the Top inhibition [4,5]. These results are interesting from
a SAR point of view, when comparing these structures to ARC 111 (4),
which exhibits a good Top1 inhibition correlated with a good cyto-
toxicity. This type of observation has been previously reported in
analogous series and outlines the low level of tuning in these
chemically related families [12]. Because of the DNA binding activity
of these series of compounds, we evaluated the suppressor activity of
the compounds on CPT-induced DNA cleavages (Fig. 7). Addition of
increasing concentrations of the evaluated compounds toTop1 in the
Table 2
Cytotoxicities of C6-substituted Analogues (Serie B).
For Top2 experiments, a supercoiled plasmid DNA was used and
the cleaved DNA products were resolved by gel electrophoresis on
agarose (Fig. 6) [10]. In the presence of the reference Top2 inhibitor
etoposide, supercoiled plasmid DNA (Sc) is cleaved to form linear
(Lin) and nicked (Nck) DNAs after resolution. A weaker effect is
observed with nitidine 1 [11]. For all the substituted tetracycles no
significant Top2 poisoning effect was observed. As previously noted
in these aza-series, topoisomerases inhibition does not constitute the
PhPOCl2
160°C
Y
Y
Y
Z
Z
Z
R1
R2
R1
R2
R1
R2
NH
N
N
R
O
Cl
HN
Compound R1, R2
Y, Z
Amino chain
IC50 mM (HT-29)
Nitidine 1
2.75 ꢀ 0.15
1.02 ꢀ 0.29
3.14 ꢀ 0.99
5.47 ꢀ 0.48
0.97 ꢀ 0.20
3.33 ꢀ 0.96
5.77 ꢀ 0.51
29-k7121 (OCH3)2 Y ¼ CH, Z ¼ N NCH2CH2N(CH3)2
30-k7116 (OCH3)2 Y ¼ CH, Z ¼ N NCH2CH2CH2N(CH3)2
31-k7119 (OCH3)2 Y ¼ CH, Z ¼ N NCH2CH2CH2N(C2H5)
32-k7166 (OCH3)2 Y ¼ N, Z ¼ CH NCH2CH2N(CH3)2
33-k7164 (OCH3)2 Y ¼ N, Z ¼ CH NCH2CH2CH2N(CH3)2
34-k7165 (OCH3)2 Y ¼ N, Z ¼ CH NCH2CH2CH2N(C2H5)
35-k7887 OCH2O Y ¼ CH, Z ¼ N NCH2CH2N(CH3)2
36-k7968 OCH2O Y ¼ CH, Z ¼ N NCH2CH2CH2N(CH3)2
37-k7894 OCH2O Y ¼ N, Z ¼ CH NCH2CH2N(CH3)2
38-k7772 OCH2O Y ¼ N, Z ¼ CH NCH2CH2CH2N(CH3)2
1 ) PhPOCl2, Δ
NaH, NaI,
Y
Y
Y
Z
Z
Z
DMF, Δ
2 ) R3NH2
R1
R2
R1
R2
R1
R2
N
NH
N
R3
R3-Cl
HN
O
O
R3
5 R1 = R2 : OCH3, Y = CH, Z = N
6 R1 = R2 : OCH3, Y = N, Z = CH
7 R1, R2 = OCH2O, Y = CH, Z = N
8 R1, R2 = OCH2O, Y = N, Z = CH
0.070 ꢀ 0.021
7.62 ꢀ 1.03
0.11 ꢀ 0.01
1.73 ꢀ 0.09
see table 2
compounds 29-39
see table 1
compounds 17-28
39-k7970 OCH2O Y ¼ N, Z ¼ CH NCH2CH2CH2N(C2H5)2 1.83 ꢀ 0.42
Scheme 6. Introduction of the aminoalkyl chains.

2120
C. Genès et al. / European Journal of Medicinal Chemistry 46 (2011) 2117e2131
Fig. 1. Cell-cycle effect. Increasing concentrations (as indicated on the top of the plots, in
m
M) of the various drugs were added to HT-29 colon carcinoma cells for 48 h prior to IP
staining and FACS analysis.
presence of CPT evidenced a concentration-dependant inhibition of
CPT-induced poisoning effects. Particularly, the most cytotoxic
compounds 23 and 35 are those presenting the highest inhibition of
CPT-induced poisoning. A correlation between cytotoxic activities
and the efficiency of the inhibition of CPT-induced poisoning was
established for all tested compounds (Fig. 8). From this graph, it is
clear that all strongly cytotoxic compounds (in bold, bellow the
was incubated with increasing concentrations of the different
substituted tetracycles. After digestion by a DNase I and resolution
by gel electrophoresis, some sequence preferences at 50-CTA sites
were observed. It is noteworthy that benzo[c]phenanthrolines
substituted by an aminoalkylamine chain on cycle A (position 1 or
2) present some preferential DNA intercalation sequences (50-CT).
Compounds presenting the highest abilities to interact with this
sequence belong to the most cytotoxic series (methylenedioxy-
substituted in the N5-series, compounds 23e38) whereas the
horizontal dotted lane fixed at 0.5 mM) are those presenting the
highest inhibition of CPT-induced Top1-poisoning. Similarly, all
dimethoxy-substituted derivatives in the A or B series (N5- or C6-
substituted compounds) are cytotoxic at higher concentrations and
are weaker inhibitors of CPT-induced poisoning. Such results suggest
a correlation between the inhibition of CPT-induced poisoning and
the cytotoxic effect of these series.
weakest (>3
mM) cytotoxic compounds 30, 31 and 36 failed to
bind this sequence.
4. Conclusion
Aza-analogues of the natural alkaloids nitidine (1) and fagar-
onine (2) were substituted on cycle C by different aminoalkylamino
chain, in order to improve their biological activities. The anti-
proliferative profile of the natural compounds has been amended,
particularly in the case of methylenedioxy bearing benzo[c]phe-
nanthrolinone derivatives. The pharmacological studies outline
that these new analogues display a good DNA affinity, but appear to
3.2.3. Sequence-specific DNA binding
The DNA relaxation profiles of the different tetracycles also
suggested some intercalation. Consequently, a DNase I foot-
printing analysis was carried out to explore the potency of
compounds 17e39 to bind DNA in a sequence-specific manner
(Figs. 9and10). A radio-labelled 265-bp DNA restriction fragment

C. Genès et al. / European Journal of Medicinal Chemistry 46 (2011) 2117e2131
2121
Fig. 2. UV/visible spectrometry. The various indicated compounds (20
m
M) were incubated alone (top lanes), with a fixed concentration of CT-DNA (200
m
M) or with increasing
concentrations of CT-DNA (from 1 to 200
mM, top to bottom). Nitidine 1 (grey box) was used as a reference compound.
be weak substrates for Top1 and Top2 as previously observed for
related benzophenanthroline derivatives. It is noteworthy that the
more cytotoxic compounds (methylenedioxy-substituted in the A
series) do not display efficient DNA intercalation potencies (Figs. 3
and 4) but inhibit CPT-induced Top1-poisoning effects and present
common abilities to display specific DNA binding propensities.
In conclusion, from a structure-activity viewpoint, the replace-
ment of the benzenic A ring in the natural BZP alkaloids by a pyri-
dine in the benzophenanthroline and benzophenanthrolinone
series resulted in a significant increase in the cytotoxicity associ-
ated with a loss of the topoisomerase affinity.
5. Experimental section
5.1. Chemistry
5.1.1. General
All reactions were carried out in a dried glassware under an
argon atmosphere. All solvents were purchased with an analytical
grade from SDS. CH2Cl2 was distilled from CaH2. Methanol, aceto-
nitrile and DMF were dried over molecular sieves. All other
commercially available reagents were used as received. Flash
chromatography was performed with silica gel CHROMATOGEL 60

2122
C. Genès et al. / European Journal of Medicinal Chemistry 46 (2011) 2117e2131
Fig. 3. Topoisomerase 1-induced DNA relaxation. Effect of compounds YeY on the relaxation of a supercoiled plasmid DNA by human Top1. Native supercoiled pUC19 vector
(130 ng, lanes DNA) was incubated with 4 units of Top1 in the absence (lanes 0) or presence of tested compound at the indicated concentration ( M). Samples were separated by
m
electrophoresis on a 1% agarose gel, which was stained post-electrophoresis with ethidium bromide after DNA migration. Gels were photographed under UV light. Nck, nicked;
Sc, supercoiled; Rel, relaxed; Topo, topoisomer products.
(particle size 20e45
m
or 35e70
m
) supplied by SDS. Yield refers to
heated at 110 ꢂC for 3 h. After disappearance of the starting material
(TLC monitoring), the mixture was cooled to r.t. and quenched by
addition of a few drops of water. The solvent was removed under
vacuum and the crude product was dissolved in 1 M aqueous HCl
solution (50 mL). The aqueous layer was washed with CH2Cl2
(3 ꢃ 50 mL), basified with a 30% aqueous NaOH solution and
extracted with CH2Cl2 (6 ꢃ 75 mL). The organic layers were
combined, dried over MgSO4 and concentrated under vacuum. The
chromatography and spectroscopically pure compounds, unless
otherwise noted. 1H NMR spectra were recorded on a BRUCKER AC
300 MHz spectrometer or a BRUCKER AVANCE 400 MHz spec-
trometer. Chemical shifts are reported in ppm and corrected to dH
7.26 for CDCl3 as internal reference, coupling constants (J) in Hz. 13
C
NMR spectra were recorded on a BRUCKER AC 300 spectrometer at
75 MHz. Chemical shifts are reported in ppm and corrected to dC
77.16 for CDCl3 as internal reference. The assignments are based
upon 1D and 2D spectra recorded using the following pulse
sequences from the Bruker standard pulse program library: DEPT,
COSY, HSQC, HMBC and NOESY. Mass spectra were measured with
a ZQ 2000 Waters mass spectrometer (ESI). High resolution mass
spectra were obtained on a Q-ToF1 ESI mass spectrometer (Waters).
Infra-red spectra were recorded on a Nicolet FT-IR spectrometer
residue was purified by chromatography on silica gel (20/45
m,
eluent CH2Cl2/MeOH/NH4OH 95/4.35/0.65 v/v/v).
5.1.2.1. 5-[20-(dimethylamino)ethyl]-8,9-dimethoxy
benzo[c][1,8]
phenanthrolin-6(5H)-one (17). (49% yield) IR (CH2Cl2, cmꢁ1) 3403,
2934, 2854, 2778, 2356, 2343,1731,1645,1615,1591,1523,1486,1455,
1417, 1329, 1274, 1209, 1035, 835, 789, 757, 723. 1H NMR (300 MHz,
and wavelengths (
on a Beckman DU 640 and wavelengths (
y
) are given in cmꢁ1. UV spectra were recorded
) are given in nm.
CDCl3)
d
9.29 (s, 1H), 8.84 (d, J ¼ 5.6 Hz, 1H), 8.72 (d, J ¼ 5.6 Hz, 1H),
l
8.32 (d, J ¼ 8.9 Hz,1H), 7.85 (d, J ¼ 8.9 Hz,1H), 7.77 (s,1H), 7.72 (s,1H),
4.91 (t, J ¼ 5.9 Hz, 2H), 4.14 (s, 3H), 4.10 (s, 3H), 3.01 (t, J ¼ 5.9 Hz, 2H),
5.1.2. General procedure for the preparation of the N5
dialkylaminoalkylbenzo[c]phenantholinone
2.50 (s, 2 ꢃ 3H); 13C NMR (75 MHz, CDCl3)
d 157.9,153.0,151.3,150.3,
144.2, 137.7, 135.0, 130.4, 128.0, 122.9, 121.4, 120.9, 117.4, 115.1, 104.6,
102.4, 63.7, 57.8, 56.3, 56.2, 45.8 (2C). MS (ESþ) m/z 378 [M þ H]þ.
HRMS (ESþ) m/z calcd for C22H24N3O3 [M þ H]þ 378.1818, found
378.1807.
At 0 ꢂC, to a solution of a benzo[c]phenanthrolin-6-one (80 mg),
NaI (1.5 equiv.) and the appropriate dialkylaminoalkylchloride
hydrochloride (1.1 equiv.) in DMF (20 mL) was added NaH (60%
mineral oil suspension, 3 equiv.) in small portions over 5 min. The
mixture was allowed to warm to r.t. with stirring for 45 min. Then
the flask was transferred into a preheated oil bath (65 ꢂC), and
5.1.2.2. 5-[30-(dimethylamino)propyl]-8,9-dimethoxy benzo[c][1,8]
phenanthrolin-6(5H)-one (18). (5% yield) IR (CH2Cl2, cmꢁ1) 3400,

C. Genès et al. / European Journal of Medicinal Chemistry 46 (2011) 2117e2131
2123
Fig. 4. Circular dichroism. A fixed concentration of CT-DNA vas incubated with he various indicated compounds at a fixed or at increasing concentrations. Nitidine 1 (grey box) was
used as a reference compound.
2915, 1646, 1615, 1591, 1523, 1485, 1448, 1426, 1275, 1093, 1034; 1H
NMR (300 MHz, CDCl3)
5.1.2.3. 5-[20-(diethylamino)ethyl]-8,9dimethoxy benzo[c][1,8]phe-
nanthrolin-6(5H)-one (19). (48% yield) IR (CH2Cl2, cmꢁ1) 3379,
2957, 2921, 2851, 1731, 1714, 1592, 1523, 1455, 1423, 1329, 1273,
d
9.25 (s,1H), 8.83 (d, J ¼ 5.8 Hz,1H), 8.66 (d,
J ¼ 5.8 Hz,1H), 8.36 (d, J ¼ 9.0 Hz,1H), 7.90 (d, J ¼ 9.0 Hz,1H), 7.80 (s,
1H), 7.70 (s,1H), 4.84 (t, J ¼ 6.2 Hz, 2H), 4.14 (s, 3H), 4.11 (s, 3H), 3.02 (t,
J ¼ 7.5 Hz, 2H), 2.63 (s, 2 ꢃ 3H), 2.48e2.40 (m, 2H); 13C NMR (75 MHz,
1209, 1162, 1121, 1092, 1037, 789; 1H NMR (300 MHz, CDCl3)
d 9.24
(s, 1H), 8.83 (d, J ¼ 5.8 Hz, 1H), 8.66 (d, J ¼ 5.8 Hz, 1H), 8.33 (d,
J ¼ 9.1 Hz, 1H), 7.85 (d, J ¼ 9.1 Hz, 1H), 7.77 (s, 1H), 7.71 (s, 1H), 4.93
(t, J ¼ 6.0 Hz, 2H), 4.13 (s, 3H), 4.07 (s, 3H), 3.22 (t, J ¼ 6.0 Hz, 2H),
2.86 (q, J ¼ 7.2 Hz, 2x2H), 1.21 (t, J ¼ 7.2 Hz, 2 ꢃ 3H); 13C NMR
CDCl3)
d 157.8, 153.1, 151.0, 150.4, 143.6, 137.4, 135.1, 130.3, 128.0,
122.8, 121.6, 121.1, 117.6, 115.0, 104.3, 102.4, 63.6, 56.6, 56.2 (2C), 44.2
(2C), 25.7; MS (ESþ) m/z 392 [M þ H]þ; HRMS (ESþ) m/z calcd for
C23H26N3O3 [Mþ H]þ 392.1974, found392.1983;U.V. lmax (nm) (log
e)
(75 MHz, CDCl3) d 157.8, 152.9, 151.0, 150.3, 143.7, 137.5, 135.0, 130.3,
(CH2Cl2) 196 (3.7), 222 (4.1), 229 (4.1), 281 (4.4), 361 (3.4).
128.0, 122.7, 121.5, 121.0, 117.6, 115.0, 104.4, 102.4, 63.5, 56.2, 56.1,

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C. Genès et al. / European Journal of Medicinal Chemistry 46 (2011) 2117e2131
Fig. 5. Topoisomerase 1 poisoning assays. Potential cleavage of the 117-mer DNA fragment by Top1 was studied in the presence of the compounds YeY. The 30-end labelled
fragment (0) was incubated in the absence (lane TopoI) or presence of the tetracycles at the indicated concentrations ( M). Camptothecin (CPT) was used as a positive control at
concentrations of 20e50 M Top1 cleavage reactions were analyzed on an 8% denaturing polyacrylamide gel, compared to the guanine track labelled (G).
m
m
50.9, 47.9 (2C), 11.2 (2C); MS (ESþ) m/z 405 [M þ H]þ; HRMS (ESþ)
m/z calcd for C24H28N3O3 [M þ H]þ 406.2131, found 406.2112; U.V.
5.1.2.4. 5-[20-(dimethylamino)ethyl]-8,9-dimethoxy
benzo[c][1,7]
phenanthrolin-6(5H)-one (20). (23% yield) IR (CH2Cl2, cmꢁ1) 3370,
2943, 1614, 1592, 1525, 1496, 1463, 1426, 1416, 1328, 1272, 1208,
1179, 1116, 1059, 1031, 1001, 820, 758; 1H NMR (300 MHz, CDCl3)
lmax (nm) (log
e) (CH2Cl2) 214 (3.8), 222 (4.3), 229 (4.3), 281 (4.6),
342 (3.6), 359 (3.5).

C. Genès et al. / European Journal of Medicinal Chemistry 46 (2011) 2117e2131
2125
5.1.2.6. 5-[20-(diethylamino)ethyl]-8,9-dimethoxy benzo[c][1,7]phe-
nanthrolin-6(5H)-one (22). (35% yield) IR (CH2Cl2, cmꢁ1) 3381,
2966, 2925, 1659, 1592, 1524, 1428, 1272, 1116, 1087, 1060, 1037,
786; 1H NMR (300 MHz, CDCl3)
d
9.47 (dd, J ¼ 8.4, 1.5 Hz, 1H), 9.00
(dd, J ¼ 4.3, 1.5 Hz, 1H), 8.55 (d, J ¼ 9.3 Hz, 1H), 8.09 (d, J ¼ 9.3 Hz,
1H), 7.86 (s, 1H), 7.74 (s, 1H), 7.59 (dd, J ¼ 8.4, 4.3 Hz, 1H), 4.95 (t,
J ¼ 6.1 Hz, 2H), 4.17 (s, 3H), 4.10 (s, 3H), 3.21 (t, J ¼ 6.1 Hz, 2H), 2.86
(q, J ¼ 7.2 Hz, 2 ꢃ 2H), 1.23 (t, J ¼ 7.2 Hz, 2 ꢃ 3H); 13C NMR (75 MHz,
CDCl3)
d 158.1, 153.0, 150.3, 149.8, 148.6, 138.8, 133.0, 130.6, 126.6,
125.5, 123.6, 121.2, 118.2, 114.4, 104.4, 102.2, 64.4, 56.1 (2C), 51.2,
48.1 (2C), 11.9 (2C); MS (ESþ) m/z 405 [M þ H]þ; HRMS (ESþ) m/z
calcd for C24H28N3O3 [M þ H]þ 406.2131, found 406.2126; U.V. lmax
(nm) (loge) (CH2Cl2) 222 (4.4), 282 (4.6).
5.1.2.7. 5-[20-(dimethylamino)ethyl]-8,9-methylene
[1,8]phenanthrolin-6(5H)-one (23). (14% yield) IR (CH2Cl2, cmꢁ1
3363, 2911, 2845, 2763, 1620, 1576, 1489, 1467, 1260, 1195, 1032,
939, 836, 759; 1H NMR (300 MHz, CDCl3)
9.30 (s, 1H), 8.87 (d,
dioxybenzo[c]
)
d
J ¼ 5.7 Hz, 1H), 8.74 (d, J ¼ 5.7 Hz, 1H), 8.31 (d, J ¼ 9.0 Hz, 1H), 7.87
(d, J ¼ 9.0 Hz, 1H), 7.86 (s, 1H), 7.76 (s, 1H), 6.18 (s, 2H), 4.91 (t,
J ¼ 5.8 Hz, 2H), 2.99 (t, J ¼ 5.8 Hz, 2H), 2.48 (s, 2 ꢃ 3H); 13C NMR
(75 MHz, CDCl3) d 158.3, 151.6, 151.3, 148.6, 144.2, 137.9, 135.0, 132.2,
128.1, 122.8, 121.5, 121.2, 117.5, 116.5, 102.7, 102.0, 100.4, 64.5, 58.2,
46.1 (2C); MS (ESþ) m/z 362 [M þ H]þ; HRMS (ESþ) m/z calcd for
C21H20N3O3 [M þ H]þ 362.1505, found 362.1487; U.V. lmax (nm)
(loge) (CH2Cl2) 281 (4.6), 342 (3.4), 360 (3.4).
5.1.2.8. 5-[30-(dimethylamino)propyl]-8,9-methylene dioxybenzo[c]
[1,8]phenanthrolin-6(5H)-one (24). (13% yield) IR (CH2Cl2, cmꢁ1
3386, 2946, 2911, 2816, 2764, 1625, 1585, 1493, 1470, 1455, 1332,
1261,1233,1197,1036, 838, 763; 1H NMR (300 MHz, CDCl3)
9.30 (s,
)
d
1H), 8.88 (d, J ¼ 5.7 Hz, 1H), 8.74 (d, J ¼ 5.7 Hz, 1H), 8.31 (d,
J ¼ 9.0 Hz, 1H), 7.88 (d, J ¼ 9.0 Hz, 1H), 7.86 (s, 1H), 7.74 (s, 1H), 6.19
(s, 2H), 4.82 (t, J ¼ 6.4 Hz, 2H), 2.62 (t, J ¼ 7.1 Hz, 2H), 2.35 (s, 6H),
Fig. 6. Topoisomerase 2 poisoning assays. Gel shows the effect of tetracycles YeY on
the cleavage of a supercoiled plasmid DNA by topoisomerase 2. Native supercoiled
pUC19 plasmid DNA (350 ng) (lanes DNA) was incubated with 10 units topoisomerase
2.19 (td, J ¼ 7.1, 6.4 Hz, 2H); 13C NMR (75 MHz, CDCl3)
d 158.4, 151.5,
151.3, 148.6, 144.2, 138.0, 135.0, 132.2, 128.1, 122.7, 121.5, 121.1, 117.5,
116.6, 102.5, 102.0, 100.5, 64.8, 56.8, 45.6 (2C), 27.4; MS (ESþ) m/z
376 [M þ H]þ; HRMS (ESþ) m/z calcd for C22H22N3O3 [M þ H]þ
2 in the absence (lanes 0) or presence of the indicated drugs at 100
to the reference compound etoposide (50 M). Reactions were stopped with sodium
dodecylsulfate and treatment with proteinase K. DNA samples were separated by
electrophoresis on 1% agarose gels containing ethidium bromide (1 g/mL) and then
photographed under UV light. Nck, nicked; Lin, linear; Sc, supercoiled DNA.
mM, and compared
m
m
376.1661, found 376.1659; U.V. lmax (nm) (loge) (CH2Cl2) 215 (4.0),
222 (4.6), 230 (4.3), 281 (4.7), 343 (3.6), 362 (3.5).
5.1.2.9. 5-[20-(diethylamino)ethyl]-8,9-methylene dioxybenzo[c][1,8]
phenanthrolin-6(5H)-one (25). (22% yield) IR (CH2Cl2, cmꢁ1) 3385,
2917, 2840, 1620, 1587, 1470, 1328, 1263, 1195, 1129, 1035; 1H NMR
d
9.45 (dd, J ¼ 8.1, 1.8 Hz, 1H), 8.99 (dd, J ¼ 4.1, 1.8 Hz, 1H), 8.53 (dd,
J ¼ 9.2, 1.8 Hz, 1H), 8.08 (d, J ¼ 9.2 Hz, 1H), 7.85 (s, 1H), 7.74 (s, 1H),
7.56 (dd, J ¼ 8.1, 4.1 Hz, 1H), 4.91 (t, J ¼ 6.0 Hz, 2H), 4.14 (s, 3H), 4.10
(s, 3H), 2.99 (t, J ¼ 6.0 Hz, 2H), 2.46 (s, 6H); 13C NMR (75 MHz,
(300 MHz, CDCl3)
d
9.31 (s, 1H), 8.89 (d, J ¼ 5.7 Hz, 1H), 8.74 (d,
J ¼ 5.7 Hz,1H), 8.32 (d, J ¼ 9.0 Hz,1H), 7.90 (d, J ¼ 9.0 Hz,1H), 7.89 (s,
1H), 7.75 (s,1H), 6.20 (s, 2H), 4.89 (t, J ¼ 6.3 Hz, 2H), 3.13 (t, J ¼ 6.3 Hz,
2H), 2.79 (q, J ¼ 7.1 Hz, 2x2H), 1.18 (t, J ¼ 7.1 Hz, 2 ꢃ 3H); 13C NMR
CDCl3)
d 158.1, 152.9, 150.3, 149.7, 148.6, 138.8, 133.0, 130.6, 126.6,
125.4, 123.5, 121.2, 118.2, 114.4, 104.5, 102.2, 64.4, 58.2, 56.1 (2C),
46.2 (2C); MS (ESþ) m/z 378 [M þ H]þ; HRMS (ESþ) m/z calcd for
C22H24N3O3 [M þ H]þ 378.1818, found 378.1818; U.V. lmax (nm)
(75 MHz, CDCl3) d 158.2, 151.7, 151.3, 148.7, 144.3, 138.0, 135.0, 132.4,
128.1, 123.0, 121.6, 121.2, 117.5, 116.6, 102.6, 102.1, 100.6, 64.7, 51.2,
48.0 (2C), 11.8 (2C); MS (ESþ) m/z 390 [M þ H]þ; HRMS (ESþ) m/z
calcd for C23H24N3O3 [M þ H]þ 390.1818, found 390.1815; U.V. lmax
(loge) (CH2Cl2) 222 (4.68), 282 (4.98), 338 (3.98), 353 (3.65).
(nm) (loge) (CH2Cl2) 219 (4.3), 222 (4.3), 228 (4.0), 281 (4.3).
5.1.2.5. 5-[30-(dimethylamino)propyl]-8,9-dimethoxy benzo[c][1,7]
phenanthrolin-6(5H)-one (21). (13% yield) IR (CH2Cl2, cmꢁ1) 3369,
2944, 1660, 1614, 1592, 1525, 1496, 1428, 1415, 1332, 1272, 1208,
5.1.2.10. 5-[20-(dimethylamino)ethyl]-8,9-methylene dioxybenzo[c]
[1,7]phenanthrolin-6(5H)-one (26). (17% yield) IR (CH2Cl2, cmꢁ1
3371, 2915, 2854, 2357, 2342, 1658, 1478, 1457, 1257, 1031; 1H NMR
(300 MHz, CDCl3)
)
1178, 1059, 1037, 820, 788; 1H NMR (300 MHz, CDCl3)
d 9.47 (ddd,
J ¼ 8.3,1.8, 0.7 Hz,1H), 8.99 (d, J ¼ 4.3,1.8 Hz,1H), 8.53 (d, J ¼ 9.2 Hz,
1H), 8.08 (dd, J ¼ 9.2, 0.7 Hz, 1H), 7.84 (s, 1H), 7.72 (s, 1H), 7.58 (d,
J ¼ 8.3, 4.3 Hz, 1H), 4.84 (t, J ¼ 6.5 Hz, 2H), 4.16 (s, 3H), 4.10 (s, 3H),
2.63 (t, J ¼ 7.4 Hz, 2H), 2.35 (s, 2 ꢃ 3H), 2.27e2.20 (m, 2H); 13C NMR
d
9.46 (dd, J ¼ 8.4, 1.4 Hz, 1H), 9.00 (dd, J ¼ 4.3,
1.4 Hz, 1H), 8.48 (d, J ¼ 9.3 Hz, 1H), 8.07 (d, J ¼ 9.3 Hz, 1H), 7.90 (s,
1H), 7.77 (s, 1H), 7.57 (dd, J ¼ 8.4, 4.3 Hz, 1H), 6.19 (s, 2H), 4.92 (t,
J ¼ 5.9 Hz, 2H), 3.00 (t, J ¼ 5.9 Hz, 2H), 2.48 (s, 2 ꢃ 3H); 13C NMR
(75 MHz, CDCl3)
d
158.3, 152.9, 150.3, 149.8, 148.6, 138.9, 133.1,
(75 MHz, CDCl3) d 158.3,151.6,150.5,148.7,148.2,139.0,133.0,132.6,
130.6, 126.6, 125.3, 123.6, 121.1, 118.1, 114.5, 104.3, 102.2, 64.6, 56.9,
56.1 (2C), 45.6 (2C), 27.4; MS (ESþ) m/z 392 [M þ H]þ; HRMS (ESþ)
m/z calcd for C23H26N3O3 [M þ H]þ 392.1974, found 392.1978; U.V.
126.5, 125.6, 123.7, 121.2, 118.6, 115.8, 102.6, 102.0, 100.3, 64.3, 58.1,
46.0 (2C); MS (ESþ) m/z 362 [M þ H]þ; HRMS (ESþ) m/z calcd for
C21H20N3O3 [M þ H]þ 362.1505, found 362.1492; U.V. lmax (nm)
lmax (nm) (log
e
) (CH2Cl2) 222 (4.5), 282 (4.7).
(loge) (CH2Cl2) 222 (4.2), 239 (4.2), 281 (4.6).

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C. Genès et al. / European Journal of Medicinal Chemistry 46 (2011) 2117e2131
Fig. 7. Inhibition of CPT-induced poisoning of Top1. The 30-end-labelled 117 bp DNA fragment was incubated alone (0) or with Top1 (4 units), in the presence of Top1-poisoning
compound campthotecin (CPT, 20
m
M) together with increasing concentrations (
mM) of the indicated compounds. Top1 cleavage reactions were analyzed on a 8% denaturing
polyacrylamide gel, compared to the guanine track labelled (G).
5.1.2.11. 5-[30-(dimethylamino)propyl]-8,9-methylene dioxybenzo[c]
1.4 Hz, 1H), 8.48 (d, J ¼ 9.2 Hz, 1H), 8.08 (d, J ¼ 9.2 Hz, 1H), 7.91 (s,
[1,7]phenanthrolin-6(5H)-one (27). (6% yield) IR (CH2Cl2, cmꢁ1
)
1H), 7.75 (s, 1H), 7.59 (dd, J ¼ 8.3, 4.3 Hz, 1H), 6.20 (s, 2H), 4.84 (t,
J ¼ 6.3 Hz, 2H), 2.74 (t, J ¼ 7.6 Hz, 2H), 2.43 (s, 2 ꢃ 3H), 2.28 (qu,
3374, 2916, 2845, 1606, 1470, 1447, 1250, 1135, 1035; 1H NMR
(300 MHz, CDCl3)
d
9.48 (dd, J ¼ 8.3, 1.4 Hz, 1H), 9.01 (dd, J ¼ 4.3,
J ¼ 2H); 13C NMR (75 MHz, CDCl3)
d 158.3, 151.6, 150.5, 148.7, 148.2,

C. Genès et al. / European Journal of Medicinal Chemistry 46 (2011) 2117e2131
2127
1H, J ¼ 9.0 Hz), 7.97 (s, 1H), 7.86 (s, 1H), 4.23 (s, OCH3), 4.19 (s,
OCH3). 13C NMR (75 MHz, CDCl3)
153.7, 151.1, 149.7, 145.0, 138.4,
135.2, 132.4, 130.8, 125.8, 123.3, 120.7, 106.5, 102.3, 56.4, 56.3. MS
(CI, NH3) m/z 325, 327 [M þ H]þ. HRMS (ESþ) m/z calcd for
C18H13ClN2O2 [M þ H]þ 324.0666, found 324.0668.
Inhibition of CPT-poisoning
Survival
50
10
50
10
d
1
5.1.3.3. 6-Chloro-8,9-methylenedioxybenzo[c][1,7]
phenanthroline. (74% yield). IR 2250 (
n
CN), 1477, 1736, 1470, 1296,
9.60 (dd,
CeCl) 956, 826 cmꢁ1 1H NMR (400 MHz, CDCl3)
d
0.1
1036 (
n
1H, J ¼ 8.0 Hz, 1 Hz), 9.05 (d, 1H, J ¼ 4.0 Hz, 1 Hz), 8.60 (d, 1H,
1
J ¼ 9.0 Hz), 8.22 (d, 1H, J ¼ 9.0 Hz), 7.99 (s, 1H); 7.82 (s, 1H), 7.68 (dd,
1H, J ¼ 8.0 Hz, 4.0 Hz), 6,24 (s, 2H). 13C NMR (75 MHz, CDCl3)
d 155.1,
(OCH3)2
OCH2O
(OCH3)2
OCH2O
149.9, 147.5 (2C), 142.3, 135.7, 133.7, 128.2, 126.5, 127.2, 123.8, 121.8,
121.1, 121.0, 104.0, 102.4, 99.9. MS (CI, NH3) m/z 309, 311 [M þ H]þ.
HRMS (ESþ) m/z calcd for C17H9ClN2O2 [M þ H]þ 308.0353, found
308.0351.
N5-substituted
C6-substituted
Fig. 8. Correlation analysis between the cytotoxic activity (IC50 for survival on HT-29
cells as determined in Tables 1 and 2) and the inhibition for CPT-induced Top1-
poisoning (IC50 calculated from quantification from Fig. 7). Compounds in bold
5.1.3.4. 6-Chloro-8,9-methylenedioxybenzo[c][1,8]
correspond to the most cytotoxic compounds (bellow 0.5
horizontal dashed lane).
mM as delimited by the
phenanthroline. (57% yield). IR 2912, 2334 (
n
CN), 1477, 1263, 1042
9.35 (s, 1H), 8.74 (d,
(n
CeCl), 957 cmꢁ1 1H NMR (400 MHz, CDCl3)
d
1H, J ¼ 6.0 Hz), 8.32 (d,1H, J ¼ 9.0 Hz), 8.11 (d,1H, J ¼ 6.0 Hz), 8.01 (s,
139.0,133.1,132.6,126.5, 125.6, 123.7,121.2,118.6,115.7, 102.4, 102.0,
100.3, 64.3, 56.7, 45.1, 26.8 (2C); MS (ESþ) m/z 376 [M þ H]þ;
[M þ H]þ; HRMS (ESþ) m/z calcd for C22H22N3O3 [M þ H]þ 376.1661,
1H), 7.87 (d, 1H, J ¼ 9.0 Hz), 7.74 (s, 1H), 4.17 (s, OCH3), 4,10 (s,
OCH3). 13C NMR (75 MHz, CDCl3)
d 153.7, 152.5 (2C), 150.7, 144.7,
131.9, 131.3, 129.2, 128.2, 125.6, 125.6, 120.8, 118.9, 117.1,104.3, 102.2,
100.0. MS (IC, NH3) m/z 309, 311 [M þ H]þ. HRMS (ESþ) m/z calcd for
C17H9ClN2O2 [M þ H]þ 308.0353, found 308.0357.
found 376.1657; U.V. lmax (nm) (loge) (CH2Cl2) 214 (3.9), 222 (4.3),
232 (4.2), 282 (4.5).
5.1.2.12. 5-[20-(diethylamino)ethyl]-8,9-methylene
[1,7]phenanthrolin-6(5H)-one (28). (17% yield) IR (CH2Cl2, cmꢁ1
dioxybenzo[c]
5.1.4. General procedure for the preparation of the C6
dialkylaminoalkylbenzo[c]phenanthroline
)
3379, 2959, 2910, 2845, 2355, 2333, 1603, 1527, 1467, 1440, 1325,
The chlorinated benzo[c]phenanthroline was refluxed in an
excess of the appropriate dialkylaminoalkylamine. After the reac-
tion was completed (TLC monitoring) the excess of dia-
lkylaminoalkylamine was removed under vacuum. The residue was
dissolved in CH2Cl2 and washed successively with an aqueous NaOH
solution (10% m/v) and water. The organic layer was dried over
MgSO4, filtered and concentrated in vacuo. The residue was purified
1293, 1255, 1184, 1124, 1032, 934, 825; 1H NMR (300 MHz, CDCl3)
d
9.44 (dd, J ¼ 8.3, 1.5 Hz, 1H), 9.00 (dd, J ¼ 4.3, 1.5 Hz, 1H), 8.43 (d,
J ¼ 9.2 Hz, 1H), 8.05 (d, J ¼ 9.2 Hz, 1H), 7.85 (s, 1H), 7.70 (s, 1H), 7.57
(dd, J ¼ 8.3, 4.3 Hz, 1H), 6.18 (s, 2H), 4.88 (t, J ¼ 6.2 Hz, 2H), 3.14 (t,
J ¼ 6.2 Hz 2H), 2.80 (q, J ¼ 7.1 Hz, 2x2H), 1.18 (t, J ¼ 7.1 Hz, 2 ꢃ 3H);
13C NMR (75 MHz, CDCl3)
d 158.2, 151.5, 150.4, 148.7, 148.1, 138.9,
133.0, 132.5, 126.5,125.6, 123.6, 121.1,118.5,115.7, 102.4, 101.9,100.2,
64.4, 51.3, 48.0 (2C), 11.9 (2C); MS (ESþ) m/z 390 [M þ H]þ; HRMS
(ESþ) m/z calcd for C23H23N3O3Na [M þ Na]þ 412.1637, found
by chromatography on silica gel (20/45 m, CH2Cl2/MeOH/NH4OH 90/
8.75/1.25 v/v/v) to afford the corresponding compound.
412.1656; U.V. lmax (nm) (log
e
) (CH2Cl2) 239 (4.5), 282 (4.7).
5.1.4.1. 6-[(30-(Dimethylamino)propyl)amino]-8,9-dimethoxybenzo
[c][1,8]phenanthroline (29). Yield 56%. IR 3354 (
2361, 1588, 1543, 1482, 1272 (
CN), 1209, 1035 cmꢁ1 1H NMR
(400 MHz, CDCl3)
y NH), 2923 (y CH),
5.1.3. General procedure for the C6 chlorination of benzo[c]
phenanthroline core
y
d
9.26 (s, 1H), 8.92 (d, 1H, J ¼ 6.0 Hz), 8.68 (d, 1H,
Benzo[c]phenanthroline dissolved in PhPOCl3 was heated at
160 ꢂC for 3 h. Then the reaction mixture was cooled to rt and poured
into ice. pH was adjusted to 8e9 with an aqueous ammonia solution
and the solution was left for 2 h. The precipitate formed was then
filtered, washed with water and dried. Purification by flash chro-
J ¼ 6.0 Hz), 8.32 (d, 1H, J ¼ 9.0 Hz), 7.84 (s, 1H), 7.74 (d, 1H,
J ¼ 9.0 Hz), 7.40 (s, 1H), 6.32 (bs, NH), 4.14 (s, OCH3), 4.10 (s, OCH3),
3.98 (q, 2H, J ¼ 6.0 Hz), 2.83 (t, 2H, J ¼ 6.0 Hz), 2.43 (s, 6H). 13C NMR
(75 MHz, CDCl3) d 153.0, 152.1, 151.2, 149.9, 143.6, 139.5, 134.9, 129.1,
128.4, 121.5, 120.4, 118.5, 117.6, 114.2, 103.1, 58.3, 56.3, 56.0, 45.3,
45.1, 39.0. MS (ESþ) m/z : 377 [M þ H]þ. HRMS (ESþ) m/z calcd for
C23H24N4O2 [M þ H]þ 377.1899, found 377.1903.
matography on silica gel (20/45 m, eluent CH2Cl2/MeOH 95/5 v/v)
afforded the corresponding chloro compound as a white-grey solid.
5.1.3.1. 6-Chloro-8,9-dimethoxybenzo[c][1,7] phenanthroline. (52 %
5.1.4.2. 6-[(30-(Dimethylamino)propyl)amino]-8,9-dimethoxybenzo
yield). IR 1647, 1615, 1493 (
(400 MHz, CDCl3)
y
CN), 1258, 1150, 732 cmꢁ1 1H NMR
[c][1,8]phenanthroline (30). Yield 87%. IR 3307 (
2356, 1619 ( NH),1588,1545,1482, 1272 (
CN),1209,1028 cmꢁ1 1H
NMR (400 Hz, CDCl3)
y NH), 2927 (y CH),
d
9.42 (dd, 1H, J ¼ 8.0 Hz, 1 Hz), 9.01 (dd, 1H,
d
y
J ¼ 4.0 Hz, 1 Hz), 8.45 (d, 1H, J ¼ 9.0 Hz), 8.15 (d, 1H, J ¼ 9.0 Hz), 7.74
d
9.21 (s,1H), 8.90 (d,1H, J ¼ 6.0 Hz), 8.65 (d,1H,
(s, 1H), 7.64 (s, 1H), 7.60 (dd, 1H, J ¼ 8.0 Hz, 4.0 Hz), 4.13 (s, OCH3),
J ¼ 6.0 Hz), 8.20 (d, 1H, J ¼ 9.0 Hz), 7.75 (bs, NH), 7.70 (s, 1H), 7.63 (d,
1H, J ¼ 9.0 Hz), 7.12 (s, 1H), 4.07 (s, 3H), 4.00 (s, 3H), 3.90 (td, 2H,
J ¼ 6.0 Hz, 1.0 Hz), 2.59 (t, 2H, J ¼ 6.0 Hz), 2.35 (s, 6H), 2.01e1.93 (m,
4.09 (s, OCH3). 13C NMR (75 MHz, CDCl3)
d 153.8, 150.8, 149.7, 148.3,
139.4, 133.1, 130.5, 128.7, 126.7, 123.2, 122.1, 120.9, 120.7, 106.5,
102.2, 56.4, 56.3. MS (IC, NH3) m/z 325, 327 [M þ H]þ. HRMS (ESþ)
m/z calcd for C18H13ClN2O2 [M þ H]þ 324.0666, found 324.0670.
2H).13C NMR (75 MHz, CDCl3)
d 153.4,151.8,151.1,149.7,143.3,139.8,
134.8, 129.0, 128.4, 121.5, 119.8, 118.1, 117.8, 114.3, 103.1, 102.8, 59.9,
56.0, 55.8, 45.7 (2C) 42.6, 25.1. MS (ESþ) m/z 391 [M þ H]þ. HRMS
(ESþ) m/z calcd for C23H26N4O2 [M þ H]þ 391.2056, found 391.2064.
5.1.3.2. 6-Chloro-8,9-dimethoxybenzo[c][1,8] phenanthroline. (54%
yield). IR 2927, 2844 (
CCl); 973 cmꢁ1 1H NMR (400 MHz, CDCl3)
J ¼ 6.0 Hz), 8.84 (d, 1H, J ¼ 6.0 Hz), 8.50 (d, 1H, J ¼ 9.0 Hz), 8.09 (d,
y
CN), 1655, 1620, 1521, 1487, 1165, 1016 (
y
d
9.38 (s, 1H), 9.01 (d, 1H,
5.1.4.3. 6-[(30-(Dimethylamino)propyl)amino]-8,9-dimethoxybenzo
[c][1,8]phenanthroline (31). Yield 68%. IR 3346 (y NH), 2966 (y CH),

2128
C. Genès et al. / European Journal of Medicinal Chemistry 46 (2011) 2117e2131
Fig. 9. Sequence-specific DNA binding. Appropriate concentrations of the various benzophenanthrolines were incubated with the 265 bp 30-end-radio-labelled DNA fragment prior
to DNA digestion using DNase I (0.01 unit/ml). The digestion reaction was stopped by freeze drying and lyophilisation of the samples. DNA samples were separated by electro-
phoresis on a 8% denaturing polyacrylamide gel. Each resolved band on the autoradiograph was assigned to a particular bond within the DNA fragment from comparison with its
position relative to the guanines sequencing standard (G-track).
1617 (
d
NH), 1588, 1544, 1482, 1422, 1385, 1273 (
y
CN), 1209, 1164,
5.1.4.4. 6-[(30-(Dimethylamino)propyl)amino]-8,9-dimethoxybenzo
1033, 838, 792 cmꢁ1 1H NMR (400 MHz, CDCl3)
d
9.23 (s, 1H), 8.91
[c][1,7]phenanthroline (32). Yield 48%. IR 3345 (
2854, 1614 ( NH), 1587, 1525, 1494, 1463, 1429, 1380, 1270 (
1208, 1059, 819 cmꢁ1 1H NMR (400 MHz, CDCl3)
y
NH), 2926 (
y
CH),
CN),
(d, 1H, J ¼ 6.0 Hz), 8.67 (d, 1H, J ¼ 6.0 Hz), 8.25 (d, 1H, J ¼ 9.0 Hz),
7.76 (bs, 2H), 7.68 (d, 1H, J ¼ 9.0 Hz), 7.21 (s, 1H), 4.10 (s, 3H), 4.03 (s,
3H), 3.95e3.94 (m, 2H), 2.72 (t, 2H, J ¼ 6.0 Hz), 2.66 (q, 4H,
J ¼ 6.0 Hz), 2.00e1.98 (m, 2H), 1.09 (t, 6H, J ¼ 6.0 Hz). 13C NMR
d
y
d
9.50 (d, 1H,
J ¼ 8.0 Hz, 1 Hz), 8.96 (dd, 1H, J ¼ 6.0 Hz, 1.0 Hz), 8.48 (d, 1H,
J ¼ 9.0 Hz), 7.94 (d, 1H, J ¼ 9.0 Hz), 7.84 (s, 1H), 7.52 (dd, 1H,
J ¼ 8.0 Hz, 6.0 Hz), 7.32 (s, 1H), 6.21 (bs, NH), 4.14 (s, OCH3), 4.09 (s,
OCH3), 3.97 (q, 2H, J ¼ 6.0 Hz), 2.83 (t, 2H, J ¼ 6.0 Hz), 2.41 (s, 6H).
(75 MHz, CDCl3) d 153.3, 151.9, 151.1, 149.7, 143.4, 139.8, 134.8, 129.1,
128.4, 121.5, 119.9, 118.1, 117.8, 114.3, 103.1 (2C) 56.1, 56.0, 52.8, 47.4
(2C), 43.0, 24.9, 11.6 (2C). MS (ESþ) m/z 419 [M þ H]þ. HRMS (ESþ)
m/z calcd for C25H30N4O2 [M þ H]þ 419.2369, found 419.2401.
13C NMR (75 MHz, CDCl3)
d 153.0, 152.2, 150.0, 149.5, 148.9, 140.4,
133.1, 129.4, 126.3, 123.7, 123.2, 120.6, 116.0, 113.4, 103.0, 102.9, 58.3,

C. Genès et al. / European Journal of Medicinal Chemistry 46 (2011) 2117e2131
2129
Fig. 10. Sequence-specific DNA binding. Densitometric analysis for each compounds were obtained from quantification of the band inhibition from Fig. 9. Grey boxes correspond to
the portions of DNA that are protected by the evaluated compounds from DNase I cleavage.
56.3, 56.0, 45.3 (2C), 39.0. MS (ESþ) m/z 377 [M þ H]þ. HRMS (ESþ)
m/z calcd for C23H24N4O2 [M þ H]þ 377.1899, found 377.1906.
(s, 1H), 4.11 (s, 3H), 4.01 (s, 3H), 3.94 (td, 2H, J ¼ 6.0 Hz, 1.0 Hz), 2.71
(t, 2H, J ¼ 6.0 Hz), 2.65 (q, 4H, J ¼ 8.0 Hz), 1.99e1.95 (m, 2H), 1.16 (t,
6H, J ¼ 8.0 Hz). 13C NMR (75 MHz, CDCl3)
d 153.4, 152.0, 149.9, 149.2,
5.1.4.5. 6-[(30-(Dimethylamino)propyl)amino]-8,9-dimethoxybenzo
148.9, 140.9, 133.3, 129.5, 126.4, 123.8, 122.6, 120.5, 115.5, 113.6,
103.0, 102.9, 56.1, 56.0, 53.1, 47.5 (2C), 43.3, 25.0, 11.8 (2C). MS (ESþ)
m/z 419 [M þ H]þ. HRMS (ESþ) m/z calcd for C25H30N4O2 [M þ H]þ
419.2369, found 419.2401.
[c][1,7]phenanthroline (33). Yield 54%. IR 3304 (
2849, 2765, 2357, 1611 ( NH), 1587, 1548, 1494, 1428, 1382, 1271 (
CN), 1208, 1179, 1058, 818, 790 cmꢁ1 1H NMR (400 MHz, CDCl3)
y NH), 2932 (y CH),
d
y
d
9.51 (dd, 1H, J ¼ 9.0 Hz, 1 Hz), 8.94 (dd, 1H, J ¼ 6.0 Hz, 1 Hz), 8.43
(d, 1H, J ¼ 10.0 Hz), 7.91 (d, 1H, J ¼ 10.0 Hz), 7.86 (bs, NH), 7.81 (s,
1H), 7.52 (dd, 1H, J ¼ 9.0 Hz, 6.0 Hz), 7.16 (s, 1H), 4.12 (s, OCH3), 4.03
(s, OCH3), 3.95 (q, 2H, J ¼ 6.0 Hz), 2.61 (t, 2H, J ¼ 6.0 Hz), 2.39 (s, 6H),
5.1.4.7. 6-[(20-(Dimethylamino)ethyl)amino]-8,9-methylene dioxy-
benzo[c][1,8]phenanthroline (35). Yield 14%. IR 3358 (
2779, 1626 ( CO), 1580 ( NH), 1538, 1504, 1470, 1378 (
1263, 1230, 1040 (y
OCO), 840, 758 cmꢁ1 1H NMR (400 MHz, CDCl3)
y
NH), 2917,
y
d
y CN), 1305,
2.02e1.96 (m, 2H). 13C NMR (75 MHz, CDCl3)
d
153.4, 151.9, 149.9,
149.3, 148.9, 140.9, 133.3, 129.3, 126.4, 123.8, 122.6, 120.5, 115.5,
113.5, 102.9, 102.7, 59.9, 56.0, 55.8, 45.7 (2C), 42.7, 25.1. MS (ESþ) m/
z 391 [M þ H]þ. HRMS (ESþ) m/z calcd for C24H26N4O2 [M þ H]þ
391.2056, found 391.2072.
d
9.25 (s, 1H), 8.91 (d, 1H, J ¼ 5.7 Hz), 8.69 (d, 1H, J ¼ 5.7 Hz), 8.24 (d,
1H, J ¼ 9.0 Hz), 7.85 (s, 1H), 7.74 (d, 1H, J ¼ 9.0 Hz), 7.37 (s, 1H), 6.18
(bs, NH), 6.13 (s, 2H), 3.93 (q, 2H, J ¼ 5.5 Hz), 2.79 (t, 2H, J ¼ 5.5 Hz),
2.40 (s, 6H). 13C NMR (75 MHz, CDCl3)
d 153.1, 151.2, 150.7, 148.4,
143.7, 139.6, 134.8, 131.0, 128.4, 121.6, 120.5, 118.8, 117.6, 115.3, 102.0,
101.0, 100.6, 58.0, 45.2 (2C), 38.9. MS (ESþ) m/z 361 [M þ H]þ. HRMS
(ESþ) m/z calcd for C21H20N4O2 [M þ H]þ 361.1586, found 361.1602.
5.1.4.6. 6-[(30-(Dimethylamino)propyl)amino]-8,9-dimethoxybenzo
[c][1,7]phenanthroline (34). Yield 55%. IR 3345 (
2928, 2845, 1587, 1548, 1494, 1429, 1381, 1272 (
814, 787 cmꢁ1 1H NMR (400 MHz, CDCl3)
y
y
NH), 2965 (
CN), 1209, 1058,
9.53 (dd, 1H, J ¼ 8.0 Hz,
y CH),
d
5.1.4.8. 6-[(30-(Dimethylamino)propyl)amino]-8,9-methylenediox-
1.0 Hz), 8.95 (dd, 1H, J ¼ 6.0 Hz, 1.0 Hz), 8.44 (d, 1H, J ¼ 9.0 Hz), 7.91
(d, 1H, J ¼ 9.0 Hz), 7.81 (bs, 2H), 7.51 (dd, 1H, J ¼ 8.0 Hz, 6.0 Hz), 7.18
ybenzo[c][1,8]phenanthroline (36). Yield 5%. 1H NMR (400 MHz,
CDCl3)
d
9.26 (s, 1H), 8.92 (d, 1H, J ¼ 5.6 Hz), 8.69 (d, 1H, J ¼ 5.6 Hz),

2130
C. Genès et al. / European Journal of Medicinal Chemistry 46 (2011) 2117e2131
8.29 (d, 1H, J ¼ 9.0 Hz), 7.91 (s, 1H), 7.87 (bs, NH), 7.73 (d, 1H,
J ¼ 9.0 Hz), 7.35 (s, 1H), 6.18 (s, 2H), 4.05e3.96 (m, 2H), 2.74 (t, 2H,
J ¼ 6.0 Hz), 2.49 (s, 6H), 2.11e2.09 (m, 2H). 13C NMR (75 MHz, CDCl3)
5.2.3. Topoisomerase 1-induced DNA relaxation assay (Fig. 1)
The experiments were conducted as presented elsewhere [15].
Supercoiled pUC19 plasmid DNA (130 ng) was incubated with 4
units of human recombinant topoisomerase 1 (TopoGen) at 37 ꢂC
for 45 min in 20 mL of relaxation buffer (50 mM Tris pH 7.8, 50 mM
KCl, 10 mM MgCl2, 1 mMDTT,1 mM EDTA, 1 mM ATP) in the pres-
d
153.6, 151.2, 150.7, 148.5, 143.6, 139.5, 134.7, 131.0, 128.6, 121.7,
120.2, 118.6, 117.6, 115.6, 101.9, 101.0 (2C), 57.6, 44.6 (2C), 41, 24.5.
MS (ESþ) m/z 375 [M þ H]þ. HRMS (ESþ) m/z calcd for C22H22N4O2
[M þ H]þ 375.1743, found 375.1789.
ence of graded concentrations (from 0.1 to 5
mM) of the tested
compounds. The reactions were stopped by the addition of SDS
(0.25%) and proteinase K (250 mg/mL) followed by an incubation of
30 min at 50 ꢂC. Then 3 mL of the electrophoresis dye mixture was
added to each DNA samples and loaded on a 1% agarose gel. After
resolution at room temperature for 2 h at 120 V gels run were
stained using an ethidium bromide containing bath, washed and
photographed under UV light.
5.1.4.9. 6-[(20-(Dimethylamino)ethyl)amino]-8,9-methylene dioxy-
benzo[c][1,7]phenanthroline (37). Yield 17%. IR 3347 (
2840, 2356, 2337, 1625 ( CO), 1581 ( NH), 1547, 1498, 1474, 1450,
1430, 1369 ( CN), 1120, 1044 (
OCO) cmꢁ1 1H NMR (400 MHz,
CDCl3)
y NH), 2917,
y
d
y
y
.
d
9.51 (dd, 1H, J ¼ 8.0 Hz, 1.5 Hz), 8.97 (dd, 1H, J ¼ 4.3 Hz,
1.5 Hz), 8.42 (d, 1H, J ¼ 9.0 Hz), 7.93 (d, 1H, J ¼ 9.0 Hz), 7.90 (s, 1H),
7.52 (dd, 1H, J ¼ 8.0 Hz, 4.3 Hz), 7.40 (s, 1H), 6.19 (bs, NH), 6.16 (s,
2H), 3.94 (q, 2H, J ¼ 5.7 Hz), 2.81 (t, 2H, J ¼ 5.7 Hz), 2.41 (s, 6H). 13C
5.2.4. Topoisomerase 1 inhibition assays
NMR (75 MHz, CDCl3)
d
153.1150.8, 150.2, 149.0, 148.0, 140.6, 133.1,
Agarose gel experiments were performed as specified in 5.2.3.,
unless the DNA samples were run on an ethidium bromide con-
taining agarose gel (1%) in the same condition as above, before they
were washed and photographed under UV light. In order to identify
the sequence selectivity of the cleavage sites induced by top-
oisomerase I poisoning effect, the localisation of DNA cleavage sites
that could be generated with the various tested compounds was
addressed using a 30-end-labelled 117 base pairs DNA fragment as
already published with the following modifications [16]. The
cleavage reactions to identify DNA poisoning effect were conducted
in the presence of CPT (as a positive reference) or increasing
concentrations of the tested drugs; whereas gels performed to
compete for CPT poisoning inhibition were conducted in the
131.3, 126.2, 123.9, 123.4, 120.7, 116.4, 114.7, 101.9, 100.9, 100.7, 58.1,
45.1 (2C), 38.6. MS (ESþ) m/z 361 [M þ H]þ. HRMS (ESþ) m/z calcd
for C21H20N4O2 [M þ H]þ 361.1586, found 361.1590.
5.1.4.10. 6-[(30-(Dimethylamino)propyl)amino]-8,9-methylenediox-
ybenzo[c][1,7]phenanthroline (38). Yield 17%. IR 3286 (
2846, 2347, 1622 ( CO), 1601, 1582 ( NH), 1544, 1539, 1473, 1449,
1429, 1366 ( CN), 1258, 1238, 1045 (
OCO) cmꢁ1 1H NMR
(400 MHz, CDCl3)
y NH), 2911,
y
d
y
y
.
d
9.51 (dd, 1H, J ¼ 8.3 Hz, 1.5 Hz), 8.95 (dd, 1H,
J ¼ 4.3 Hz,1.5 Hz), 8.37 (d,1H, J ¼ 9.1 Hz), 7.89 (d,1H, J ¼ 9.1 Hz), 7.84
(s, 1H),, 7.80 (bs, NH), 7.52 (dd, 1H, J ¼ 8.3 Hz, 4.3 Hz), 7.16 (s, 1H),
6.14 (s, 2H), 3.92 (q, 2H, J ¼ 5.7 Hz), 2.63 (t, 2H, J ¼ 5.7 Hz), 2.41 (s,
6H), 2.08e2.06 (m, 2H). 13C NMR (75 MHz, CDCl3)
d
153.7, 150.5,
presence of a fixed concentration of CPT (20 mM) together with
150.1, 149.0, 147.9, 141.1, 133.3, 131.2, 126.3, 123.9, 122.8, 120.5, 115.9,
114.9,101.7,100.9,100.3, 59.9, 45.6 (2C) 42.8, 25.2. MS (ESþ) m/z 375
[M þ H]þ. HRMS (ESþ) m/z calcd for C22H22N4O2 [M þ H]þ 375.1743,
found 375.1795.
increasing concentration of the tested drugs at the concentrations
specified in the figures, then stopped and treated as published
elsewhere [16].
5.2.5. Topoisomerase 2 inhibition assays (Fig. 2B)
Supercoiled pUC19 plasmid DNA (350 ng) was incubated with
50 mM of the various tetracyclic derivatives or etoposide, as control,
5.1.4.11. 6-[(30-(Diethylamino)propyl)amino]-8,9-methylene dioxy-
benzo[c][1,7]phenanthroline (39). Yield 5%. 1H NMR (400 MHz,
CDCl3)
d
9.52 (dd, 1H, J ¼ 8.3 Hz, 1.7 Hz), 8.95 (dd, 1H, J ¼ 4.3 Hz,
prior to the addition of 10 units of human topoisomerase 2
(TopoGen) at 37 ꢂC for 45 min in the appropriate cleavage buffer.
SDS (0.25%) and proteinase K (250 mg/mL) were then added to stop
the reaction during 30 min at 50 ꢂC. DNA samples were loaded on
1% agarose gels containing ethidium bromide for 2 h at 120 V in TBE
buffer. After migration, gels were washed and photographed under
UV light.
1.7 Hz), 8.40 (d,1H, J ¼ 9.1 Hz), 8.09 (bs, NH), 7.90 (s,1H), 7.89 (d,1H,
J ¼ 9.1 Hz), 7.52 (dd, 1H, J ¼ 8.3 Hz, 4.3 Hz), 7.38 (s, 1H), 6.16 (s, 2H),
4.02e3.93 (m, 2H), 2.83e2.73 (m, 6H), 2.07e2.05 (m, 2H), 1.18 (t,
6H). 13C NMR (75 MHz, CDCl3)
d 153.4, 150.6, 150.1, 149.0, 148.3,
141.0, 133.3, 131.2, 126.4, 124.0, 122.8, 120.5, 116.0, 114.8, 101.8,
100.9, 100.8, 49.7, 46.8 (2C) 46.0, 24.5, 11.2 (2C). MS (ESþ) m/z 403
[M þ H]þ. HRMS (ESþ) m/z calcd for C24H26N4O2 [M þ H]þ
403.2056, found 403.2092.
5.2.6. DNase I footprinting (Fig. 3)
Experiments were performed as previously described and using
a 265 bp 30-end-labelled DNA fragment obtained from double
digestion of the pBS plasmid (Stratagene, La Jolla, CA) at EcoRI and
PvuII restriction sites followed by radiolabelling of one strand by
5.2. Biology
5.2.1. Cytotoxicity (Tables 1 and 2)
incorporation of a
-[32P]-dATP (3000 Ci/mmol, GE Healthcare,
HT-29 colon carcinoma cells were grown as specified and the
cytotoxicity was assessed as previously described [13,14].
Buckinghamshire, England) at the EcoRI restriction site using AMV
reverse transcriptase (Ozyme, France). Appropriate concentrations
of the various substituted tetracycles were incubated with the 265-
bp radio-labelled DNA fragment for 15 min at 37 ꢂC to ensure
equilibrium prior to DNA digestion using DNase I (0.01 unit/ml) in
20 mM NaCl, 2 mM MgCl2, 2 mM MnCl2 (pH 7.3), for 3 min. The
digestion reaction was stopped by freeze drying and lyophilisation
of the samples. The DNA pellets were dissolved in 5 mL of dena-
turing loading buffer (80% formamide solution containing tracking
dyes), heated at 90 ꢂC for 4 min and chilled in ice for another 4 min
prior to be loaded on a 8% denaturing polyacrylamide gel for 90 min
at 65W in TBE buffer. The data were collected using a Molecular
Dynamics STORM 860 PhosphorImager and analyzed using the
ImageQuant version 4.1 software. Each resolved band on the
5.2.2. Cell cycle analysis
For flow cytometric analyses of DNA content were obtained as
described in Lemster et al. [13], with the following modifications.
Cells in exponential growth phase (300,000 per well) were plated
for 24 h before being treated with graded concentrations (0.1, 1 and
2.5
mM) of the various compounds for 48 h. Cells were then
collected and fixed as described and analyzed on a Becton Dick-
inson FACScan flow cytometer using the LYSYS II software. Excita-
tion and emission wavelengths for PI were 488 nm and 620 nm
(channel Fl-3), respectively. Each experiment was performed in
duplicate.

C. Genès et al. / European Journal of Medicinal Chemistry 46 (2011) 2117e2131
2131
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Elise Prost and Pascale Leproux (UMR CNRS 8638, Université
Paris Descartes) are gratefully acknowledged for their technical
assistance (NMR and Mass Spectrometry).
Gaelle Lenglet thanks the Université de Lille 2/Conseil Régional
Nord/Pas-de-Calais and the Institut pour la Recherche sur le Cancer
de Lille (IRCL) for a PhD fellowship.
Marie-Hélène David-Cordonnier thanks the Ligue Nationale
Contre le Cancer (Comité du Nord) and the Institut pour la
Recherche sur le Cancer de Lille (IRCL) for grants and Marie-Paule
Hildebrand for technical assistance.
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