Synthesis and biological evaluation of dialkylaminoalkylamino benzo[c][1,7] and [1,8]phenanthrolines as antiproliferative agents

Article abstract of DOI:10.1016/j.ejmech.2010.02.043

Benzo[c][1,7] and [1,8]phenanthroline substituted by dialkylaminoalkyl side chains at position C2 and C1, respectively, were synthesized and their biological activity evaluated. These compounds displayed more potent cytotoxicity toward L1210 cells than th

Full text of DOI:10.1016/j.ejmech.2010.02.043

European Journal of Medicinal Chemistry 45 (2010) 2547e2558
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 dialkylaminoalkylamino benzo[c][1,7]
and [1,8]phenanthrolines as antiproliferative agents
Tuba S¸ erbetçi a, Constance Genès a, Sabine Depauw b c, Soizic Prado a, François-Hugues Porée a,
,
,
,
,
*
Marie-Paule Hildebrand b c, Marie-Hélène David-Cordonnier b c, Sylvie Michel a, François Tillequin a
a Laboratoire de Pharmacognosie de l'Université Paris Descartes, U.M.R./C.N.R.S. nꢀ 8638, Faculté de Pharmacie, 4 Avenue de l'Observatoire, 75006 Paris, France
b INSERM U837-JPARC (Jean-Pierre Aubert Research Center), Team “Molecular and Cellular Targeting for Cancer Treatment”, Université de Lille 2, IMPRT-Institut Fédératif de
Recherche 114, France
c Institut de Recherches sur le Cancer de Lille (IRCL), Place de Verdun, 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][1,7] and [1,8]phenanthroline substituted by dialkylaminoalkyl side chains at position C2 and C1,
respectively, were synthesized and their biological activity evaluated. These compounds displayed more
potent cytotoxicity toward L1210 cells than the parent unsubstituted compounds, associated with strong
DNA interaction. The moderate TopoI inhibitory activity induced by the novel compounds suggests that
other cellular targets should be responsible for the antiproliferative activity.
Received 15 January 2010
Received in revised form
18 February 2010
Accepted 19 February 2010
Available online 25 February 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Benzo[c][1,7]phenanthroline
Benzo[c][1,8]phenanthroline
Cytotoxicity
DNA interaction
1. Introduction
there is a great interest in the development of non-camptothecin
topoisomerase I inhibitors as anticancer agents [7].
DNA topoisomerases constitute a superfamily of enzymes which
interfere with DNA [1]. These enzymes are essential in every living
cell and represent the Nature's solution to the topological problems
associated with manipulating double-stranded helical DNA during
essential cellular processes, such as replication, transcription,
recombination, as well as chromosome compaction and segrega-
tion [2]. The main classification of these enzymes is based on their
reaction mechanism, i.e. their ability to create a single-stranded
(type I) or double-stranded (type II) nick in DNA [3]. Among
these enzymes, the human DNA topoisomerase IB (TopoI) is of
particular interest. It catalyses the major DNA topoisomerase I
activity and represents the cellular target for the camptothecin-
derived anticancer drugs irinotecan and topotecan currently used
in the clinic, but also to many other cellular poisons [4,5]. However,
the camptothecin derivatives suffer from several drawbacks,
including poor water solubility, lactone instability, prolonged
infusion times, and facile apparition of multidrug resistance due to
transporter-mediated cellular efflux in cancer cells [6]. As a result,
We previously reported the preparation of non-charged bio-
isosters of the benzo[c]phenanthridine alkaloids nitidine 1 and
fagaronine 2, in the benzo[c][1,7]- and [1,8] phenanthroline (1,7-
BZP and 1,8-BZP) series (Scheme 1). Biological evaluations of these
compounds showed interesting cytotoxic activities associated with
a moderate TopoI inhibition [8]. In a continuation of our work, we
report here the synthesis and biological activities of new series of
1,7-BZP and 1,8-BZP bearing at positions C2 and C1 on cycle A,
respectively, different dialkylaminoalkyl substituents. These side
chains, which mimick natural DNA ligands such as spermine and
spermidine, were chosen due to the excellent results reported with
such substituents in several related series including indén-
oisoquinoléines [9], dibenzo[c,h] [1,6]naphthyridinones [10],
camptothécine [11], and acronycine analogs [12].
2. Chemistry
The synthetic route followed to prepare the different
compounds was partially reported previously. Briefly, the strategy
involved six steps with a benzyne mediated cyclization as the key
step. To clarify the lecture, the synthetic plan is divided in two
parts: one for the 1,7-BZP series (Scheme 2) and one for the 1,8-BZP
* Corresponding author. Tel.: þ33 1 53 73 98 10; fax: þ33 1 40 46 96 58.
E-mail address: francois.tillequin@parisdescartes.fr (F. Tillequin).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.043

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T. S¸ erbetçi et al. / European Journal of Medicinal Chemistry 45 (2010) 2547e2558
Scheme 1.
series (Scheme 3). Condensation of the 2-bromo- veratraldehyde 7
or piperonal 8 with the 5-amino-quinoline 9 (Scheme 2) or iso-
quinoline 10 (Scheme 3) gave access to the corresponding Schiff
bases. After reduction of the imine function with NaBH4, treatment
with an excess of LDA permitted to ensure the cyclization of cycle C
[13]. A spontaneous air oxidation step achieved the preparation of
the two different phenanthroline cores [14]. Selective introduction
exhibited cytotoxicities within the micromolar range. Moreover all
compounds presented an increased activity when compared to the
parent compounds 3e6. As previously observed in analogous
series, methylenedioxy substituted compounds displayed more
potent antiproliferative activity than their dimethoxy counterparts.
It should also be noted that no straightforward correlation was
observed between the side chain length and the cytotoxicity.
of aminoalkyl side chains in
a position to the nitrogen atom on
cycle A was realized in three steps. Oxidation with m-CPBA afforded
the BZP N-oxide, which were further converted into the chloro
derivatives by reaction with POCl3 [15]. Finally, direct heating of the
chloro BZP in the desired dialkylaminoalkylamine afforded, after
careful column chromatography, the corresponding substituted
BZP in low to moderate yield (6e52%).
3.2. DNA binding properties and inhibition of topoisomerases
I and II
3.2.1. UVevisible and fluorescence spectra
In order to address the DNA binding properties of the novel 1,7-
BZP and 1,8-BZP derivatives, the UVevisible absorbance (Fig. 1) and
fluorescence spectra (Fig. 2) of the various compounds in the
absence and presence of calf thymus DNA were studied [17]. The
binding of the drugs to DNA resulted in a decrease of absorbance in
the DNA-compound complex spectra (hypochromic shift) at
maximumwavelengths dependingon the series, 265 nm for 1,8-BZP,
275 nm for dimethoxy-1,7-BZP, and 285 nm for methylenedioxy-
1,7-BZP, associated with a small bathochromic shift (see horizontal
arrows, shifts are around 10 nm). Concerning the fluorescence
3. Biological evaluation
3.1. Evaluation of the cytotoxicity
The study of the cytotoxic properties of new compounds (17e24
and 31e36) was carried out in vitro on the L1210 murine leukemic
cell line [16]. The results (IC50) are reported in Table 1. Both series
Scheme 2. Benzo[c][1,7]phenanthroline series. Reagents and conditions: (a) (i) toluene, reflux; (ii) NaBH4, MeOH, 0 ꢀC; (b) (i) LDA, THF, ꢁ78 ꢀC to r.t.; (ii) air oxidation; (c) (i)
mCPBA, CH2Cl2, 0 ꢀC; (iii) POCl3,
d; (d) H2N(CH)nN(R3)2.

T. S¸ erbetçi et al. / European Journal of Medicinal Chemistry 45 (2010) 2547e2558
2549
Scheme 3. Benzo[c][1,8]phenanthroline series. Reagents and conditions: (a) (i) toluene, reflux; (ii) NaBH4, MeOH, 0 ꢀC; (b) (i) LDA, THF, ꢁ78 ꢀC to r.t.; (ii) air oxidation; (c) (i)
mCPBA, CH2Cl2, 0 ꢀC; (iii) POCl3,
d; (d) H2N(CH)nN(R3)2.
spectral measurement, DNA binding activities of BZP series were
evidenced bya blue shift. In 1,7-BZP series this effect is accompanied
by a decrease of the fluorescence intensity. In 1,8-BZP series the
observed blue shift is associated with an increase or a decrease of the
peak intensity forcompounds bearing a methylenedioxysubstituent
or a dimethoxy substituent, respectively.
bearing a methylenedioxy substituent, a better DNA binding
propensity is obtained using a diethylaminopropylamine side
chain. In the 1,8-BZP series, DNA stabilization also depends on the
nature of the side chain. A better relative affinity is obtained when
methylenedioxy-bearing compounds are substituted with
a dimethyl aminopropylamine side chain. It appears from these
data that both series of BZP display significant DNA binding
properties.
3.2.2. DNA melting temperature
The relative DNA affinity of these compounds was assessed
using CT-DNA melting temperature studies (Table 2). The ability of
the drugs to protect calf thymus DNA (CTa DNA, 42% GC bp) against
thermal denaturation was used as an indication of their capacity to
bind to DNA and to stabilize the DNA double helix. The induced
variation of melting temperature evidenced some strong DNA
stabilizing agents (17, 24, 36) with an increase of more than 10 ꢀC of
CT-DNA melting temperature. Others compounds are medium or
3.2.3. Circular dichroism measurement
The binding mode of the various BZP analogs was then studied
using circular dichroism (Fig. 3). CD spectra of the CT-DNA obtained
in the presence of a given concentration of the tested compound
(pointed lanes) were compared to the spectra of the CT-DNA alone
(dashed lanes) and that of the compound alone (plain lanes). As
evidenced from Fig. 3, most of the compounds increased the peak
intensities of CT-DNA (negative peak at 245 nm and positive peak at
280 nm), suggesting that they change the super-helicity of DNA.
With some compounds, a strong negative induced CD (ICD) below
300 nm was observed, suggesting DNA intercalation, whereas an
interesting positive ICD was observed at ca. 350 nm with several
methylenedioxy-1,7-BZP derivatives, including 22, 24, 28 and 36.
Such positive ICD should be attributed to some minor groove
binding of the amino side chain in the methylenedioxy-1,7-BZP
series.
low binders with
D
Tm being 4 to 9 ꢀC. TM value could not be
determined for some compounds (ND) due to their poor stability at
high temperature, leading to a modification of their absorbance
value at DNA wavelength (260 nm). Without focusing on those
latter compounds, BZP analogs inducing the highest stabilization of
the DNA helix are also those that induced the highest changes in
UV/visible absorbance spectra in the absence or presence of CT-
DNA (Fig. 1, see compounds 17, 24 and 36). Particularly, in
dimethoxy-1,7-BZP, compound 17, bearing a dimethylaminoethyl-
amine chain branched at position 2 efficiently stabilizes the DNA
helix, whereas substitution with a longer dimethylaminopropyl-
amine chain results in a loss of DNA stabilization. With compounds
3.2.4. Topoisomerases I and II inhibition assays
In numerous cases efficient DNA binders, structurally related to
1,7-BZP and 1,8-BZP, have been shown to inhibit DNA dependant
enzymes, such as topoisomerases I and/or II [11]. The DNA relaxa-
tion induced by topoisomerase I was first studied (Fig. 4). Incuba-
tion of increasing concentrations of the various drugs with
supercoiled plasmid DNA in the presence of topoisomerase I
permitted to characterize the strongest DNA intercalators (17, 21,
22, 23, 35 and 36), evidenced by formation of relaxed plasmid DNA.
Compounds bearing a methylenedioxy substituent appeared to be
more potent DNA intercalators than their dimethoxy counterparts
suggesting that the additional oxirane ring present in the methyl-
enedioxy compounds should increase their DNA intercalation
potency. Also, it should be noted that at the lower concentrations
Table 1
Cytotoxicities against L1210 cell line.
Compound
cytotoxicity
Compound
Cytotoxicity
(IC50 in mM)
(IC50 in
mM)
17
18
19
20
21
22
23
24
1.73
2.85
0.96
1.26
0.32
0.51
1.74
0.83
31
32
33
34
35
36
29
3.51
1.80
1.77
3.70
ND
0.29
4.30
6.60
Fagaronine
(2)
5
3
4
7.10
8.80
6.60
4.40
(1e5
mM), which are within the cytotoxic values of the various
6
drugs, no significant inhibition of topoismerase I was observed.

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T. S¸ erbetçi et al. / European Journal of Medicinal Chemistry 45 (2010) 2547e2558
Fig. 1. UV spectra forcompounds 17e24, 31e33, and 35e36 (20 mM) in theabsence (dashedlanes) or presence of CT-DNA (plain lanes) (20 mM). Spectrawere recorded in BPE buffer, pH 7.1.
A conventional DNA cleavage assay was used to assess the
effects of the new compounds on the catalytic activity of human
topoisomerases I and II (Fig. 5). In these experiments, supercoiled
plasmid DNA was treated with either topoisomerase I or II in the
presence of two concentrations of test drug, CPT and etoposide,
respectively, and the DNA relaxation products were then resolved
by gel electrophoresis on agarose gel [18]. Fig. 5a evidences that, in
contrast with CPT used as a positive control, none of those
compounds present TopoI poisoning effects based both on the lack
of generated nicked forms from supercoiled circular DNA and the
generation of cleaved DNA from a radio-labeled DNA fragment (Fig.
6). In the same way, a weak TopoII poisoning effect was obtained in
comparison with the reference compound etoposide (Fig. 5b). As
previously noted with the unsubstituted parent compounds, top-
potency to bind DNA in a sequence-specific manner using DNase I
footprinting analysis (Fig. 7). The nature of the sequence preferen-
tially recognized by these compounds was determined using
a radiolabeled 265-bp DNA restriction fragment as substrate.
Comparison of the DNase I digested DNA lanes (lanes “0”) with those
obtained from DNA previously incubated with increasing concen-
trations of either of the two compounds evidenced a global decrease
in the intensities of all bands. Interestingly, fine quantification of the
bands by densitometric analysis suggested some sequence prefer-
ences (grey bars) using both 22 and 24. The protected sequences
contained in both cases a CT dinucleotide with any base pairs
surrounding it to cover a larger sequence. This result suggests that
both 22 and 24 intercalate preferentially at 50-CT dinucleotide with
their side chains lying in the DNA groove, thus resulting in
a widening of the covered sequence.
oisomerases inhibition does not constitute
responsible for the cytotoxic activity.
a primary event
4. Conclusion
3.2.5. Sequence-specific DNA binding
Compounds 22 and 24 (and, to a lesser extend, 21 and 23) gave
positive induced circular dichroism in the presence of DNA, that
could evidenced for DNA groove binding (Fig. 3). The DNA relaxation
profiles of those compounds also suggested some intercalation.
Consequently, compounds 22 and 24 were selected to explore their
In order to improve the antiproliferative profile of 1,7- and 1,8-
BZP, dialkylaminoalkyl side chains were introduced on cycle A at
position C2 or C1, depending on the series. This substitution of the
basic BZP core resulted in a significant improvement of the
cytotoxicity. However, the TopoI inhibitory activity induced by the

T. S¸ erbetçi et al. / European Journal of Medicinal Chemistry 45 (2010) 2547e2558
2551
Fig. 2. Fluorescence spectra for compounds 17e24, 31e33, and 35e36. Fluorescence spectral measurement were recorded using 20
m
M of the fluorescent in the presence (plain
lanes) or absence (dashed lanes) of 200
mM of CT-DNA The excitation wavelengths were 268 nm for compound 35, 275 nm for compounds 31, 32, 33 and 36, and 285 nm for all other
evaluated compounds. The emission spectra were scanned at the wavelength specified in the abscissa of the figures.
novel compounds remains low, suggesting that other cellular
targets should be responsible for the antiproliferative activity. It is
interesting to underline that similar discrepancies between cyto-
toxicity and TopoI inhibition have been previously observed in
several related series, including camptothecin, anthracenes, aro-
mathecins and indénoisoquinoléines [19]. It is also worth noting
that in the indenoisoquinoline and dibenzo[c,h][1,6]naphthyridine
series, the aminoalkyl residue were introduced in cycle B or C [7].
From a structure-activity viewpoint, we had previously shown
that the dioxygenated aromatic ring A of natural benzo[c]phenan-
thridines, such as nitidine and fagaronine, could be replaced by
a pyridin unit without loss of activity. Introduction of a dia-
lkylaminoalkyl chain in the pyridin cycle A results in an increase of
the antiproliferative activity of about one order of magnitude when
compared to the parent compounds.
Table 2
CT-DNA melting temperature study.
Compound
D
Tm CT-DNA (ꢀC)a
Compound
D
Tm CT-DNA (ꢀC)a
5. Experimental section
17
18
19
20
21
22
23
24
12
0
5
ND
4
ND
5
31
32
33
34
35
36
37
9
ND
9
ND
7
5.1. Chemistry
5.1.1. General
14
ND
All reactions were carried out in a dried glassware underan argon
atmosphere. All solvents were purchased with an analytical grade
fromSDS. CH2Cl2 was distilled fromCaH2. Methanol, acetonitrile and
DMF were dried over molecular sieves. All other commercially
11
a
complex
Variation in melting temperature
D
Tm (TdmrugeDNA
ꢁ TmDNA alone). Drug/CT
DNA ratio ¼ 1.

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Fig. 3. Circular dichroism spectra for compounds 17e24, 31e33, and 35e36 (50 mM) alone (plaine lanes), with CT-DNA (200 mM) (pointed lanes) or CT-DNA (200 mM) alone (dashed
lanes). The spectra were recorded from 500 to 230 nm with a resolution of 0.1 nm.
available reagents were used as received. Flash chromatography was
performed with silica gel CHROMATOGEL 60 (particle size
5.1.3. General procedure for the preparation of benzo[c]
phenanthrolines-N-oxides
20e45
mm or 35e70
mm) supplied by SDS. Yield refers to chroma-
To
a
solution of the benzo[c]phenanthroline (191 mg,
tography 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 spectrometer.
Chemical shifts are reported in ppm and corrected to dH 7.26 for
CDCl3 as internal reference, coupling constants (J) in Hertz. 13C 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).
Infrared spectra were recorded on a Nicolet FT-IR spectrometer and
0.656 mmol)inanhydrousCH2Cl2 (10 ml)wasaddedmCPBA(113 mg,
0.656 mmol) at rt. After 3 h the precipitate formed was filtered,
washed with a minimum of CH2Cl2 then with water and dried. The
corresponding N-oxide was used without further purification.
5.1.3.1. 8,9-Dimethoxybenzo[c][1,7]phenanthroline-N-oxide. Yield
79%. IR 2960 (
n
CH), 2920, 1642, 1519 (
n
CNO), 1478, 1427, 1276,
9.33
1178, 1168, 873, 771 cmꢁ1
.
1H NMR (400 MHz, CDCl3)
d
(s, 1H), 9.27 (d, 1H, J ¼ 8.0 Hz), 8.86 (d, 1H, J ¼ 8.0 Hz), 8.76 (d, 1H,
J ¼ 9.0 Hz), 8.68 (d, 1H, J ¼ 6.0 Hz), 7.95 (s, 1H), 7.58 (dd, 1H,
J ¼ 6.0, 8.0 Hz), 7.46 (s, 1H), 4.20 (s, 3H), 4.09 (s, 3H). 13C NMR
(75 MHz, CDCl3)
d 153.6, 151.4, 150.9, 140.9, 140.0, 136.5, 130.3,
128.2, 125.0, 123.8, 123.2, 121.9, 121.6, 117.5, 107.2, 101.7. 56.3
(2 C). MS (ESþ) m/z 329 [M þ Na]þ . HRMS (ESþ) m/z calcd for
C18H15N2O3 [M þ H]þ 307.1083, found 307.1081.
wavelengths (y .
) are given in cmꢁ1
5.1.2. General procedure for the preparation of benzo[c]
phenanthrolines cores 12, 13, 27 and 28
See Ref. [8].
5.1.3.2. 8,9-Methylenedioxybenzo[c][1,7]phenanthroline-N-oxidepar-
a>Yield 74%. IR 3093, 2920 (
n
CH), 1598, 1473 (
n CNO), 1444, 1265
(n
CN), 1211, 1158, 1032, 946, 821, 780 cmꢁ1
.
1H NMR (400 MHz,

T. S¸ erbetçi et al. / European Journal of Medicinal Chemistry 45 (2010) 2547e2558
2553
Fig. 4. Topoisomerase I-induced DNA relaxation. Effect of compounds 17e24 and 31e36 on the relaxation of plasmid DNA by human topoisomerase I. Native supercoiled pUC19
(130 ng, lane DNA) was incubated with 4 units of topoisomerase I in the absence (lane 0) or presence of tested compound at the indicated concentration (1e50 M). Samples were
m
separated by electrophoresis on a 1% agarose gel, which was stained with ethidium bromide after DNA migration. Gels were photographed under UV light: Nck, nicked; Sc,
supercoiled; Rel, relaxed; Topo, topoisomer products.
CDCl3)
d
9.30 (s, 1H), 9.26 (d, 1H, J ¼ 8.5 Hz), 8.90 (d, 1H, J ¼ 9.3 Hz),
5.1.3.4. 8,9-Methylenedioxybenzo[c][1,8]phenanthroline-N-oxide.
Yield 84%. IR 3079, 2909 ( CH),1701,1618 ( CNO),1575,1478 ( CNO),
1444, 1256, 1158, 1044, 946 (OCH2O), 844, 746 cmꢁ1 1H NMR
(300 MHz, CDCl3)
8.70 (dd, 1H, J ¼ 6.0, 1.0 Hz), 8.68 (d, 1H, J ¼ 9.3 Hz), 8.01 (s, 1H), 7.56
(dd, 1H, J ¼ 8.5, 6.0 Hz), 7.47 (s, 1H), 6.24 (s, 2H). MS (ESþ) m/z 291
[M þ H]þ, 313 [M þ Na]þ . HRMS (ESþ) m/z calcd for C17H11N2O3
[M þ H]þ 291.0770, found 291.0766.
n
n
n
.
d
9.29 (s, 1H), 9.15 (d, 1H, J ¼ 6.0 Hz), 8.93 (d, 1H,
J ¼ 2.0 Hz), 8.52(d,1H, J ¼ 9.0 Hz), 8.42(dd,1H, J ¼ 6.0, 2.0 Hz), 8.09(s,
1H), 7.85 (d,1H, J ¼ 9.0 Hz), 7.22 (s,1H), 6.24 (s, 2H).13C NMR (75 MHz,
5.1.3.3. 8,9-Dimethoxybenzo[c][1,8]phenanthroline-N-oxide. Yield
86%. IR 3070, 2948 (
1273 (
CDCl3)
CDCl3) d 152.3,151.8,149.2,137.0,136.3,136.1,132.7,130.4,130.3,129.7,
n
CH), 2920, 1733, 1610 (
n
CNO), 1519, 1406,
128.2, 125.0, 123.3, 121.8, 105.4, 102.6, 100.0. MS (ESþ) m/z 291
[M þ H]þ, 313 [M þ Na]þ. HRMS (ESþ) m/z calcd for C17H11N2O3
[M þ H]þ 291.0770, found 291.0768.
n
CN), 1227, 1157, 1090, 799 cmꢁ1 1H NMR (400 MHz,
.
d
9.34 (s, 1H), 9.19 (d, 1H, J ¼ 7.0 Hz), 9.07 (d, 1H,
J ¼ 1.5 Hz), 8.61 (d, 1H, J ¼ 9.0 Hz), 8.49 (dd, 1H, J ¼ 7.0, 1.5 Hz),
7.91 (s, 1H), 7.89 (d, 1H, J ¼ 9.0 Hz), 7.47 (s, 1H), 4.21 (s, 3H), 4.14
5.1.4. General procedure for the chlorination of benzo[c]
phenanthrolines-N-oxides
(s, 3H). 13C NMR (75 MHz, CDCl3)
d 153.1, 151.5, 151.0, 138.2, 137.3,
136.0, 133.3, 130.5, 128.9, 124.2, 122.8, 122.7, 121.8, 121.5, 107.4,
101.6, 56.2 (2 C). MS (ESþ) m/z 307 [M þ H]þ. HRMS (ESþ) m/z
calcd for C18H15N2O3 [M þ H]þ 307.1083, found 307.1079.
Benzo[c]phenanthroline-N-oxide dissolved in POCl3 was heated
at 160 ꢀC. After the reaction was completed (TLC determination) the
solution was cooled to rt and poured into ice. pH was adjusted to

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T. S¸ erbetçi et al. / European Journal of Medicinal Chemistry 45 (2010) 2547e2558
Fig. 6. Topoisomerase I poisoning. Cleavage of the 117-mer DNA fragment by top-
oisomerase I in the presence of the benzo[c]phenanthrolines 17e21 and 33. The 30-end
labeled fragment (0) was incubated in the absence (lane TopoI) or presence of the
alkaloids at 10 or 50 mM. Camptothecin (CPT) was used at 20e50 mM. Topoisomerase I
cleavage reactions were analyzed on a 8% denaturing polyacrylamide gel, compared to
the guanine track labeled (G).
MS (ESþ) m/z 309/311 [M þ H]þ. HRMS (ESþ) m/z calcd for
C17H10N2O2Cl [M þ H]þ 309.0431, found 309.0427.
Fig. 5. Topoisomerase I and II inhibition. (a) Effect of benzo[c]phenanthrolines 17e36
on the relaxation of plasmid DNA by topoisomerase I at 50
and 50 M. Native supercoiled pUC19 plasmid DNA (130 ng) (lane DNA) was incubated
with 4 units topoisomerase I in the absence (lane 0) or presence of drug at the indi-
cated concentration (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. Gel (b) shows the effect of benzo[c]phenanthrolines 17e36 on the relaxation of
plasmid DNA by topoisomerase II at 100 M, compared to Etop at 20e100 M. Native
mM compared to CPT at 20
m
5.1.4.3. 1-Chloro-8,9-dimethoxybenzo[c][1,8]phenanthroline (29).
m
Yield 82%. IR 2954 (
n
CH), 2918, 2852, 1609, 1517 (
n
CN), 1411,
9.30
1269, 1115, 987, 812 cmꢁ1
.
1H NMR (400 MHz, CDCl3)
d
m
(s, 1H), 9.03 (d, 1H, J ¼ 5.5 Hz), 8.53 (d, 1H, J ¼ 5.5 Hz), 8.52 (d, 1H,
m
m
J ¼ 9.2 Hz), 8.36 (d, 1H, J ¼ 9.2 Hz), 7.88 (s, 1H), 7.43 (s, 1H), 4.19
supercoiled pUC19 plasmid DNA (350 ng) (lane DNA) was incubated with 10 units
topoisomerase I in the absence (lane TopoII) or presence of drug at the indicated
(s, 3H), 4.12 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 153.6, 151.1, 150.9
(2 C), 143.4, 138.7, 135.7, 128.1, 126.0, 123.7, 123.5, 122.5, 117.6,
concentration (100
treatment with proteinase K. DNA samples were separated by electrophoresis on 1%
agarose gels containing ethidium bromide (1 g/mL) and then photographed under
mM). Reactions were stopped with sodium dodecylsulfate and
117.5, 107.4, 102.0, 56.2 (2 C). MS (ESþ) m/z 325/327 [M þ H]þ
.
HRMS (ESþ) m/z calcd for C18H14N2O2Cl [M þ H]þ 325.0744,
m
UV light. Nck, nicked; Rel, relaxed; Sc, supercoiled DNA.
found 325.0741.
5.1.4.4. 1-Chloro-8,9-methylenedioxybenzo[c][1,8]phenanthroline
7e8 with an aqueous ammonia solution and the solution was left
for 30 min. The precipitate formed was then filtered washed with
water and dried. Purification by flash chromatography on silica gel
(eluent CH2Cl2/MeOH 100:0 to 99.5:0.5) afforded the correspond-
ing chloro compound as a white solid.
(30). Yield 68%. IR 3134, 2925, 2860, 1640, 1610, 1266, 1210, 1103,
1036, 939, 840, 669 cmꢁ1. 1H NMR (300 MHz, CDCl3)
d 9.32 (s, 1H),
9.10 (d, 1H, J ¼ 6.0 Hz), 8.58 (d, 1H, J ¼ 6.0 Hz), 8.56 (d, 1H,
J ¼ 9.0 Hz), 8.45 (d, 1H, J ¼ 9.0 Hz), 8.03 (s, 1H), 7.49 (s, 1H), 6.27 (s,
2H). MS (ESþ) m/z 309/311 [M þ H]þ. HRMS (ESþ) m/z calcd for
C17H10N2O2Cl [M þ H]þ 309.0431, found 309.0429.
5.1.4.1. 2-chloro-8,9-Dimethoxybenzo[c][1,7]phenanthroline (15). Yield
80%. IR 3183 (
n CH), 1659, 1618, 1570, 1490, 1431, 1274, 1157, 1078, 821,
5.1.5. General procedure for the preparation of dialkylamino
alkylbenzo[c]phenanthroline
800, 738 cmꢁ1. 1H NMR (300 MHz, CDCl3)
d
9.58 (d, 1H, J ¼ 8.6 Hz),
9.33 (s, 1H), 8.72 (d, 1H, J ¼ 9.3 Hz), 8.18 (d, 1H, J ¼ 9.3 Hz), 7.94 (s, 1H),
The chlorinated benzo[c]phenanthroline (70 mg, 0.241 mmol)
was heated in a excess of the appropriate dialkylaminoalkylamine
(7 ml)overnight. Thereactionmixturewasconcentrated invacuoand
purified by chromatography on silica gel (eluent: CH2Cl2/MeOH/
NH4OH 100:0:0 to 90:10:0.5) to afford the corresponding compound.
7.64 (d, 1H, J ¼ 8.6 Hz), 7.46 (s, 1H), 4.21 (s, 3H), 4.16 (s, 3H). 13C NMR
(75 MHz, CDCl3)
d 153.4, 151.4, 151.0, 150.6, 148.1, 140.1, 136.1, 128.5,
127.2, 126.3, 124.8, 122.9, 122.7, 120.8, 107.3, 101.5, 56.2 (2 C). MS (ESþ)
m/z 325/327 [M þ H]þ, 347/349 [M þ Na]þ . HRMS (ESþ) m/z calcd for
C18H14N2O2Cl [M þ H]þ 325.0744, found 325.0739.
5.1.5.1. 2-[(20-(Dimethylamino)ethyl)amino]-8,9-dimethoxybenzo[c]
5.1.4.2. 2-Chloro-8,9-methylenedioxybenzo[c][1,7]phenanthroline
(16). Yield 56%. IR 3033, 2094, 1655, 1605, 1578, 1473, 1442, 1306,
1253,1197, 1122, 1038, 947, 910, 827 cmꢁ1. 1H NMR (300 MHz, CDCl3)
[1,7]phenanthroline (17). Yield 38%. IR 3366 (
2655, 2637, 1616 ( NH), 1587, 1522, 1497, 1458, 1270 (
1038, 822 cmꢁ1. 1H NMR (400 MHz, CDCl3)
n
NH), 2930 (
CN), 1155,
9.19 (d, 1H, J ¼ 9.0 Hz),
n CH),
d
n
d
d
9.60 (d, 1H, J ¼ 8.6 Hz), 9.28 (s, 1H), 8.65 (d, 1H, J ¼ 9.0 Hz), 8.17 (d,
1H, J ¼ 9.0 Hz), 7.99 (s,1H), 7.65 (d,1H, J ¼ 8.6 Hz), 7.47 (s,1H), 6.24 (s,
2H). 13C NMR (75 MHz, CDCl3)
151.9, 151.5, 151.2, 150.9, 148.8,142.3,
136.1, 130.5, 127.4, 127.2, 125.2, 125.0, 124.2, 122.8, 105.1, 102.1, 99.8.
9.16 (s, 1H), 8.44 (d, 1H, J ¼ 9.2 Hz), 7.88 (d, 1H, J ¼ 9.2 Hz), 7.78
(s,1H), 7.29 (s,1H), 6.84 (d, 1H, J ¼ 9.0 Hz), 4.77 (bs, NH), 4.11 (s, 3H),
4.04 (s, 3H), 3.62 (t, 2H, J ¼ 6.0 Hz), 2.62 (t, 2H, J ¼ 6.0 Hz), 2.30
d
(s, 6H). 13C NMR (75 MHz, CDCl3)
d 157.8, 152.9, 150.2, 149.0, 148.2,

T. S¸ erbetçi et al. / European Journal of Medicinal Chemistry 45 (2010) 2547e2558
2555
Fig. 7. Sequence-specific DNA binding. Appropriate concentrations of the various benzophenanthrolines were incubated with the 265 bp 30-end-radio-labeled DNA fragment prior
to DNA digestion using DNase I (0.01 unit/ml). The digestion reaction was stopped by freeze drying and lyophilization of the samples. DNA samples were separated by electro-
phoresis on an 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).
140.7, 133.7, 128.8, 126.3, 123.0, 121.5, 120.6, 118.0, 111.3, 107.1, 101.1,
58.1, 56.0 (2C), 45.1 (2 C), 38.8. MS (DIC/NH3) m/z 376 [M]þ. HRMS
(ESþ) m/z calcd for C22H25N4O2 [M þ H]þ 376.1978, found 376.1974.
J ¼ 7.0 Hz). 13C NMR (75 MHz, CDCl3)
d 157.9, 152.9, 150.3, 149.3,
148.4, 140.9, 133.7, 128.8, 126.6, 123.0, 121.6, 120.7, 117.9, 111.2, 107.2,
101.2, 56.1, 53.4 (2C), 46.6 (2 C), 39.0, 11.6 (2 C). MS (DIC/NH3) m/z
404 [M]þ. HRMS (ESþ) m/z calcd for C24H29N4O2 [M þ H]þ 405.2291,
found 405.2286.
5.1.5.2. 2-[(30-(Dimethylamino)propyl)amino]-8,9-dimethoxybenzo
[c][1,7]phenanthroline (18). Yield 19%. IR 3328, 2922, 1681, 1654,
1615, 1541, 1457, 1258, 1149, 1036, 825, 765, 740 cmꢁ1
.
1H NMR
5.1.5.4. 2-[(30-(Diethylamino)propyl)amino]-8,9-dimethoxybenzo[c]
(400 MHz, CDCl3)
d
9.23 (d, 1H, J ¼ 8.0 Hz), 9.21 (s, 1H), 8.49 (d, 1H,
[1,7]phenanthroline (20). Yield 42%. IR 3355 (
CH), 1615 ( NH), 1537, 1471, 1385, 1271 ( CN), 1224, 1150, 824, 732,
707 cmꢁ1. 1H NMR (400 MHz, CDCl3)
9.16 (d, 1H, J ¼ 8.0 Hz), 9.14
n NH), 3053, 2969 (n
J ¼ 9.0 Hz), 7.89 (d, 1H, J ¼ 9.0 Hz), 7.85 (s, 1H), 7.35 (s, 1H), 6.85
(d, 1H, J ¼ 8.0 Hz), 5.85 (bs, NH), 4.15 (s, 3H), 4.08 (s, 3H), 3.63 (t, 2H,
J ¼ 6.0 Hz), 2.51 (t, 2H, J ¼ 6.0 Hz), 2.31 (s, 6H), 1.89 (quint, 2H,
d
n
d
(s, 1H), 8.42 (d, 1H, J ¼ 9.0 Hz), 7.85 (d, 1H, J ¼ 9.0 Hz), 7.76 (s, 1H),
7.28 (s, 1H), 6.81 (d, 1H, J ¼ 8.0 Hz), 6.31 (bs, NH), 4.10 (s, 3H), 4.01
(s, 3H), 3.64 (t, 2H, J ¼ 6.0 Hz), 2.63 (q, 4H, J ¼ 7.0 Hz), 2.52 (t, 2H,
J ¼ 6.0 Hz), 1.89 (quint, 2H), 1.12 (t, 6H, J ¼ 7.0 Hz). 13C NMR
J ¼ 6.0 Hz). 13C NMR (75 MHz, CDCl3)
d 158.0, 153.0, 150.3, 149.4,
148.7, 141.1, 133.9, 128.9, 126.5, 123.1, 121.7, 120.9, 118.0, 110.8, 107.2,
101.3, 58.þ2, 56.1 (2 C), 45.5 (2 C), 41.0, 26.9. MS (ESþ) m/z 391
[M þ H]
.
HRMS (ESþ) m/z calcd for C23H27N4O2, [M þ H]þ
(75 MHz, CDCl3) d 157.9, 152.9, 150.3, 149.3, 148.4, 140.8, 133.6,
391.2134, found 391.2130.
128.8, 126.4, 122.9, 121.6, 120.6, 117.9, 111.3, 107.1, 101.2, 56.0 (2C),
51.7, 46.7 (2 C), 41.1, 25.7, 11.2 (2 C). MS (DIC/NH3) m/z 418 [M]þ.
HRMS (ESþ) m/z calcd for C25H31N4O2 [M þ H]þ 419.2447, found
419.2445.
5.1.5.3. 2-[(20-(Diethylamino)ethyl)amino]-8,9-dimethoxybenzo[c]
[1,7]phenanthroline (19). Yield 41%. IR 3355 (
2660, 1653, 1570, 1520, 1418, 1259, 1205, 1077, 819 cmꢁ1
(400 MHz, CDCl3)
J ¼ 9.0 Hz), 7.90 (d, 1H, J ¼ 9.0 Hz), 7.86 (s, 1H), 7.36 (s, 1H), 6.86
(d, 1H, J ¼ 8.0 Hz), 5.79 (bs, NH), 4.18 (s, 3H), 4.11 (s, 3H), 3.66 (q, 2H,
J ¼ 6.0 Hz), 2.85 (t, 2H, J ¼ 6.0 Hz), 2.68 (q, 4H, J ¼ 7.0 Hz), 1.12 (t, 6H,
n
NH), 2922 (
n CH),
.
1H NMR
d
9.23 (d, 1H, J ¼ 8.0 Hz), 9.22 (s, 1H), 8.50 (d, 1H,
5.1.5.5. 2-[(20-(Dimethylamino)ethyl)amino]-8,9-methylenedioxybenzo
[c][1,7]phenanthroline (21). Yield 27%. IR 3407 (
1665, 1545, 1473, 1400, 1364, 1257 (
CN), 1101, 1037, 821, 750 cmꢁ1
1H NMR (300 MHz, CDCl3)
9.20 (d,1H, J ¼ 8.0 Hz), 9.12 (s, 1H), 8.38
n NH), 2923 (n CH),
n
.
d

2556
T. S¸ erbetçi et al. / European Journal of Medicinal Chemistry 45 (2010) 2547e2558
(d, 1H, J ¼ 9.0 Hz), 7.86 (d, 1H, J ¼ 9.0 Hz), 7.84 (s, 1H), 7.31 (s, 1H),
6.84 (d, 1H, J ¼ 8.0 Hz), 6.72 (bs, NH), 6.12 (s, 2H), 3.63 (q, 2H,
J ¼ 6.0 Hz), 2.42 (t, 2H, J ¼ 6.0 Hz), 2.22 (t, 6H, J ¼ 6.0 Hz). 13C NMR
J ¼ 6.0 Hz), 2.59 (t, 2H, J ¼ 6.0 Hz), 2.39 (s, 6H), 1.40 (quint, 2H,
J ¼ 6.0 Hz). 13C NMR (75 MHz, CDCl3)
d 155.8, 152.9 (2C), 150.3,
143.5, 139.6, 137.1, 128.1, 122.9, 122.8, 120.3, 119.1, 116.5, 107.2, 106.4,
101.7, 59.6, 56.1(2C), 45.5 (2 C), 45.5, 25.3. MS (DIC/NH3) m/z 390
[M]þ. HRMS (ESþ) m/z calcd for C23H27N4O2, [M þ H]þ 391.2134,
found 391.2131.
(75 MHz, CDCl3) d 157.8, 151.6, 150.5, 148.2, 147.6, 141.0, 133.8, 130.9,
126.4, 123.3, 122.9, 120.5, 118.7, 111.4, 104.9, 101.8, 99.4, 57.8, 45.0 (2
C), 41.0. MS (ESþ) m/z 361 [M þ H]þ. HRMS (ESþ) m/z calcd for
C21H21N4O2, [M þ H]þ 361.1665, found 361.1661.
5.1.5.11. 1-[(20-(Diethylamino)ethyl)amino]-8,9-dimethoxybenzo[c]
5.1.5.6. 2-[(30-(Dimethylamino)propyl)amino]-8,9-methylenedioxy
[1,8]phenanthroline (33). Yield 46%. IR 3373 (
1669, 1615 ( NH), 1518, 1483, 1417, 1271 ( CN), 1164, 1068, 827,
788 cmꢁ1 1H NMR (300 MHz, CDCl3)
9.31 (s, 1H), 8.43 (d, 1H,
n NH), 2969 (n CH),
benzo[c][1,7]phenanthroline (22). Yield 31%. IR 3345 (
CH), 2853, 1612, 1544, 1470, 1254 (
CN), 1038, 825, 725 cmꢁ1. 1H
NMR (300 MHz, CDCl3)
n
NH), 2923
d
n
(n
n
.
d
d
9.21 (d, 1H, J ¼ 8.0 Hz), 9.12 (s, 1H), 8.39
J ¼ 9.0 Hz), 8.34 (d, 1H, J ¼ 5.0 Hz), 8.28 (d, 1H, J ¼ 5.0 Hz), 8.03
(d, 1H, J ¼ 9.0 Hz), 7.91 (s, 1H), 7.41 (s, 1H), 6.56 (bs, NH), 4.18 (s, 3H),
4.11 (s, 3H), 3.72 (t, 2H, J ¼ 6.0 Hz), 2.90 (t, 2H, J ¼ 6.0 Hz), 2.71
(q, 4H, J ¼ 7.0 Hz), 1.14 (t, 6H, J ¼ 7.0 Hz). 13C NMR (75 MHz, CDCl3)
(d, 1H, J ¼ 9.0 Hz), 7.86 (d, 1H, J ¼ 9.0 Hz), 7.84 (s, 1H), 7.31 (s, 1H),
6.83 (d, 1H, J ¼ 8.0 Hz), 6.13 (s, 2H), 5.74 (bs, NH), 3.59 (t, 2H,
J ¼ 6.0 Hz), 2.48 (t, 2H, J ¼ 6.0 Hz), 2.29 (s, 6H), 1.86 (quint, 2H,
J ¼ 6.0 Hz). 13C NMR (75 MHz, CDCl3)
d
158.1, 151.5, 150.5, 148.4,
d 155.3, 153.8, 150.3 (2 C), 143.1, 139.9, 137.2, 128.2, 123.0 (2C), 120.4,
147.6, 141.1,134.0, 130.9, 126.5, 123.3, 122.9, 120.5, 118.6, 110.7,104.9,
119.5, 116.5, 107.1 (2C), 101.8, 56.1 (2C), 51.5, 46.9 (2 C), 39.2, 11.2
(2 C). MS (DIC/NH3) m/z 404 [M]þ. HRMS (ESþ) m/z calcd for
C24H29N4O2, [M þ H]þ 405.2291, found 405.2289.
101.7, 99.4, 57.9, 45.0 (2 C), 40.7, 26.9. MS (ESþ) m/z 375 [M þ H] þ
,
397 [M þ Na] þ. HRMS (ESþ) m/z calcd for C22H23N4O2, [M þ H]þ
375.1821, found 375.1815.
5.1.5.12. 1-[(30-(Diethylamino)propyl)amino]-8,9-dimethoxybenzo[c]
5.1.5.7. 2-[(20-(Diethylamino)ethyl)amino]-8,9-methylenedioxybenzo
[1,8]phenanthroline (34). Yield 41%. IR 3341 (
1614 ( NH), 1584, 1518, 1484, 1416, 1337, 1270 (
827, 746 cmꢁ1. 1H NMR (400 MHz, CDCl3)
9.26 (s, 1H), 8.30 (d, 1H,
n
NH), 2968 (n CH),
[c][1,7]phenanthroline (23). Yield 16%. IR 3384 (
2870, 1654, 1541, 1457, 1259 (
CN), 1116, 1038, 936 (OCH2O) cmꢁ1
1H NMR (400 MHz, CDCl3)
n
NH), 2924 (
n
CH),
d
n
CN), 1164, 1071,
n
.
d
d
9.24 (d, 1H, J ¼ 8.0 Hz), 9.18 (s, 1H), 8.44
J ¼ 9.0 Hz), 8.28 (d, 1H, J ¼ 5.0 Hz), 8.25 (d, 1H, J ¼ 5.0 Hz), 7.99 (bs,
NH), 7.92 (d, 1H, J ¼ 9.0 Hz), 7.87 (s, 1H), 7.38 (s, 1H), 4.17 (s, 3H),
4.09 (s, 3H), 3.74 (q, 2H, J ¼ 6.0 Hz), 2.72 (t, 2H, J ¼ 6.0 Hz), 2.69
(q, 4H, J ¼ 7.0 Hz), 1.94 (quint, 2H, J ¼ 6.0 Hz), 1.15 (t, 6H, J ¼ 7.0 Hz).
(d, 1H, J ¼ 9.0 Hz), 7.91 (s, 1H), 7.88 (d, 1H, J ¼ 9.0 Hz), 7.37 (s, 1H),
6.90 (d, 1H, J ¼ 8.0 Hz), 6.17 (s, 2H), 5.88 (bs, NH), 3.70 (q, 2H,
J ¼ 6.0 Hz), 2.90 (t, 2H, J ¼ 6.0 Hz), 2.73 (q, 4H, J ¼ 7.0 Hz), 1.19
(s, 6H). 13C NMR (75 MHz, CDCl3)
d
158.1, 151.6, 150.0, 148.6, 147.5,
13C NMR (75 MHz, CDCl3)
d 155.9, 153.0 (2C), 150.3, 143.5, 139.7,
141.3, 133.8, 131.1, 126.4, 123.5, 123.2, 120.5, 117.0, 110.9, 104.9, 102.þ0,
137.2, 128.2, 123.0 (2C), 120.9, 119.0, 116.6, 107.3, 107.6, 101.9, 56.3
(2 C), 51.2, 46.9 (2 C), 45.0, 24.8, 11.2 (2 C). MS (ESþ) m/z 419
[M þ H]þ. HRMS (ESþ) m/z calcd for C25H31N4O2, [M þ H]þ 419.2447,
found 419.2447.
99.2, 58.1, 46.6 (2 C), 39.0, 11.6 (2 C). MS (ESþ) m/z 389 [M þ H]
.
HRMS (ESþ) m/z calcd for C23H24N4O2, [M þ H]þ 389.1978, found
389.1975.
5.1.5.8. 2-[(30-(Diethylamino)propyl)amino]-8,9-methylenedioxybenzo
5.1.5.13. 1-[(20-(Dimethylamino)ethyl)amino]-8,9-methylenedioxy
[c][1,7]phenanthroline (24). Yield 8%. IR 3317 (
2853, 1613, 1544, 1451, 1254 (
CN), 1226, 1192, 1038, 826, 745 cmꢁ1
1H NMR (300 MHz, CDCl3)
9.21 (d, 1H, J ¼ 8.0 Hz), 9.16 (s, 1H), 8.43
n
NH), 2924 (
n
CH),
benzo[c][1,8]phenanthroline (35). Yield 17%. IR 3413 (
CH), 2858, 1655, 1623 ( NH), 1585, 1509, 1466, 1267 (
1047, 951 (OCH2O), 845, 738 cmꢁ1.1H NMR (400 MHz, CDCl3)
n
NH), 2951
CN), 1197,
9.16 (s,
n
.
(n
d
n
d
d
(d, 1H, J ¼ 9.0 Hz), 7.89 (s, 1H), 7.87 (d, 1H, J ¼ 9.0 Hz), 7.35 (s, 1H),
6.82 (d, 1H, J ¼ 8.0 Hz), 6.20 (bs, NH), 6.16 (s, 2H), 3.66 (t, 2H,
J ¼ 6.0 Hz), 2.71 (t, 2H, J ¼ 6.0 Hz), 2.66 (q, 4H, J ¼ 7.0 Hz), 1.92
(quint, 2H, J ¼ 6.0 Hz), 1.08 (t, 6H, J ¼ 7.0 Hz). 13C NMR (75 MHz,
1H), 8.28 (d, 1H, J ¼ 9.0 Hz), 8.25 (d, 1H, J ¼ 6.0 Hz), 8.19 (d, 1H,
J ¼ 6.0 Hz), 8.08 (d, 1H, J ¼ 9.0 Hz), 7.88 (s, 1H), 7.36 (s, 1H), 6.51 (bs,
NH), 6.17 (s, 2H) 3.70 (t, 2H, J ¼ 6.0 Hz), 2.60 (t, 2H, J ¼ 6.0 Hz), 2.38
(s, 6H). 13C NMR (75 MHz, CDCl3)
d 155.3, 151.7, 150.5, 148.7, 143.2,
CDCl3)
d
158.1, 151.5, 150.5, 148.6, 147.5, 141.2, 133.8, 130.9, 126.7,
139.5, 137.0, 130.2, 124.3, 123.6, 120.6, 119.6, 116.6, 107.1, 105.0, 102.0,
100.0, 58.1, 45.0 (2 C), 38.8. MS (ESþ) m/z ¼ 361 [M þ H]þ. HRMS (ESþ)
m/z calcd for C21H21N4O2, [M þ H]þ 361.1665, found 361.1663.
123.2, 122.9, 120.6, 118.5, 111.2, 105.0, 101.7, 99.4, 53.4, 46.8 (2 C),
41.3, 25.9, 11.4 (2 C). MS (ESþ) m/z 403 [M þ H]þ. HRMS (ESþ) m/z
calcd for C24H27N4O2, [M þ H]þ 403.2134, found 403.2130.
5.1.5.14. 1-[(30-(Dimethylamino)propyl)amino]-8,9-methylenediox-
5.1.5.9. 1-[(20-(Dimethylamino)ethyl)amino]-8,9-dimethoxybenzo[c]
ybenzo[c][1,8]phenanthroline (36). Yield 6%. IR 3453 (
CH), 1635 ( NH), 1533, 1493, 1471, 1262 ( CN), 1118, 1045, 946
(OCH2O), 843, 739 cmꢁ1. 1H NMR (300 MHz, CDCl3)
9.22 (s, 1H),
n NH), 2925 (n
[1,8]phenanthroline (31). Yield 52%. IR 3370 (
1666, 1519, 1386, 1270 (
CN), 1038, 830, 782 cmꢁ1
(300 MHz, CDCl3)
n
NH), 2946 (
n
CH),
d
n
n
.
1H NMR
d
d
9.33 (s, 1H), 8.42 (d, 1H, J ¼ 9.0 Hz), 8.37 (d, 1H,
8.30e8.26 (m, 2H), 8.26 (d, 1H, J ¼ 9.0 Hz), 7.90 (s, 1H), 7.82 (d, 1H,
J ¼ 9.0 Hz), 7.72 (bs, NH), 7.38 (s, 1H), 6.19 (s, 2H), 3.75 (t, 2H,
J ¼ 6.0 Hz), 2.51 (t, 2H, J ¼ 6.0 Hz), 2.21 (s, 6H), 1.95 (quint, 2H,
J ¼ 5.0 Hz), 8.32 (d, 1H, J ¼ 5.0 Hz), 8.02 (d, 1H, J ¼ 9.0 Hz), 7.94
(s, 1H), 7.46 (s, 1H), 6.17 (bs, NH), 4.20 (s, 3H), 4.13 (s, 3H), 3.73
(q, 2H, J ¼ 6.0 Hz), 2.74 (t, 2H, J ¼ 6.0 Hz), 2.38 (s, 6H). 13C NMR
J ¼ 6.0 Hz). 13C NMR (75 MHz, CDCl3)
d 155.8, 151.5, 150.4, 148.5,
(75 MHz, CDCl3)
d
155.4, 153.1, 150.5, 150.4, 143.2, 139.6, 137.2, 128.1,
143.6, 140.0, 137.1, 130.2, 124.3, 123.3, 120.4, 119.3, 116.5, 106.5,
105.0, 102.0, 99.9, 59.6, 45.5 (2 C), 42.8, 25.4. MS (ESþ) m/z 375
[M þ H]þ. HRMS (ESþ) m/z calcd for C22H21N4O2, [M þ H]þ 375.1821,
found 375.1818.
123.0 (2C), 120.4, 119.3, 116.4, 107.3, 107.2, 101.8, 58.1, 56.2 (2C), 45.1
(2 C), 39.2. MS (ESþ) m/z 377 [M þ H]þ. HRMS (ESþ) m/z calcd for
C22H25N4O2, [M þ H]þ 377.1978, found 377.1975.
5.1.5.10. 1-[(30-(Dimethylamino)propyl)amino]-8,9-dimethoxybenzo
5.1.5.15. 1-[(30-(Diethylamino)propyl)amino]-8,9-methylenedioxy
[c][1,8]phenanthroline (32). Yield 50%. IR 3371 (
1615 ( NH), 1584, 1484, 1416, 1270 ( CN), 1164, 1070, 827,
788 cmꢁ1 1H NMR (400 MHz, CDCl3)
9.25 (s, 1H), 8.30 (d, 1H,
n
NH), 2939 (
n
CH),
benzo[c][1,8]phenanthroline (37). Yield 9%. IR 3373 (
CH), 2853, 1647, 1617 ( NH), 1540, 1471, 1375, 1261 (
(OCH2O), 828 cmꢁ1. 1H NMR (300 MHz, CDCl3)
9.24 (s,1H), 8.31 (d,
n
NH), 2923 (
n
d
n
d
n CN), 1037, 942
.
d
d
J ¼ 9.0 Hz), 8.28e8.24 (m, 2H), 7.84 (s, 1H), 7.81 (d, 1H, J ¼ 9.0 Hz),
1H, J ¼ 9.0 Hz), 8.29 (d, 1H, J ¼ 6.0 Hz), 8.19 (d, 1H, J ¼ 6.0 Hz), 8.02
7.66 (bs, NH), 7.37 (s, 1H), 4.15 (s, 3H), 4.08 (s, 3H), 3.74 (t, 2H,
(d,1H, J ¼ 9.0 Hz), 7.97 (s,1H), 7.42 (s, 1H), 6.35 (bs, NH), 6.21 (s, 2H),

T. S¸ erbetçi et al. / European Journal of Medicinal Chemistry 45 (2010) 2547e2558
2557
3.77 (t, 2H, J ¼ 6.0 Hz), 2.76 (q, 4H), 1.99 (t, 2H, J ¼ 6.0 Hz) 1.77e1.72
(m, 2H), 1.19 (t, 6H). MS (ESþ) m/z 403 [M þ H]þ. HRMS (ESþ) m/z
calcd for C24H27N4O2, [M þ H]þ 403.2134, found 403.2132.
a PTC-424S/L peltier type cell changer (Jasco). The spectra were
recorded from 500 to 230 nm with a resolution of 0.1 nm.
5.2.6. Topoisomerase I-mediated DNA relaxation and DNA cleavage
assays (Figs. 4 and 5a)
5.2. Biology
The experiments were conducted as presented elsewhere [20].
Briefly, supercoiled pUC19 plasmid DNA (130 ng) was incubated
with 4 units of human recombinant topoisomerase I (TopoGen) at
5.2.1. Cytotoxicity (Table 1)
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 L-glutamine,
100 U/mL penicillin, 100 lg/mL streptomycin and 10 mM HEPES
buffer (pH 7.4). The cytotoxicity was measured by microculture
tetrazolium assay essentially as described. Cells were exposed for
48 h to nine graded concentrations in triplicate of the test drug.
Results are expressed as IC50 (mean, n 1/43), which is defined as
the drug concentration inhibiting the absorbance by 50% with
respect to that of untreated cells.
37 ꢀC for 45 min in 20
m
L of relaxation buffer (50 mM Tris pH 7.8,
50 mM KCl, 10 mM MgCl2, 1 mM DTT, 1 mM EDTA, 1 mM ATP) in the
presence of graded concentrations (from 0.5 to 20 M) of the tested
compounds. The reactions were stopped by the addition of SDS
(0.25%) and proteinase K (250 g/mL) followed by an incubation of
30 min at 50 ꢀC. Then 3
L of the electrophoresis dye mixture was
m
m
m
added to each DNA samples and loaded on a 1% agarose gel con-
taining (topoisomerase DNA cleavage gel) or not (inhibition of the
relaxation of DNA) ethidium bromide (1 mg/mL) at room tempera-
ture for 2 h at 120 V. Gels run without ethidium bromide were then
stained post-electrophoresis using an ethidium bromide-
containing bath. Both gels were washed prior to be photographed
under UV light.
5.2.2. UVevisible absorption spectrometry (Fig. 1)
CT-DNA were obtained from Sigma. After dissolution, CT-DNA
was deproteinized using sodium dodecyl sulfate and then dia-
lyzed against 1 mM sodium cacodylate (pH 7.0). The concentrations
of DNA were determined from their molar extinction coefficients of
6600 Mꢁ1 cmꢁ1. Tested compounds, as well as the reference
molecules camptothecin and etoposide (Sigma), were dissolved in
DMSO as 10 mM solutions. Further dilutions were made in the
appropriate aqueous binding or reaction buffers.
5.2.7. Topoisomerase II cleavage assay (Fig. 5b)
Supercoiled pUC19 plasmid DNA (350 ng) was incubated with 50
m
M of the various benzophenanthroline derivatives or etoposide, as
control, (20 or 50 M) prior to the addition of 10 units of human
topoisomerase II (TopoGen) at 37 ꢀC for 45 min in the appropriate
m
cleavage buffer. SDS (0.25%) and proteinase K (250 mg/mL) were then
UVevisible spectrum were measured using an Uvikon 943
added to stop the reaction during 30 min at 50 ꢀC. 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 pho-
tographed under UV light.
spectrophotometer. Samples containing 20
were prepared in 1 mL of BPE buffer (6 mM Na2HPO4, 2 mM
NaH2PO4, 1 mM EDTA, pH 7.1) in the presence or absence of 20
mM of the tested drugs
mM
of CT-DNA and transferred in a quartz cuvette of 10 mm pathlength.
The various spectra were recorded from 230 nm to 500 nm and are
referenced against a cuvette containing the same DNA concentra-
tion in the same buffer.
5.2.8. DNase I footprinting (Fig. 7)
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
5.2.3. Melting temperature studies (Table 2)
For melting temperature measurements, 20
mM of CT-DNA were
incorporation of a
- [32P]-dATP (3000 Ci/mmol, GE Healthcare,
incubated alone (control Tm) or with increasing concentrations of
the various benzophenanthroline derivatives 1 mL of BPE buffer
(base pair/drug ratio of 1). The absorbency at 260 nm was measured
in quartz cells every min over the range of 20e100 ꢀC with an
increment of 1 ꢀC per min using an Uvikon XL spectrophotometer
coupled with a peltier temperature controller. The Tm values were
deduced from the midpoint of the hyperchromic transition of
double strand to single strand DNA.
Buckinghamshire, England) at the EcoRI restriction site using AMV
reverse transcriptase (Ozyme, France). Appropriate concentrations
of the various benzophenanthrolines were incubated with the 265-
bp radio-labeled DNA fragment for 15 min at 37 ꢀC 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 lyophilization
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 ꢀC 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 65 W 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
autoradiograph was assigned to a particular bond within the DNA
fragment from comparison with its position relative to the
guanines sequencing standard (G-track) classically obtained using
DMS and piperidine treatment of the same DNA fragment.
5.2.4. Fluorescence spectroscopy (Fig. 2)
Fluorescence spectral measurement were recorded using 20
of the fluorescent drugs incubated in 1 mL of BPE buffer in the
presence or absence of 200 M of CT-DNA in a quartz cuvette of
mM
m
10 mm pathlength using a SPEX Fluorolog spectrofluorimeter
(Jobin Ivon, Longjumeau, France). The excitation wavelengths were
268 nm for compound 35, 275 nm for compounds 31, 32, 33 and 36,
and 285 nm for all other evaluated compounds. The emission
spectra were scanned at the wavelength specified in the abscissa of
the figures.
5.2.5. Circular dichroism (Fig. 3)
Acknowledgements
The CD spectra from samples containing 50
drugs incubated in 1 mL of sodium cacodylate (1 mM, pH 7.0)
without (control) or with 200 M (base pairs) of CT-DNA were
mM of the various
Elise Prost and Pascale Leproux (UMR CNRS 8638, Université
Paris Descartes) are gratefully acknowledged for their technical
assistance (NMR and Mass Spectrometry). Marie-Hélène David-
Cordonnier thanks the Ligue Nationale Contre le Cancer, Comité
m
collected in a quartz cell of 10 mm path length using a J-810 Jasco
spectropolarimeter at a fixed 20 ꢀC temperature controlled by

2558
T. S¸ erbetçi et al. / European Journal of Medicinal Chemistry 45 (2010) 2547e2558
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3943e3953.
du Nord and the Institut pour la Recherche sur le Cancer de Lille
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