Synthesis, cytotoxicity, DNA interaction, and topoisomerase II inhibition properties of novel indeno[2,1-c]quinolin-7-one and indeno[1,2-c]isoquinolin-5, 11-dione derivatives

Article abstract of DOI:10.1021/jm800017u

Indeno[2,1-c]quinolin-7-ones and 6H-indeno[1,2-c]isoquinolin-5,11-diones, bearing two cationic aminoalkyl side chains, were synthesized and evaluated for DNA interaction, topoisomerases inhibition, and cytotoxicity against human cancer cell lines. They displayed strong interaction with DNA and one indeno[1,2-c]isoquinolin-5,11-dione bearing side chains at N-6 and C-8 positions (6a) was a potent human topoisomerase II inhibitor with high cytotoxicity toward HL60 cells. An increased topoisomerase II inhibition is found with (a) a cationic aminoalkyl side chain at the C-8 rather than at the C-9 position, (b) a dimethylaminoethoxy side chain at the C-8 position introduced on the N-6 monosubstituted derivative, going with suppression of topoisomerase I poisoning, and (c) a dimethylaminoethyl rather than a dimethylaminopropyl side chain at the N-6 position. The cytotoxicity was only partially reduced when using the topoisomerase II-mutated mitoxantrone-resistant HL60/MX2 cell line, suggesting that additional targets are involved in their mechanism of action. These indeno[1,2-c]isoquinolin-5,11-dione derivatives represent new DNA-topoisomerase II interfering anticancer molecules.

Full text of DOI:10.1021/jm800017u

J. Med. Chem. 2008, 51, 3617–3629
3617
Synthesis, Cytotoxicity, DNA Interaction, and Topoisomerase II Inhibition Properties of Novel
Indeno[2,1-c]quinolin-7-one and Indeno[1,2-c]isoquinolin-5,11-dione Derivatives
Adina Ryckebusch,*,§,† Deborah Garcin,§ Amélie Lansiaux,‡ Jean-François Goossens, Brigitte Baldeyrou,‡ Raymond Houssin,§
Christian Bailly,‡,# and Jean-Pierre Hénichart§
UMR 8009 “Chimie Organique et Macromoléculaire”, Laboratoire de Chimie Organique Physique, Bâtiment C3(2), UniVersité des Sciences et
Technologies de Lille, 59655 VilleneuVe d’Ascq, France, Institut de Chimie Pharmaceutique Albert Lespagnol, EA 2692, UniVersité de Lille 2,
BP 83, 59006 Lille, France, INSERM U-837 et Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret (COL), IRCL, Place de
Verdun, 59045 Lille, France, and Laboratoire de Chimie Analytique, EA 4034, Faculté des Sciences Pharmaceutiques et Biologiques,
UniVersité de Lille 2, BP 83, 59006 Lille, France
ReceiVed January 8, 2008
Indeno[2,1-c]quinolin-7-ones and 6H-indeno[1,2-c]isoquinolin-5,11-diones, bearing two cationic aminoalkyl
side chains, were synthesized and evaluated for DNA interaction, topoisomerases inhibition, and cytotoxicity
against human cancer cell lines. They displayed strong interaction with DNA and one indeno[1,2-c]isoquinolin-
5,11-dione bearing side chains at N-6 and C-8 positions (6a) was a potent human topoisomerase II inhibitor
with high cytotoxicity toward HL60 cells. An increased topoisomerase II inhibition is found with (a) a
cationic aminoalkyl side chain at the C-8 rather than at the C-9 position, (b) a dimethylaminoethoxy side
chain at the C-8 position introduced on the N-6 monosubstituted derivative, going with suppression of
topoisomerase I poisoning, and (c) a dimethylaminoethyl rather than a dimethylaminopropyl side chain at
the N-6 position. The cytotoxicity was only partially reduced when using the topoisomerase II-mutated
mitoxantrone-resistant HL60/MX2 cell line, suggesting that additional targets are involved in their mechanism
of action. These indeno[1,2-c]isoquinolin-5,11-dione derivatives represent new DNA-topoisomerase II
interfering anticancer molecules.
Introduction
events finally inducing apoptosis or other types of cell death.8
DNA topoisomerases are targets for several clinically prescribed
anticancer agents: (i) topoisomerase I poisons irinotecan (Camp-
to) and topotecan (Hycamtin) derived from camptothecin9 and
(ii) topoisomerase II poisons including etoposide, doxorubicin,
and mitoxantrone. Despite their antitumor potency, the develop-
ment of resistance mechanisms and the toxicity or reversibility
of the covalent complex formation have limited the use of these
molecules. Novel molecules are being sought to overcome such
limitations and/or expand the anticancer spectrum of the above-
cited inhibitors.
A significant number of DNA-targeted anticancer agents
contain a planar intercalating polycycle bearing one aminoalkyl
side chain. This is particularly the case for two classes of
compounds: (i) indeno[2,1-c]quinolin-7-ones represented by 1a
(TAS-1031) and (ii) indeno[1,2-c]isoquinolin-5,11-diones rep-
resented by 2 (MJ-III-652) and 3 (NSC7273573) (Chart 1). These
compounds display marked cytotoxic properties and, for some
of them, potent antitumor activities in xenograft models.
Indenoquinolinone 1a has been advanced to phases I and II
clinical trials,4 and several indeno[1,2-c]isoquinolin-5,11-diones
are currently in preclinical development.5 Although the cytotoxic
mechanism of these drugs has not been clearly elucidated, they
probably act through the inhibition of DNA religation by
topoisomerases and are considered as topoisomerase “poisons”
with the stabilization of covalent enzyme-DNA complexes.
In the cell, DNA topoisomerases control the topological state
of DNA.6,7 There are two classes of topoisomerase: (i) type I
enzymes under- and overwind DNA by generating transient
single-stranded breaks in the DNA double helix and (ii) type II
enzymes resolve DNA knots and tangles by creating transient
double-stranded breaks. Topoisomerase inhibitors designated as
“poisons” interact with DNA and/or topoisomerases to form
stable ternary complexes, termed “cleavable complexes”, caus-
ing permanent DNA damage that triggers a series of cellular
Indenoquinolinone 1a was initially developed as a dual
topoisomerase I/topoisomerase II poison,10 but it was subse-
quently requalified as a topoisomerase II poison, with a very
minor activity against topoisomerase I. Compound 1a was
shown to intercalate into the DNA double helix, and the
structural constraints imposed by the intercalative binding block
the topoisomerase I-catalyzed DNA relaxation but not the
topoisomerase catalytic activity.11,12 At the cellular level,
apoptosis induced by compound 1a is firmly established. There
is a correlation between the G2 cell cycle arrest and changes in
mitochondrial membrane potential,13 hydrogen peroxide forma-
tion,14 and p-300-dependent activation of transcription factor
Sp1, enhanced by cellular acidosis.15
Indenoisoquinolin-5,11-diones form a class of potent non-
camptothecin topoisomerase I poisons endowed with high
cytotoxic properties on several tumor cell lines.16,17 Some
compounds have been described as both topoisomerase I and
II inhibitors and DNA intercalators: the carboxylic acid deriva-
tive 4 (MJ-238,18,19 Chart 1) and bisindenoisoquinolin-5,11-
dione 3.3 The representative indenoisoquinolin-5,11-dione 220
was shown to form more stable DNA-topoisomerase I cleavage
complexes than camptothecin and maintains a high cytotoxicity
on camptothecin-resistant cells, which makes it a good candidate
* To whom correspondence should be addressed. Phone: +33-3-2043-
4440. Fax: +33-3-2033-6309. E-mail: Adina.Ryckebusch@ensc-lille.fr.
§
Institut de Chimie Pharmaceutique Albert Lespagnol, Université de
Lille 2.
†
Current address: Laboratoire de Chimie Organique Physique, Université
des Sciences et Technologies de Lille.
‡
Inserm U-837, COL, IRCL.
Laboratoire de Chimie Analytique, Université de Lille 2.
Current address: Institut de Recherche Pierre Fabre, Toulouse, France.
#
10.1021/jm800017u CCC: $40.75
2008 American Chemical Society
Published on Web 05/29/2008

3618 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Ryckebusch et al.
Chart 1. Structures of Indenoquinolinone 1a, Indenoisoquinolin-5,11-diones 2 and 4, and Bisindenoisoquinolin-5,11-dione 3
Chart 2. Structures of Series I and Series II Compounds
(Indenoquinolin-7-ones 1a–c and 5a–c and
Chart 3. Reference Indenoisoquinolin-5,11-diones 8a,b
Indenoisoquinolin-5,11-diones 6a–g and 7a,b)
diates 1a–c for comparison and indenoquinolinone 1a as a
reference molecule.
In series II, a cationic side chain was maintained at the N-6
position, as this element has been recognized as important for
antiproliferative activity.18,20 Recently, studies performed with
simplified indenoisoquinolin-5,11-diones have shown that linkers
with lengths of between two and four atoms are optimal for
topoisomerase I inhibition and cytotoxicity,25 and therefore,
analogues bearing an ethyl or a propyl chain on the lactam
nitrogen were designed. A 2,3-dimethoxy sequence was intro-
duced on cycle A of some derivatives, as this was reported in
previous series20 to increase the biological activity. However,
more recent studies have shown that simplified isoquinolines
lacking the 2,3-dimethoxy sequence but bearing an aminoalkyl
group on the lactam nitrogen could also be potent cytotoxics.26
To make a comparison, two simplified indenoisoquinolin-5,11-
diones (Chart 3) were used: compound 8b bearing a dimethyl-
aminopropyl side chain on the lactam nitrogen, previously
described as a potent topoisomerase I inhibitor,26 and its shorter
side chain counterpart 8a.
for preclinical development.2,16 Molecular modeling calculations
supported by X-ray diffraction studies performed with the
carboxylic acid derivative 4 suggest not only that the inde-
noisoquinolin-5,11-dione skeleton binds to DNA through an
intercalation mode by π-π stacking with the DNA base pairs
flanking the topoisomerase I cleavage site but also that the C-11
ketone is implicated in a network of hydrogen bonds with
surrounding amino acid residues, notably Arg364.19,21–23
Because DNA affinity appears to be the key determinant
involved in the mechanism of indenoquinolin-7-ones and
indenoisoquinolin-5,11-diones,3 we decided to synthesize new
derivatives (Chart 2) incorporating a second cationic side chain
on the tetracyclic scaffolds.
Not only did we hypothesize that the additional side chain
would increase DNA binding through ionic interactions with
the negatively charged phosphate backbone, but we also
considered that an increased DNA affinity could reduce the
dissociation rates of topoisomerase-DNA cleavage complexes.
It was also expected that chemical modifications would increase
the ionization state of the compounds at physiological pH to
enhance their aqueous solubility. We assessed the contribution
of these structural modifications to their character as topoi-
somerase I and II poisons, DNA interaction, and cytotoxicity
to be able to elaborate structure–activity relationships in both
series of molecules.
Chemistry
The synthesis of 3-hydroxyindenoquinolin-7-ones 1a–c was
performed in six steps (Scheme 1) according to a described
procedure.24 The key intermediate 13 was obtained by
Claisen-Dieckmann cyclization of diethyl phthalate 9 followed
by the reaction of ester 10 with 3-methoxyaniline and cyclization
of amide 11 (polyphosphoric acid) into quinolin-6,7-dione 12.
Chlorination of compound 12 (POCl3) yielded 6-chloroquinoline
13. Compounds 1a–c were obtained after nucleophilic aromatic
substitution of the 6-chloro carbon with appropriate ω-ami-
noalkylamine (compounds 14a–c) and O-demethylation (hy-
drobromic acid). The 3-aminoalkoxy chain was then introduced
by nucleophilic substitution with appropriate ω-halogenoalkyl-
amine to give disubstituted quinolines 5a–c.
In series I, the published biological data24 on 3-hydroxylated
analogues of compound 1a indicate good cytotoxicity on P388
leukemia cells for derivatives bearing a two-carbon saturated
chain, either a dimethylamine or a pyrrolidine at the N-6 position
and a hydroxyl at the C-3 position. An amino side chain was
introduced at the 3-OH substituent, using the hydroxy interme-
Considering indenoisoquinolin-5,11-diones 6a–g (Scheme
2), the first aminoalkyl side chain was immediately introduced

Quinolin-7-one and Isoquinolin-5,11-dione DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3619
Scheme 1. Synthesis of Indenoquinolin-7-ones 1a–c and 5a–c (Series I)a
a Reagents and conditions: (i) Na, EtOAc, EtOH, 80 °C, 5 h; (ii) m-anisidine, toluene, reflux, 30 min; (iii) PPA, 120 °C, 4 h; (iv) POCl3, DMF, reflux,
4 h; (v) appropriate aminoalkylamine, pyridine, 100 °C, 18 h; (vi) 47% HBr, AcOH, 100 °C, 65 h; (vii) appropriate chloroalkylamine, Cs2CO3, NaI, DMF,
80 °C, 3 h.
Scheme 2. Synthesis of Indenoisoquinolin-5,11-diones 6a–g (Series II)a
a Reagents and conditions: (i) appropriate alkyl chloride, K2CO3, DMF, 80 °C, 5 h; (ii) R3NH2, MgSO4, CHCl3, room temp, 6 h; (iii) MeOH or THF,
room temp, 1 h; (iv) SOCl2, 80 °C, 12 h; (v) AlCl3, dry CH2Cl2, 0 °C, 3 h.
via an etheroxide linkage on 3- or 4-hydroxybenzaldehyde
15 or 16 (compounds 17–19), whereas the second aminoalkyl
substituent on the isoquinolone nitrogen resulted from the
subsequent formation of imines 20a–e using an appropriate
ω-aminoalkylamine R3NH2. The sequence of the target
compounds 6a–g was pursued using a two-step procedure18
from appropriate imines 20a–e; condensation with homoph-
thalic anhydride 21 or 2227 provided carboxylic acids 23a–g
and was followed by a reaction of the cis stereoisomer alone
with thionyl chloride to give the final indenoisoquinolin-5,11-
diones 6a–g, implying two-electron oxidation followed by
an intramolecular Friedel–Crafts cyclization (AlCl3).28 It was
to be noted that isoquinolones 23a–g were obtained as a
cis-trans diastereoisomeric mixture that could not be sepa-
rated by fractionated crystallization or silica gel chromatog-
raphy. The low yields observed for compounds 6a and 6b
can be attributable to the formation of their corresponding
regioisomers as byproducts (relative to substituents R1 and
R2) at the intramolecular cyclization step, whereas those
observed for dimethoxy derivatives 6f and 6g can be
explained by their demethylation in the conditions required
by the Friedel–Crafts cyclization.

3620 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Ryckebusch et al.
Scheme 3. Synthesis of 9-Hydroxyindenoisoquinolin-5,11-diones 7a,b (Series II)a
a Reagents and conditions: (i) benzoyl chloride, TEA, dry CH2Cl2, 0 °C, 3 h; (ii) R3NH2, MgSO4, CHCl3, room temp, 6 h; (iii) THF, room temp, 1 h; (iv)
SOCl2, 80 °C, 12 h; (v) AlCl3, CH2Cl2, 0 °C, 3 h; (vi) LiOH, CH2Cl2/MeOH 1:1, room temp,1 h.
Table 1. Interaction of Series I and Series II Compounds with DNA
Scheme 4. Synthesis of Indenoisoquinolin-5,11-diones 8a,b
(Series II)a
compd
∆Tm (°C)a
Kapp (106 M-1 b
)
Series I, Method 1c
1a
1b
1c
5a
5b
5c
10
4
7
24
24
27
1.54 ( 0.12
0.51 ( 0.04
0.88 ( 0.07
1.58 ( 0.14
0.46 ( 0.05
ndd
Series II, Method 2e
a Reagents and conditions: (i) (CH3)2N(CH2)2NH2 or (CH3)2N(CH2)3NH2,
CHCl3, room temp, 18 h.
6a
6b
6c
6d
6e
6f
6g
7a
7b
8a
8b
15
17
17
19
24
19
22
5
1.92 ( 0.21
9.61 ( 0.81
36.74 ( 2.17
24.53 ( 1.14
36.11 ( 1.95
37.21 ( 2.35
46.80 ( 2.86
2.57 ( 0.19
6.94 ( 0.07
0.86 ( 0.07
6.79 ( 0.52
drug–DNA complex
Synthesis of 9-hydroxyindenoisoquinolin-5,11-diones 7a and
7b was performed using a similar strategy (Scheme 3), starting
from 4-hydroxybenzaldehyde 16, whose aromatic hydroxyl was
protected as benzoyl ester, resistant to acidic conditions at the
cyclization step but removable in alkaline conditions.
Reference compounds 8a,b were synthesized from benz[d]in-
deno[1,2-b]pyran-5,11-dione 2829 (Scheme 4) according to the
procedure previously reported for 8b.26
8
5.9
10.8
a Variation in melting temperature ∆Tm (Tm
-
TmDNA alone). Drug/CT DNA ratio ) 0.25. b Apparent binding constant
measured by fluorescence. c Intrinsic fluorescence. [Ligand] ) 1 µM. d nd:
not determined. e Competition with ethidium bromide (EB). EB/DNA ratio
) 1.26.
Results
DNA Interaction. DNA Thermal Denaturation. 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
of the conventional fluorescence quenching assay based on DNA
competition between the intercalating drug ethidium bromide
and the tested molecules.
drug–DNA complex
helix. The variations of the Tm values (∆Tm ) Tm
– TmDNA alone) are presented in Table 1. In series I, ∆Tm values
for compounds 5a–c reach 24 °C (drug/DNA ratio ) 0.25)
whereas ∆Tm values range from 4 to 10 °C for compounds 1a–c.
Introduction of a second cationic side chain (compounds 5a–c)
was found to strongly stabilize DNA against heat denaturation
when compared with their respective hydroxylated single side
chain counterparts 1a–c.
In series I, the first step to validate the method was to relate
the Kapp value calculated from DNA titration and variation in
intrinsic fluorescence measurement for compound 1a to the
previous results.12 In the second step, Kapp was calculated for
the five derivatives 1a–c, 5a,b, using the same procedure. The
two side chain derivatives 5a,b displayed binding constants
similar to those of their hydroxyl counterparts 1a,b, whereas
they provided better stabilization of the DNA complex against
heat denaturation, as mentioned above. These results led to the
assumption that despite a comparable affinity for DNA,
compounds 1a,b would display a specific mode of binding that
would be less efficient at stabilizing the duplex structure of
DNA.
A similar behavior was noted in series II where compounds
6c and 6e, bearing two cationic side chains, were considerably
more efficient at stabilizing the duplex structure of DNA than
their 9-hydroxylated counterparts 7a and 7b. This could be
attributable to additional electrostatic interactions with DNA
phosphodiester groups.
In series II, the presence of an additional amino side chain at
C-8 (compounds 6a,b) or at C-9 (compounds 6c,e) increased
both DNA affinity constant and duplex stabilization when
compared to their one side chain counterparts 8a,b. This could
be attributable to additional stabilizing interactions, potentially
between DNA phosphate backbone and cationic side chains.
Moreover, moving the side chain from C-9 (compounds 6c,e)
to C-8 (compounds 6a,b) decreased activity for DNA by 18-
and 4-fold, respectively. Increasing the length of the N-6 side
Fluorescence Measurements. Binding affinities for com-
pounds in both series were quantified by means of fluorescence
using two different methods (Table 1). In series I, the intrinsic
fluorescence of the compounds was exploited to determine the
apparent binding constant (Kapp). In series II, since weak
fluorescence was observed upon DNA titration, use was made
a Abbreviations: CT, calf thymus; GC, guanine-cytosine; Tm, melting
temperature; TkLC, thick layer chromatography.

Quinolin-7-one and Isoquinolin-5,11-dione DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3621
Figure 1. Effect of compounds 1a,c, 6a, 8a,b on the relaxation of plasmid DNA by human topoisomerase I. Native supercoiled pLAZ (130 ng,
lane DNA) was incubated with 4 units of topoisomerase I in the absence (lane Topo I) or presence of tested compound at the indicated concentration
(1-20 µM). Camptothecin (CPT) was used at 20 µM. DNA samples were 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.
chain (compound 6e vs 6c, n ) 3 vs n ) 2) or introducing a
2,3-dimethoxy sequence (compound 6f vs 6c, n ) 2) had no
effect on DNA affinity. When both modifications were carried
out simultaneously by adding a methyl group on the N-6
aminoalkyl side chain and a 2,3-dimethoxy sequence (compound
6g), a slight increase in DNA affinity was noted compared with
its counterparts 6e,f, suggesting that both modifications are
required simultaneously to enhance binding to DNA. Finally,
replacing the dimethylamino group (6c) by a bulkier cyclic
amine such as pyrrolidine (6d) decreased DNA affinity.
ential binding sequence could be determined with any of the
molecules belonging to series I or II.
Topoisomerase Inhibition. A conventional DNA relaxation
assay was used to assess the effects of the compounds on the
catalytic activity of human topoisomerases I and II. In these
experiments (Figure 2), supercoiled plasmid DNA was treated
with either topoisomerase I or II in the presence of two
concentrations of the test drug (20 and 50 µM) and the DNA
relaxation products were then resolved by gel electrophoresis
on agarose gel. In either series, no molecule promoted DNA
cleavage by topoisomerase I (data not shown). In series I, this
observation concords with that previously reported.11,12
Mode of Binding to DNA. Different binding modes (inter-
calation and/or groove binding) can account for the melting
temperature evaluation. Therefore, to gain an understanding of
the drug binding mechanism, a DNA unwinding assay was used
(Figure 1) based on the relaxation of supercoiled plasmid DNA
in the presence of topoisomerase I for three selected compounds:
1a, 1c and 6a, with compounds 8a,b as references. Supercoiled
DNA was treated with topoisomerase I in the presence of
increasing concentrations of the tested drug. DNA relaxation
products were then identified by agarose gel electrophoresis.
As seen in Figure 1, the DNA relaxed by topoisomerase I alone
(lane Topo I) generated a family of DNA topoisomers with a
slow electrophoretic mobility. The drug was then added while
topoisomerase I was maintained in the reaction mixture. When
the drug concentration was increased, the closed circular duplex
DNA was progressively supercoiled, indicating that the drug
intercalates into DNA,30 the intercalation occurring at micro-
molar concentrations.
All compounds in series I and II totally inhibit the relaxation
of the DNA. In series I, the effects obtained for 1a were more
pronounced than for 1c (compare the 5 µM lanes) and were
consistent with the intercalative mode (unwinding of closed
circular duplex DNA). In series II, intercalation is also consistent
with previous results obtained from a crystallographic study of
the cleavage complex.22 It is worth mentioning that, as for
several intercalating agents, the compounds studied here give
no circular dichroism (CD) signals in the drug absorption band
in the presence of DNA (data not shown). Therefore, CD could
no longer be used to investigate further the mode of binding to
DNA.
In contrast, inhibition of topoisomerase II was clearly detected
with these compounds. In this case, the reference drug was
etoposide, which produced a marked level of DNA double-
stranded breaks. Among all the tested molecules (Figure 2A),
five compounds were found to stabilize the drug–DNA complex.
Their ranking was 6a > 1c > 1a > 1b > 7a (data not shown
for compounds displaying no detectable DNA cleavage). A net
increase in the band corresponding to linear DNA (double-
stranded breaks) was noted with 6a, which appeared to be more
potent than compound 1a. These derivatives clearly promote
the cleavage of DNA by topoisomerase II and can be considered
as topoisomerase II poisons. The most active topoisomerase II
poisons were then compared (Figure 2B) to simplified inde-
noisoquinolin-5,11-dione reference-compounds 8a,b. The results
showed that 8a is also capable of inducing DNA cleavage but
to a lesser extent than its C-8 substituted counterpart 6a. In series
I, none of the compounds bearing two cationic amino side chains
generated DNA cleavage in the presence of topoisomerase II,
showing that the introduction of an amino side chain into
compounds 1a–c also suppressed the topoisomerase II poison
character (data not shown). In series II, results obtained from
different experiments indicated that the lactam side chain length
is critical for topoisomerase II inhibition. Indeed, aminoethyl
derivatives 6a, 6f, 7a, 8a are more potent than their aminopropyl
counterparts 6b, 6g (data not shown because no DNA cleavage
was detected), 7b, and 8b. The position of the second side chain
on the tetracycle also seems to be a critical element for
topoisomerase II inhibition, as the C-8 substituted derivative
6a is a potent topoisomerase II poison whereas its C-9
substituted counterpart 6c displayed no detectable activity. It is
interesting to note that indenoisoquinolin-5,11-diones nonsub-
stituted on cycle D and bearing an aminoalkyl side chain on
the lactam nitrogen, such as 8b, have been described as
topoisomerase I poisons.26 This work reveals that the introduc-
tion of a dimethylaminoethoxy side chain at C-8 (compound
6b) or at C-9 (compound 6e) suppresses the topoisomerase I
Sequence Selectivity. The nature of the sequence preferen-
tially recognized by the compounds was determined by the
DNase I footprinting methodology, using a radiolabeled 117-
bp DNA restriction fragment as a substrate. In all cases, no clear
footprints could be detected, whatever the drug concentration
tested. Either the affinity of the drugs for DNA was not
sufficiently high, or more likely, they formed insufficiently stable
drug–DNA complexes to inhibit DNase I cleavage. No prefer-

3622 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Ryckebusch et al.
Figure 2. Effect of compounds 1a-c, 6a,b,f, 7a,b on the relaxation of plasmid DNA by human topoisomerase II: (A) selected compounds displaying
detectable DNA breaks; (B) comparison of most potent compounds with reference compounds 8a,b. Native supercoiled pLAZ (130 ng, lane DNA)
was incubated with 4 units of topoisomerase II in the absence (lane Topo II) or presence of tested compound at the indicated concentration (20 and
50 µM). Etoposide was used at 20 (left) and 50 µM (right). DNA samples were separated by electrophoresis on a 1% agarose gel containing 1
µg/mL ethidium bromide. Gels were photographed under UV light: Nck, nicked; Sc, supercoiled; Lin, linear; Rel, relaxed.
inhibitory character. This could probably be the result of
unfavorable steric interactions in the cleavage complex with the
topoisomerase I pocket.
These results suggest that in series II, topoisomerase I or
topoisomerase II preference could be influenced by the nature
and position of the cationic side chain. Similar conclusions have
been reported for one-side chain-bearing anthraquinone31 and
acridine topoisomerase inhibitors.32
icity and topoisomerase II inhibition. This could be the result
of several contributions such as differences in cellular uptake
between compounds bearing two positive charges compared to
their monocationic counterparts and/or involvement of targets
and mechanisms different from topoisomerase II-mediated DNA
cleavage.
Conclusion
In Vitro Antiproliferative Activity. The antiproliferative
activities of the compounds were tested (Table 2) using two
human leukemia cell lines, HL60 and HL60/MX2, respectively
sensitive and resistant to the antitumor drug mitoxantrone.
To gain insight into the involvement of topoisomerase II
inhibition in the cytotoxicity of the molecules, their antiprolif-
erative activity was assessed using the HL60/MX2 cell line
resistant to mitoxantrone, which displays altered catalytic activity
and reduced levels of topoisomerase II33 (the values for
mitoxantrone and etoposide are given as references). When
evaluated on the HL60 cell line, all compounds displayed
cytotoxicity in the micromolar and submicromolar ranges. In
In this study, a second cationic side chain was introduced on
known indenoquinolin-7-ones and indenoisoquinolin-5,11-di-
ones. The designed molecules generally show an increased DNA
interaction consistent with the formation of additional electro-
static contacts. However, in both series, DNA interaction was
not found to correlate either with topoisomerase poisoning or
with the antiproliferative activity. In series I, the introduction
of a second amino side chain on indenoquinolinone 1a and on its
counterparts (1b,c) suppressed the topoisomerase II poison
character, thereby probably indicating the loss of interaction
with the enzyme in the cleavable complex. In series II, the
introduction of a second amino side chain on cycle D, as well
as a hydroxyl group, suppressed topoisomerase I inhibition
compared to the reference compounds. Nevertheless, the
introduction of a cationic side chain at C-8 position enhanced
topoisomerase II inhibition potency over C-8 nonsubstituted or
C-8 hydroxylated derivatives, presumably by promoting favor-
able interactions within the enzyme active site. The length of
the side chain on the lactam nitrogen also influenced topoi-
somerase inhibition, as an ethyl group provided better inhibition
than a propyl group. The best topoisomerase II poison was
compound 6a, displaying better potency than the reference
topoisomerase poison 1a. This compound also displayed a potent
cytotoxicity toward HL60 leukemia cells, comparable to that
of etoposide. However, the lack of correlation between cyto-
toxicity and topoisomerase II inhibition for these compounds,
together with the low relative resistance index they display
series I, compounds 1a and 1b were highly cytotoxic (IC50
)
33 and 88 nM, respectively). Their respective high resistance
index (82 and 59) suggests a significant contribution of
topoisomerase II inhibition to the cytotoxicity. Their counterparts
5a and 5b, bearing two side chains, displayed a significant loss
of cytotoxic activity that could be attributable to their lack of
topoisomerase II inhibition. In series II, the most cytotoxic
compounds were 6a,b and 7a,b, which displayed submicromolar
IC50, comparable to that of the compound 8b. When evaluated
on the HL60/MX2 cell line, the strongest topoisomerase II
poison 6a was found to behave as a weaker topoisomerase II
poison, such as 6b and 8b. The weak resistance index obtained
for compound 6a may be explained by its binding to the enzyme
at a site different from that of mitoxantrone or by specific
kinetics. No obvious relationship was found between cytotox-

Quinolin-7-one and Isoquinolin-5,11-dione DeriVatiVes
Table 2. Cytotoxicities of Series I and Series II Compounds
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3623
IC50 (µM)a
compd
HL60
HL60/MX2
RRIb
MXc
0.063 ( 0.020
0.486 ( 0.190
1.51 ( 0.08
48.5 ( 1.5
24
100
Etopd
Series I
IC50 (µM)a
compd
C-3
n
R
HL60
HL60/MX2
RRIb
1a
1b
1c
5a
5b
5c
OH
OH
OH
O(CH2)nR
O(CH2)nR
O(CH2)nR
2
2
3
2
2
3
N(CH3)2
N(CH2)4
N(CH3)2
N(CH3)2
N(CH2)4
N(CH3)2
0.033 ( 0.010
0.088 ( 0.010
2.83 ( 0.22
4.64 ( 0.47
3.01 ( 1.28
5.44 ( 0.05
2.70 ( 0.14
5.18 ( 0.11
28.5 ( 1.8
4.95 ( 0.66
3.81 ( 2.14
9.14 ( 0.58
82
59
10
1.1
1.3
1.7
Series II
IC50 (µM)a
compd
C-8
C-9
n
R
C-2,3
HL60
HL60/MX2
RRIb
6a
6b
6c
6d
6e
6f
6g
7a
7b
8a
8b
O(CH2)2R
O(CH2)2R
2
3
2
2
3
2
3
2
3
2
3
N(CH3)2
N(CH3)2
N(CH3)2
N(CH2)4
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
0.64 ( 0.03
0.87 ( 0.06
1.55 ( 0.19
1.81 ( 0.12
4.62 ( 0.44
4.88 ( 0.21
7.46 ( 1.99
0.62 ( 0.14
0.52 ( 0.01
1.05 ( 0.01
0.63 ( 0.07
5.26 ( 0.23
6.61 ( 0.68
2.55 ( 0.81
5.25 ( 0.58
4.41 ( 0.61
5.26 ( 0.19
10.80 ( 7.55
nde
8.3
7.6
1.7
2.9
1.0
1.1
1.4
nde
5.8
7.0
7.8
O(CH2)2R
O(CH2)2R
O(CH2)2R
O(CH2)2R
O(CH2)2R
OH
OCH3
OCH3
OH
2.97 ( 0.27
7.35 ( 0.49
4.92 ( 0.61
(MX-sensitive)
a IC50 values are the concentrations corresponding to 50% growth inhibition. b Relative resistance index: IC50(MX-resistant)/IC50
.
c MX: mitoxantrone.
d Etop: etoposide. e nd: not determined.
Electron Surveyor MSQ spectrometer. High performance liquid
chromatography (ODS column, elution gradient, H2O/CH3CN/
HCOOH), with a UV detector and with an APCI+ (atmospheric
pression chemical ionization) mass detector, was also used to verify
the purity of final compounds. Elemental analyses were performed
by the “Service Central d’Analyses” at the CNRS, Vernaison
(France). Commercially available reagents and solvents were used
throughout without further purification. Dichloromethane was dried
on calcium chloride.
against mitoxantrone-resistant cells, suggests that additional
mechanisms and/or targets could be involved.
Experimental Section
Chemistry. Melting points were determined with a Büchi 535
capillary melting point apparatus and remain uncorrected. Analytical
thin-layer chromatography (TLC) was performed on precoated
Kieselgel 60F254 plates (Merck). The spots were located by UV
(254 and 366 nm), and Rf values are given for guidance. Silica gel
60 230–400 mesh purchased from Merck was used for column
chromatography. Thick-layer chromatography (TkLC) was per-
formed using silica gel from Merck, from which the compounds
were extracted by the solvent system: acetone/aqueous NH4OH 8:2.
The structure of all compounds was supported by IR (FT-Bruker
Vector 22 instrument), by 1H NMR at 300 MHz on a Bruker DRX-
300 spectrometer, or by 13C NMR at 75 MHz on a Bruker AC-
300 spectrometer. Chemical shifts were reported in ppm using
tetramethylsilane as a standard. J values are in hertz, and the
splitting patterns were designated as follows: s, singlet; d, doublet;
dd, double doublet; td, triple doublet; t, triplet; dt, double triplet;
qt, quadruple triplet; q, quadruplet; qt, quintuplet; m, multiplet; b,
broad. Analytical HPLC for assessing the purity of final compounds
was performed on a Kontron 325 system equipped with a C18
reversed-phase column (Kromasil C18) and a UV diode array
detector (DAD 440L). Compounds were detected between 200 and
400 nm. Analysis was performed isocratically (eluent H2O/MeOH/
TFA 58:42:0.1). HPLC retention times (tR) were obtained at a flow
rate of 1 mL/min. LC-MS spectra were performed on a Thermo
General Procedure for the Synthesis of Compounds 14a–c.
The appropriate aminoalkylamine was added to a suspension of
compound 1324 (3.0 g, 10.1 mmol, 1 equiv) in pyridine (30 mL),
and the mixture was heated while stirring at 100 °C for 18 h. The
solvent was removed under reduced pressure, and dichloromethane
(50 mL) and water (30 mL) were added to the residue for extraction.
The organic layer was washed with 1 N sodium bicarbonate (3 ×
20 mL), dried over MgSO4, and concentrated. The crude product
was purified by column chromatography (CH2Cl2/MeOH 9:1).
3-Methoxy-6-(3-dimethylaminoethylamino)indeno[2,1-c]quin-
olin-7-one (14a). Compound 14a was obtained as a red oil (1.8 g,
51% yield) from N,N-dimethylethane-1,2-diamine (5.6 mL, 51
mmol, 5 equiv). Rf ) 0.20 (CH2Cl2/MeOH 9:1); mp 166–167 °C;
IR (neat) 3373, 2930, 2770, 1687, 1618, 1587, 1532, 1460, 1419
1
cm-1; H NMR (CDCl3) δ 8.13 (d, J ) 9.3 Hz, 1H, Ar), 7.98 (d,
J ) 7.6 Hz, 1H, Ar), 7.66 (d, J ) 7.1 Hz, 1H, Ar), 7.53 (dd, J )
7.6 Hz, 1H, Ar), 7.42 (dd, J ) 7.1 Hz, 1H, Ar), 7.35–7.32 (m, 1H,
NH), 7.07 (d, J ) 2.7 Hz, 1H, Ar), 6.93 (dd, J ) 9.3 Hz, J ) 2.7

3624 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Ryckebusch et al.
Hz, 1H, Ar), 3.96 (s, 3H, CH3), 3.80 (dt, J ) 6.0 Hz, 2H, CH2),
2.70 (t, J ) 6.0 Hz, 2H, CH2), 2.40 (s, 6H, CH3).
concentrated. The crude product was purified by column chroma-
tography (CH
2Cl2/MeOH/25% aqueous NH4OH 8:2:0.2).
3-Methoxy-6-(2-pyrrolidin-1-ylethylamino)indeno[2,1-c]quin-
olin-7-one (14b). Compound 14b was obtained as a red oil (1.7 g,
45% yield) from 2-pyrrolidin-1-ylethylamine (6.5 mL, 51 mmol, 5
equiv). Rf ) 0.25 (CH2Cl2/MeOH 9:1); IR (neat) 3372, 2930, 2778,
3-(2-Dimethylaminoethoxy)-6-(2-dimethylaminoethylamino)in-
deno[2,1-c]quinolin-7-one (5a). Compound 5a was obtained from
compound 1a (0.47 g, 1.4 mmol) and 2-chloroethyldimethylamine
hydrochloride (0.30 g, 2.1 mmol). After crystallization from ethyl
acetate, compound 5a was obtained as an orange solid (0.22 g,
38% yield). Rf ) 0.60 (CH2Cl2/MeOH/25% aqueous NH4OH 8:2:
0.2); mp 123–124 °C; IR (neat) 2772, 1692, 1618, 1601, 1585,
1532, 1461, 1423 cm-1; 1H NMR (CDCl3) δ 8.13 (d, J ) 9.1 Hz,
1H, Ar), 7.99 (d, J ) 7.3 Hz, 1H, Ar), 7.66 (d, J ) 7.3 Hz, 1H,
Ar), 7.53 (dd, J ) 7.4 Hz, 1H, Ar), 7.42 (dd, J ) 7.4 Hz, 1H, Ar),
7.34 (bs, 1H, NH), 7.07 (d, J ) 2.4 Hz, 1H, Ar), 6.99 (dd, J ) 9.1
Hz, J ) 2.4 Hz, 1H, Ar), 4.23 (t, J ) 5.5 Hz, 2H, CH2), 3.77 (dt,
J ) 6.2 Hz, 2H, CH2), 2.82 (dd, J ) 5.5 Hz, 2H, CH2), 2.66 (dd,
J ) 6.2 Hz, 2H, CH2), 2.38 (s, 6H, CH3), 2.37 (s, 6H, CH3); HPLC;
LC-MS.
1
1687, 1618, 1588, 1531, 1463, 1419 cm-1; H NMR (CDCl3) δ
8.13 (d, J ) 9.2 Hz, 1H, Ar), 7.98 (d, J ) 7.6 Hz, 1H, Ar), 7.67
(d, J ) 7.4 Hz, 1H, Ar), 7.53 (dd, J ) 7.6 Hz, 1H, Ar), 7.42 (dd,
J ) 7.4 Hz, 1H, Ar), 7.31 (m, 1H, NH), 7.07 (d, J ) 2.1 Hz, 1H,
Ar), 6.94 (dd, J ) 9.2 Hz, J ) 2.1 Hz, 1H, Ar), 3.96 (s, 3H),
3.91–3.85 (m, 2H, CH2), 2.93 (m, 2H, CH2), 2.76 (m, 4H, CH2),
1.88 (m, 4H, CH2).
3-Methoxy-6-(3-dimethylaminopropylamino)indeno[2,1-c]quin-
olin-7-one (14c). Compound 14c was obtained as a red oil (1.6 g,
43% yield) from N,N-dimethylpropane-1,3-diamine (6.4 mL, 51
mmol, 5 equiv). Rf ) 0.25 (CH2Cl2/MeOH 9:1); IR (neat) 3360,
1
2933, 1686, 1619, 1602, 1585, 1536, 1459, 1437, 1427 cm-1; H
3-(2-Pyrrolidin-1-ylethoxy)-6-(2-pyrrolidin-1-ylethylamino)in-
deno[2,1-c]quinolin-7-one (5b). Compound 5b was obtained from
compound 1b (0.50 g, 1.4 mmol) and 1-(2-chloroethyl)pyrrolidine
hydrochloride (0.36 g, 2.1 mmol). After crystallization from ethyl
acetate, compound 5b was obtained as an orange solid (0.21 g,
33% yield). Rf ) 0.60 (CH2Cl2/MeOH/25% aqueous NH4OH 8:2:
0.2); mp 116–117 °C; IR (neat) 2925, 2854, 2763, 1691, 1616,
1599, 1583, 1530, 1458, 1428 cm-1; 1H NMR (CDCl3) δ 7.96 (d,
J ) 9.1 Hz, 1H, Ar), 7.83 (d, J ) 7.4 Hz, 1H, Ar), 7.56 (d, J ) 7.3
Hz, 1H, Ar), 7.44–7.39 (m, 1H, Ar), 7.34–7.30 (m, 1H, Ar),
7.23–7.22 (m, 1H, NH), 7.07 (d, J ) 2.3 Hz, 1H, Ar), 6.98 (dd, J
) 9.0 Hz, J ) 2.3 Hz, 1H, Ar), 4.28 (t, J ) 5.8 Hz, 2H, CH2),
3.86–3.81 (m, 2H, CH2), 3.01 (t, J ) 5.6 Hz, 2H, CH2), 2.91–2.88
(m, 2H, CH2), 2.71–2.66 (m, 8H, CH2), 1.88–1.85 (m, 8H, CH2);
HPLC; LC-MS.
NMR (CDCl3) δ 8.10 (d, J ) 9.4 Hz, 1H, Ar), 7.95 (d, J ) 7.6
Hz, 1H, Ar), 7.65 (d, J ) 7.1 Hz, 1H, Ar), 7.50 (dd, J ) 7.6 Hz,
1H, Ar), 7.41 (dd, J ) 7.1 Hz, 1H, Ar), 7.26–7.24 (m, 1H, NH),
7.06 (d, J ) 2.5 Hz, 1H, Ar), 6.86 (dd, J ) 9.4 Hz, J ) 2.5 Hz,
1H, Ar), 3.96 (s, 3H, CH3), 3.76–3.70 (m, 2H, CH2), 2.55–2.48
(m, 2H, CH2), 1.93–1.89 (m, 2H, CH2).
General Procedure for the Synthesis of Compounds 1a–c.
Aqueous hydrobromic acid (47%, 40 mL) was added to a solution
of the appropriate compound 14a–c (8.7 mmol) in acetic acid (40
mL), and the mixture was refluxed for 65 h. The reaction mixture
was brought to dryness, water (30 mL) was added to the residue,
and the solution was adjusted to pH 8 with aqueous ammonia and
subsequently extracted with dichloromethane (6 × 50 mL). The
organic layer was dried over MgSO4 and concentrated. The crude
product was purified by column chromatography (CH2Cl2/MeOH/
25% aqueous NH4OH 8:2:0.2) and recrystallized from ethanol.
3-Hydroxy-6-(2-dimethylaminoethylamino)indeno[2,1-c]quin-
olin-7-one (1a). Compound 1a was obtained as a red solid (2.0 g,
69% yield) from compound 14a (3.0 g, 8.7 mmol). Rf ) 0.35
(CH2Cl2/MeOH/25% aqueous NH4OH 8:2:0.2); mp 216–217 °C;
3-(3-Dimethylaminopropoxy)-6-(3-dimethylaminopropylami-
no)indeno[2,1-c]quinolin-7-one (5c). Compound 5c was obtained
from compound 1c (0.49 g, 1.4 mmol) and 3-chloropropyldim-
ethylamine hydrochloride (0.33 g, 2.1 mmol). After crystallization
from ethyl acetate, compound 5c was obtained as an orange solid
(0.21 g, 35% yield). Rf ) 0.65 (CH2Cl2/MeOH/25% aqueous
NH4OH 8:2:0.2); mp 93–94 °C; IR (neat) 2924, 2855, 2764, 1691,
IR (neat) 3378, 1679, 1613, 1582, 1543, 1458, 1428, 1403 cm-1
;
1H NMR (CDCl3) δ 7.62 (d, J ) 9.3 Hz, 1H, Ar), 7.52–7.49 (m,
1H, NH), 7.45–7.32 (m, 3H, Ar), 7.18 (dd, J ) 7.3 Hz, 1H, Ar),
7.09 (dd, J ) 7.3 Hz, 1H, Ar), 6.74–6.71 (m, 1H, Ar), 3.62–3.59
(m, 2H, CH2), 2.69–2.66 (m, 2H, CH2), 2.38 (s, 6H, CH3); HPLC;
LC-MS.
1
1616, 1599, 1583, 1530, 1458, 1425 cm-1; H NMR (CDCl3) δ
8.10 (d, J ) 9.4 Hz, 1H, Ar), 7.96 (d, J ) 7.3 Hz, 1H, Ar), 7.65
(d, J ) 6.3 Hz, 1H, Ar), 7.51 (t, J ) 7.7 Hz, 1H, Ar), 7.41 (t, J )
7.1 Hz, 1H, Ar), 7.26–7.24 (m, 1H, NH), 7.06 (d, J ) 2.4 Hz, 1H,
Ar), 6.91 (dd, J ) 9.1 Hz, J ) 2.5 Hz, 1H, Ar), 4.17 (t, J ) 6.3
Hz, 2H, CH2), 3.72 (q, J ) 6.6 Hz, 2H, CH2), 2.49 (q, J ) 7.6 Hz,
4H, CH2), 2.37 (s, 6H, CH3), 2.36 (s, 6H, CH3), 2.09–2.06 (m, 2H,
CH2), 1.93–1.90 (m, 2H, CH2); HPLC; LC-MS.
3-Hydroxy-6-(2-pyrrolidin-1-ylethylamino)indeno[2,1-c]quin-
olin-7-one (1b). Compound 1b was obtained as an orange solid
(1.9 g, 60% yield) from compound 14b (3.2 g, 8.7 mmol)). Rf )
0.35 (CH2Cl2/MeOH/25% aqueous NH4OH 8:2:0.2); mp 126 °C
(dec); IR (neat) 3374, 2822, 1676, 1611, 1587, 1502, 1456, 1414
1
cm-1; H NMR (CDCl3) δ 7.60 (d, J ) 9.5 Hz, 1H, Ar), 7.50 (d,
General Procedure for the Synthesis of Compounds 17–19.
A suspension of appropriate hydroxybenzaldehyde 15, 16 (70 mmol,
2 equiv), and potassium carbonate (14.4 g, 101 mmol, 3 equiv) in
DMF (20 mL) was stirred at 80 °C for 30 min. The appropriate
alkyl chloride (35 mmol, 1 equiv) was added, and the reaction
mixture was heated and maintained at 80 °C for 5 h. After cooling,
the reaction mixture was diluted in water (200 mL) and extracted
with ethyl acetate (2 × 250 mL). The organic layer was separated,
washed with brine (3 × 100 mL), and dried over MgSO4 before
the solvent was evaporated to give the desired product.
J ) 6.7 Hz, 1H, Ar), 7.45 (d, J ) 6.6 Hz, 1H, Ar), 7.29–7.19 (m,
3H, Ar), 7.05 (bs, 1H, NH), 6.71 (dd, J ) 8.8 Hz, J ) 2.2 Hz, 1H,
Ar), 3.73–3.69 (m, 2H, CH2), 3.02–2.99 (m, 2H, CH2), 2.91–2.88
(m, 4H, CH2), 1.96–1.93 (m, 4H, CH2); HPLC; LC-MS.
3-Hydroxy-6-(3-dimethylaminopropylamino)indeno[2,1-c]quin-
olin-7-one (1c). Compound 1c was obtained as a red solid (2.0 g,
65% yield) from compound 14c (3.1 g, 8.7 mmol). Rf ) 0.40
(CH2Cl2/MeOH/25% aqueous NH4OH 8:2:0.2); mp 110 °C (dec);
IR (neat) 3360, 1682, 1615, 1578, 1528, 1457, 1417 cm-1; 1H NMR
(DMSO-d6) δ 8.33–8.25 (m, 2H, Ar), 7.66–7.61 (m, 2H, Ar),
7.54–7.51 (m, 1H, Ar), 7.39–7.36 (m, 1H, NH), 6.93–6.87 (m, 2H,
Ar), 3.60 (dt, J ) 6.5 Hz, 2H, CH2), 2.42–2.39 (m, 2H, CH2), 2.25
(s, 6H, CH3), 1.80–1.77 (m, 2H, CH2); HPLC; LC-MS.
General Procedure for the Synthesis of Compounds 5a–c. The
appropriate chloroalkylamine (2.1 mmol, 1.5 equiv), cesium
carbonate (0.46 g, 1.4 mmol, 1 equiv), and sodium iodide (0.31 g,
2.1 mmol, 1.5 equiv) were added to the appropriate compounds
1a–c, (1.4 mmol, 1 equiv) in DMF (15 mL). The reaction mixture
was heated to 80 °C for 3 h. After cooling, the reaction mixture
was diluted with dichloromethane (50 mL) and washed with brine
(3 × 20 mL). The organic layer was dried over MgSO4 and
3-(2-Dimethylaminoethoxy)benzaldehyde (17). Compound 17
was obtained as a brown oil (3.11 g, 46% yield) from aldehyde 15
(8.55 g, 70 mmol) and (2-chloroethyl)dimethylamine (3.76 g, 35
mmol). Rf ) 0.55 (CH2Cl2/MeOH 8:2); IR (neat) 2945, 2822, 2774,
1
1697, 1596, 1486, 1455; H NMR (CDCl3) δ 9.89 (s, 1H, CHO),
7.37–7.33 (m, 3H, Ar), 7.14–7.11 (m, 1H, Ar), 4.04 (t, J ) 5.2
Hz, 2H, CH2), 2.65 (t, J ) 5.2 Hz, 2H, CH2), 2.27 (s, 6H, CH3).
4-(2-Dimethylaminoethoxy)benzaldehyde (18). Compound 18
was obtained as a brown oil (2.96 g, 44% yield) from aldehyde 16
(8.55 g, 70 mmol) and (2-chloroethyl)dimethylamine (3.76 g, 35
mmol). Rf ) 0.50 (CH2Cl2/MeOH 8:2); IR (neat) 2900, 2815, 2725,
1
1677, 1603, 1578, 1509; H NMR (CDCl3) δ 9.89 (s, 1H, CHO),

Quinolin-7-one and Isoquinolin-5,11-dione DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3625
7.83 (d, J ) 8.7 Hz, 2H, Ar), 7.03 (d, J ) 8.7 Hz, 2H, Ar), 4.16
(t, J ) 5.7 Hz, 2H, CH2), 2.78 (t, J ) 5.7 Hz, 2H, CH2), 2.36 (s,
6H, CH3).
cm-1; 1H NMR (CDCl3) δ 8.17 (s, 1H, CH), 7.60 (dd, J ) 8.8 Hz,
J ) 2.9 Hz, 2H, Ar), 6.90 (dd, J ) 8.8 Hz, J ) 2.9 Hz, 2H, Ar),
4.07–4.02 (m, 2H, CH2), 3.58–3.53 (m, 2H, CH2), 2.71–2.66 (m,
2H, CH2), 2.29–2.28 (m, 8H, CH3, CH2), 2.18 (s, 6H, CH3).
4-[(2-Dimethylaminoethylimino)methyl]phenyl Benzoate
(25a). Compound 25a was obtained as a light-yellow oil (3.58 g,
93% yield) from aldehyde 24 (2.94 g) and N,N-dimethylethane-
1,2-diamine (1.43 mL). IR (neat) 3341, 1643, 1601, 1580, 1290
4-(2-Pyrrolidin-1-ylethoxy)benzaldehyde (19). Compound 19
was obtained as a brown oil (5.37 g, 70% yield) from aldehyde 16
(8.55 g, 70 mmol) and 1-(2-chloroethyl)pyrrolidine (4.68 g, 35
mmol). Rf ) 0.50 (CH2Cl2/MeOH 8:2); IR (neat) 2920, 2823, 2750,
1
1678, 1601, 1578, 1510; H NMR (CDCl3) δ 9.84 (s, 1H, CHO),
1
cm-1; H NMR (CDCl3) δ 8.32 (s, 1H, CH), 8.22–8.19 (m, 2H,
7.79 (d, J ) 8.7 Hz, 2H, Ar), 6.99 (d, J ) 8.7 Hz, 2H, Ar), 4.11
(t, J ) 5.7 Hz, 2H, CH2), 2.73 (t, J ) 5.7 Hz, 2H, CH2), 2.33–2.29
(m, 8H, CH2).
Ar), 7.80 (d, J ) 8.3 Hz, 2H, Ar), 7.66–7.63 (m, 1H, Ar), 7.54–7.49
(m, 2H, Ar), 7.27 (d, J ) 8.6 Hz, 2H, Ar), 3.76 (t, J ) 6.8 Hz, 2H,
CH2), 2.67 (t, J ) 6.8 Hz, 2H, CH2), 2.32 (s, 6H, CH3).
4-Formylphenyl Benzoate (24). A solution aldehyde 16 (4.0 g,
32.7 mmol, 1 equiv) and triethylamine (4.6 mL, 32.7 mmol, 1 equiv)
in dry CH2Cl2 (40 mL) was cooled to 0 °C before benzoyl chloride
(4.62 g, 32.7 mmol) was added dropwise, and the reaction mixture
was stirred for 3 h at 0 °C. The reaction mixture was diluted with
CH2Cl2 and washed with 1 M NaHCO3 (2 × 25 mL). Ester 24 was
obtained as a white powder after crystallization from ethanol (4.07
g, 55% yield). Rf ) 0.55 (cyclohexane/AcOEt 8:2); mp 91–92 °C;
IR (neat) 1738, 1699, 1597; 1H NMR (CDCl3) δ 10.04 (s, 1H,
CHO), 8.22 (d, J ) 7.4 Hz, 2H, Ar), 8.00 (d, J ) 8.5 Hz, 2H, Ar),
7.69–7.66 (m, 1H, Ar), 7.57–7.53 (m, 2H, Ar), 7.43 (d, J ) 8.5
Hz, 2H, Ar).
General Procedure for the Synthesis of Imines 20a–e and
25a,b. The appropriate amine R3NH2 (13 mmol, 1 equiv) and
MgSO4 (5.0 g) were added to a solution of aldehyde 17–19 (13
mmol, 1 equiv) in CHCl3 (25 mL), and the reaction mixture was
stirred for 6 h. The reaction mixture was filtered and the filtrate
was washed with a solution of 1 M NaHCO3 (2 × 25 mL), dried
over MgSO4, and concentrated under reduced pressure to give the
desired product.
4-[(3-Dimethylaminopropylimino)methyl]phenyl Benzoate
(25b). Compound 25b was obtained as a light-yellow oil (3.83 g,
95% yield) from aldehyde 24 (2.94 g) and N,N-dimethylpropane-
1,3-diamine (1.64 mL). IR (neat) 3340, 1641, 1601, 1578, 1291
1
cm-1; H NMR (CDCl3) δ 8.31 (s, 1H, CH), 8.23–8.19 (m, 2H,
Ar), 7.79 (d, J ) 8.1 Hz, 2H, Ar), 7.65–7.62 (m, 1H, Ar), 7.53–7.49
(m, 2H, Ar), 7.26 (d, J ) 8.5 Hz, 2H, Ar), 3.60–3.54 (m, 2H, CH2),
2.68–2.64 (m, 2H, CH2), 2.42 (s, 6H, CH3), 1.90–1.86 (m, 2H, CH2).
General Procedure for the Synthesis of Carboxylic Acids
23a–g and 26a,b. Appropriate homophtalic anhydride 21 or 22
(11.4 mmol, 1 equiv) was added to a solution of imine 20a–e (11.4
mmol, 1 equiv) in MeOH for compounds 23a–e (60 mL) or in THF
for compounds 23f,g and 26a,b (60 mL), and the mixture was stirred
at room temperature for 1 h. The reaction mixture was concentrated
under reduced pressure to give a yellow solid containing a mixture
of cis and trans diastereoisomers of acids 23a–g and 26a,b. The
cis/trans ratio was calculated by 1H NMR based on the integration
of characteristic peaks of each isomer each time it was possible.
The crude mixture of diastereoisomers was further reacted without
purification.
N-[3-(2-Dimethylaminoethoxy)benzylidene]-N′,N′-dimethyl-
ethane-1,2-diamine (20a). Compound 20a was obtained as a brown
oil (3.35 g, 98% yield) from aldehyde 17 (2.85 g) and N,N-
dimethylethane-1,2-diamine (1.43 mL). IR (neat) 1596, 1266, 1039
3-[3-(2-Dimethylaminoethoxy)phenyl]-2-(2-dimethylaminoet-
hyl)-1-oxo-1,2,3,4-tetrahydroisoquinolin-4-carboxylic Acid (23a).
Carboxylic acid 23a was obtained from anhydride 21 (1.84 g, 11.4
mmol) and imine 20a (3.0 g, 11.4 mmol). Characteristic signals:
1H NMR (CDCl3) δ 5.35 (s, Htrans), 5.22 (d, J ) 6.4 Hz, Hcis), 4.63
(d, J ) 6.4 Hz, Hcis), 3.87 (s, Htrans), cis/trans ratio ) 1:2.
3-[3-(2-Dimethylaminoethoxy)phenyl]-2-(3-dimethylamino-
propyl)-1-oxo-1,2,3,4-tetrahydroisoquinolin-4-carboxylic Acid
(23b). Carboxylic acid 23b was obtained from carboxylic anhydride
21 (1.84 g, 11.4 mmol) and imine 20b (3.16 g, 11.4 mmol).
Characteristic signals: 1H NMR (CDCl3) δ 5.43 (s, Htrans), 5.29 (d,
J ) 6.3 Hz, Hcis), 4.70 (d, J ) 6.3 Hz, Hcis), 3.76 (s, Htrans), cis/
trans ratio ) 1:2.
1
cm-1; H NMR (CDCl3) δ 8.21 (s, 1H, CH), 7.31–7.16 (m, 3H,
Ar), 6.93 (dd, J ) 6.6 Hz, J ) 1.3 Hz, 1H, Ar), 4.05 (t, J ) 7.0
Hz, 2H, CH2), 3.87 (t, J ) 5.6 Hz, 2H, CH2), 2.68 (t, J ) 5.6 Hz,
2H, CH2), 2.59 (t, J ) 7.0 Hz, 2H, CH2), 2.28 (s, 6H, CH3), 2.26
(s, 6H, CH3).
N-[3-(2-Dimethylaminoethoxy)benzylidene]-N′,N′-dimethyl-
propane-1,3-diamine (20b). Compound 20b was obtained as a
brown oil (2.63 g, 73% yield) from aldehyde 17 (2.85 g) and N,N-
dimethylpropane-1,3-diamine (1.64 mL). IR (neat) 1654, 1273, 1042
1
cm-1; H NMR (CDCl3) δ 8.21 (s, 1H, CH), 7.34–7.17 (m, 3H,
Ar), 7.00–6.94 (m, 1H, Ar), 4.08 (t, J ) 5.6 Hz, 2H, CH2), 3.61 (t,
J ) 6.9 Hz, 2H, CH2), 2.70 (t, J ) 5.6 Hz, 2H, CH2), 2.33–2.29
(m, 8H, CH3, CH2), 2.21 (s, 6H, CH3), 1.84 (qt, J ) 7.2 Hz, 2H,
CH2).
N-[4-(2-Dimethylaminoethoxy)benzylidene]-N′,N′-dimethyl-
ethane-1,2-diamine (20c). Compound 20c was obtained as a brown
oil (3.01 g, 88% yield) from aldehyde 18 (2.51 g) and N,N-
dimethylethane-1,2-diamine (1.43 mL). IR (neat) 1605, 1246, 1031
cm-1; 1H NMR (CDCl3) δ 8.23 (s, 1H, CH), 7.65 (d, J ) 8.8 Hz,
2H, Ar), 6.92 (d, J ) 8.8 Hz, 2H, Ar), 4.08 (t, J ) 5.7 Hz, 2H,
CH2), 3.69 (t, J ) 6.7 Hz, 2H, CH2), 2.72 (t, J ) 5.7 Hz, 2H,
CH2), 2.61 (t, J ) 6.7 Hz, 2H, CH2), 2.32 (s, 6H, CH3), 2.29 (s,
6H, CH3).
[4-(2-Pyrrolidin-1-ylethoxy)benzylidene]-(2-pyrrolidin-1-yl-
ethyl)amine (20d). Compound 20d was obtained as a brown oil
(2.51 g, 61% yield) from aldehyde 19 (2.85 g) and 2-pyrrolidin-
1-ylethylamine (1.65 mL). IR (neat) 2961, 1785, 1604, 1251, 1033
cm-1; 1H NMR (CDCl3) δ 8.17 (s, 1H, CH), 7.58 (d, J ) 7.6 Hz,
2H, Ar), 6.86 (d, J ) 7.6 Hz, 2H, Ar), 4.50 (t, J ) 6.2 Hz, 2H,
CH2), 3.68 (t, J ) 7.1 Hz, 2H, CH2), 2.85 (t, J ) 6.0 Hz, 2H,
CH2), 2.80 (t, J ) 7.2 Hz, 2H, CH2), 2.54–2.51 (m, 8H, CH2),
1.75–1.71 (m, 8H, CH2).
N-[4-(2-Dimethylaminoethoxy)benzylidene]-N′,N′-dimethyl-
propane-1,3-diamine (20e). Compound 20e was obtained as a
brown oil (2.45 g, 68% yield) from aldehyde 18 (2.51 g) and N,N-
dimethylpropane-1,3-diamine (1.64 mL). IR (neat) 1604, 1251, 1033
3-[4-(2-Dimethylaminoethoxy)phenyl]-2-(2-dimethylaminoet-
hyl)-1-oxo-1,2,3,4-tetrahydroisoquinolin-4-carboxylic Acid (23c).
Carboxylic acid 23c was obtained from carboxylic anhydride 21
(1.84 g, 11.4 mmol) and imine 20c (3.0 g, 11.4 mmol). Charac-
teristic signals: H NMR (CDCl3) δ 5.26 (s, Htrans), 5.13 (d, J )
6.0 Hz, Hcis), 4.57 (d, J ) 6.0 Hz, Hcis), 3.83 (s, Htrans), cis/trans
ratio ) 1:2.
1-Oxo-3-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-2-(2-pyrrolidin-
1-ylethyl)-1,2,3,4-tetrahydroisoquinolin-4-carboxylic Acid (23d).
Carboxylic acid 23d was obtained from carboxylic anhydride 21
(1.84 g, 11.4 mmol) and imine 20d (3.16 g, 11.4 mmol).
Characteristic signals: 1H NMR (CDCl3) δ 5.20 (s, Htrans), 5.15 (d,
J ) 5.6 Hz, Hcis), 4.53 (d, J ) 5.6 Hz, Hcis), 3.78 (s, Htrans), cis/
trans ratio ) 1:2.
3-[4-(2-Dimethylaminoethoxy)phenyl]-2-(3-dimethylamino-
propyl)-1-oxo-1,2,3,4-tetrahydroisoquinolin-4-carboxylic Acid
(23e). Carboxylic acid 23e was obtained from carboxylic anhydride
21 (1.84 g, 11.4 mmol) and imine 20e (3.16 g, 11.4 mmol).
Characteristic signals: 1H NMR (CDCl3) δ 5.44 (s, Htrans), 5.30 (d,
J ) 5.7 Hz, Hcis), 4.73 (d, J ) 5.7 Hz, Hcis), 3.82 (s, Htrans), cis/
trans ratio ) 1:3.
1
6,7-Dimethoxy-3-[4-(2-dimethylaminoethoxy)phenyl]-2-(2-
dimethylaminoethyl)-1-oxo-1,2,3,4-tetrahydroisoquinolin-4-car-
boxylic Acid (23f). Carboxylic acid 23f was obtained from
carboxylic anhydride 2227 (2.51 g, 11.4 mmol) and imine 20c (3.0
g, 11.4 mmol). Characteristic signals: 1H NMR (CDCl3) δ 5.18 (s,

3626 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Ryckebusch et al.
H
trans), 5.12 (d, J ) 6.0 Hz, Hcis), 4.52 (d, J ) 6.0 Hz, Hcis), 3.76
MeOH/25% aqueous NH4OH 7:3:0.2) followed by TkLC (acetone/
25% aqueous NH4OH 95:5). Rf ) 0.85 (CH2Cl2/MeOH/25%
aqueous NH4OH 8:2:0.2); mp 114–115 °C; IR (neat) 1667, 1610,
(s, Htrans), cis/trans ratio ) 1:2.
6,7-Dimethoxy-3-[4-(2-dimethylaminoethoxy)phenyl]-2-(3-
dimethylaminopropyl)-1-oxo-1,2,3,4-tetrahydroisoquinolin-4-
carboxylic Acid (23g). Carboxylic acid 23g was obtained from
carboxylic anhydride 2227 (2.51 g, 11.4 mmol) and imine 20e (3.16
g, 11.4 mmol). Characteristic signals: 1H NMR (CDCl3) δ 5.34 (s,
1
1225, 1021 cm-1; H NMR (CDCl3) δ 8.66 (d, J ) 8.1 Hz, 1H,
Ar), 8.31 (d, J ) 8.7 Hz, 1H, Ar), 7.74–7.65 (m, 2H, Ar), 7.44 (m,
1H, Ar), 7.24 (d, J ) 2.2 Hz, 1H, Ar), 6.92 (dd, J ) 8.1 Hz, J )
2.2 Hz, 1H, Ar), 4.66 (t, J ) 8.1 Hz, 2H, CH2), 4.24 (t, J ) 5.1
Hz, 2H, CH2), 2.90 (t, J ) 5.1 Hz, 2H, CH2), 2.79 (t, J ) 8.1 Hz,
2H, CH2), 2.47 (s, 6H, CH3), 2.46 (s, 6H, CH3); HPLC; LC-MS.
9-(2-Pyrrolidin-1-ylethoxy)-6-(2-pyrrolidin-1-ylethyl)-6H-in-
deno[1,2-c]isoquinolin-5,11-dione (6d). Compound 6d was ob-
tained as a red solid (263 mg, 57% yield from cis isomer) from
carboxylic acid 23d (1.42 g, 3 mmol) and aluminum chloride (2.0
g, 15 mmol) after purification by column chromatography (CH2Cl2/
MeOH/25% aqueous NH4OH 2:8:0.2). Rf ) 0.80 (CH2Cl2/MeOH/
25% aqueous NH4OH 8:2:0.2); mp 109–110 °C; IR (neat) 1660,
1612, 1250, 1021 cm-1; 1H NMR (CDCl3) δ 8.65 (d, J ) 8.1 Hz,
1H, Ar), 8.31 (d, J ) 7.4 Hz, 1H, Ar), 7.73–7.63 (m, 2H, Ar), 7.42
(t, J ) 7.4 Hz, 1H, Ar), 7.22 (d, J ) 3.0 Hz, 1H, Ar), 6.89 (dd, J
) 8.1 Hz, J ) 3.0 Hz, 1H, Ar), 4.66 (t, J ) 8.1 Hz, 2H, CH2),
4.22 (t, J ) 7.3 Hz, 2H, CH2), 2.96 (m, 4H, CH2), 2.77 (m, 8H,
CH2), 1.87 (m, 8H, CH2); HPLC; LC-MS.
H
trans), 5.25 (d, J ) 6.4 Hz, Hcis), 4.61 (d, J ) 6.4 Hz, Hcis), 3.70
(s, Htrans), cis/trans ratio ) 2:3.
3-(4-Benzoyloxyphenyl)-2-(2-dimethylaminoethyl)-1-oxo-1,2,3,4-
tetrahydroisoquinolin-4-carboxylic Acid (26a). Carboxylic acid
26a was obtained from carboxylic anhydride 21 (2.51 g, 11.4 mmol)
and imine 25a (3.37 g, 11.4 mmol). Characteristic signals: 1H NMR
(CDCl3) δ 5.41 (s, Htrans), 5.35 (bs, Hcis), 4.80 (bs, Hcis), 3.89 (s,
Htrans), cis/trans ratio ) 1:2.
3-(4-Benzoyloxyphenyl)-2-(3-dimethylaminopropyl)-1-oxo-
1,2,3,4-tetrahydroisoquinolin-4-carboxylic Acid (26b). Carboxy-
lic acid 26b was obtained from carboxylic anhydride 21 (2.51 g,
11.4 mmol) and imine 25b (3.54 g, 11.4 mmol). Characteristic
signals: 1H NMR (CDCl3) δ 5.49 (s, Htrans), 4.70 (bs, Hcis), 3.88 (s,
H
trans).
General Procedure for the Synthesis of Indenoisoquinolines
9-(2-Dimethylaminoethoxy)-6-(3-dimethylaminopropyl)-6H-
indeno[1,2-c]isoquinolin-5,11-dione (6e). Compound 6e was
obtained as an orange solid (113 mg, 36% yield from cis isomer)
from carboxylic acid 23e (1.32 g, 3 mmol) and aluminum chloride
(2.0 g, 15 mmol) after purification by column chromatography
(CH2Cl2/MeOH/25% aqueous NH4OH 7:3:0.2) followed by TkLC
(acetone/25% aqueous NH4OH 95:5). Rf ) 0.75 (CH2Cl2/MeOH/
25% aqueous NH4OH 8:2:0.2); mp 84–87 °C; IR (neat) 1656, 1608,
6a–g and 27a,b. Thionyl chloride (10 mL) was added dropwise to
the carboxylic acids 23a–g and 26a,b (3 mmol, 1 equiv) while
stirring, and the reaction mixture was refluxed at 80 °C for 12 h.
After cooling, the reaction mixture was concentrated under reduced
pressure, diluted in dry CH2Cl2 (20 mL), and cooled to 0 °C.
Aluminum chloride was added slowly, and the reaction mixture
was stirred for 3 h at 0 °C. A solution of 1 N HCl (200 mL) was
then added dropwise, and the aqueous layer was separated and
washed with CH2Cl2 (3 × 100 mL). The aqueous layer was
alkalinized with brine until pH 8 and subsequently extracted with
ethyl acetate (3 × 200 mL). The organic layer was washed with
brine (3 × 200 mL), dried over MgSO4, and concentrated under
reduced pressure to give the crude product, which was purified to
obtain indenoisoquinolines 6a–g and 27a,b.
1
1250, 1034 cm-1; H NMR (CDCl3) δ 8.65 (d, J ) 8.0 Hz, 1H,
Ar), 8.31 (d, J ) 8.3 Hz, 1H, Ar), 7.73–7.63 (m, 2H, Ar), 7.41 (m,
1H, Ar), 7.23 (d, J ) 2.5 Hz, 1H, Ar), 6.87 (dd, J ) 8.3 Hz, J )
2.5 Hz, 1H, Ar), 4.56 (t, J ) 7.9 Hz, 2H, CH2), 4.17 (t, J ) 5.6
Hz, 2H, CH2), 2.79 (t, J ) 5.5 Hz, 2H, CH2), 2.53 (t, J ) 6.7 Hz,
2H, CH2), 2.38 (s, 6H, CH3), 2.33 (s, 6H, CH3), 2.12–1.99 (m, 2H,
CH2); HPLC; LC-MS.
8-(2-Dimethylaminoethoxy)-6-(2-dimethylaminoethyl)-6H-in-
deno[1,2-c]isoquinolin-5,11-dione (6a). Compound 6a was ob-
tained as an orange solid (100 mg, 24% yield from cis isomer)
from carboxylic acid 23a (1.27 g, 3 mmol) and aluminum chloride
(2.0 g, 15 mmol) after purification by column chromatography
(CH2Cl2/MeOH/25% aqueous NH4OH 2:8:0.2) followed by crystal-
lization from ethanol. Rf ) 0.80 (CH2Cl2/MeOH/25% aqueous
NH4OH 8:2:0.2); mp 197–198 °C; IR (neat) 1652, 1610, 1273, 1042
2,3-Dimethoxy-9-(2-dimethylaminoethoxy)-6-(2-dimethylami-
noethyl)-6H-indeno[1,2-c]isoquinolin-5,11-dione (6f). Compound
6f was obtained as a red oil (91 mg, 16% yield from cis isomer)
from carboxylic acid 23f (1.45 g, 3 mmol) and aluminum chloride
(2.0 g, 15 mmol) after purification by column chromatography
(CH2Cl2/MeOH/25% aqueous NH4OH 2:8:0.2). Rf ) 0.70 (CH2Cl2/
MeOH/25% aqueous NH4OH 8:2:0.2); IR (neat) 1649, 1612, 1267,
1023 cm-1 1H NMR (CDCl3) δ 8.09 (s, 1H, Ar), 7.67 (s, 1H,
;
1
cm-1; H NMR (CDCl3) δ 8.73 (d, J ) 8.0 Hz, 1H, Ar), 8.35 (d,
Ar), 7.60 (d, J ) 8.6 Hz, 1H, Ar), 7.20 (d, J ) 2.6 Hz, 1H, Ar),
6.88 (dd, J ) 8.4 Hz, J ) 2.6 Hz, 1H, Ar), 4.65 (m, 2H, CH2),
4.21 (t, J ) 5.5 Hz, 2H, CH2), 4.07 (s, 3H, CH3), 4.00 (s, 3H,
CH3), 2.86 (t, J ) 5.5 Hz, 2H, CH2), 2.78 (m, 2H, CH2), 2.46 (s,
6H, CH3), 2.45 (s, 6H, CH3); HPLC; LC-MS.
J ) 8.0 Hz, 1H, Ar), 7.73 (t, J ) 7.6 Hz, 1H, Ar), 7.58 (d, J ) 8.0
Hz, 1H, Ar), 7.48 (t, J ) 7.3 Hz, 1H, Ar), 7.41 (s, 1H, Ar), 6.81
(dd, J ) 8.0 Hz, J ) 1.8 Hz, 1H, Ar), 4.71 (t, J ) 7.5 Hz, 2H,
CH2), 4.59 (m, 2H, CH2), 2.92–2.71 (m, 4H, CH2), 2.42 (m, 12H,
CH3); HPLC; LC-MS.
8-(2-Dimethylaminoethoxy)-6-(3-dimethylaminopropyl)-6H-
indeno[1,2-c]isoquinolin-5,11-dione (6b). Compound 6b was
obtained as an orange solid (84 mg, 20% yield from cis isomer)
from carboxylic acid 23b (1.31 g, 3 mmol) and aluminum chloride
(2.0 g, 15 mmol) after purification by column chromatography
(CH2Cl2/MeOH/25% aqueous NH4OH 2:8:0.2) followed by crystal-
lization from ethanol/isopropanol 9:1. Rf ) 0.80 (CH2Cl2/MeOH/
25% aqueous NH4OH 8:2:0.2); mp 160–162 °C; IR (neat) 1654,
1612, 1242, 1045 cm-1; 1H NMR (CDCl3) δ 8.74 (d, J ) 7.8 Hz,
1H, Ar), 8.34 (d, J ) 8.6 Hz, 1H, Ar), 7.73 (td, J ) 8.3 Hz, J )
1.3 Hz, 1H, Ar), 7.58 (d, J ) 8.0 Hz, 1H, Ar), 7.47 (td, J ) 7.2
Hz, J ) 1.0 Hz, 1H, Ar), 7.41 (d, J ) 1.8 Hz, 1H, Ar), 6.77 (dd,
J ) 8.0 Hz, J ) 1.8 Hz, 1H, Ar), 4.57 (t, 2H, J ) 7.6 Hz, CH2),
4.18 (t, J ) 5.5 Hz, 2H, CH2), 2.83 (t, J ) 5.5 Hz, 2H, CH2), 2.63
(m, 2H, CH2), 2.41 (s, 6H, CH3), 2.38 (s, 6H, CH3), 2.11 (m, 2H,
CH2); HPLC; LC-MS.
2,3-Dimethoxy-9-(2-dimethylaminoethoxy)-6-(3-dimethylami-
nopropyl)-6H-indeno[1,2-c]isoquinolin-5,11-dione (6g). Com-
pound 6g was obtained as a red oil (86 mg, 15% yield from cis
isomer) from carboxylic acid 23 g (1.50 g, 3 mmol) and aluminum
chloride (2.0 g, 15 mmol) after purification by column chroma-
tography (CH2Cl2/MeOH/25% aqueous NH4OH 2:8:0.2). Rf ) 0.50
(CH2Cl2/MeOH/25% aqueous NH4OH); IR (neat) 1648, 1613, 1268,
1024 cm-1 1H NMR (CDCl3) δ 8.09 (s, 1H, Ar), 7.67 (s, 1H,
;
Ar), 7.58 (d, J ) 8.6 Hz, 1H, Ar), 7.20 (d, J ) 2.6 Hz, 1H, Ar),
6.85 (dd, J ) 8.4 Hz, J ) 2.6 Hz, 1H, Ar), 4.58–4.52 (m, 2H,
CH2), 4.17 (t, J ) 5.5 Hz, 2H, CH2), 4.07 (s, 3H, CH3), 4.00 (s,
3H, CH3), 2.80 (t, J ) 5.5 Hz, 2H, CH2), 2.64–2.43 (m, 2H, CH2),
2.43–2.39 (m, 12H, CH3), 2.15–2.10 (m, 2H, CH2); HPLC;
LC-MS.
6-(2-Dimethylaminoethyl)-5,11-dioxo-5,11-dihydro-6H-inde-
no[1,2-c]isoquinolin-9-yl Benzoate (27a). Compound 27a was
obtained as a red oil (222 mg, 50% from cis isomer) from carboxylic
acid 26a (0.82 g, 3 mmol) and aluminum chloride (3.2 g, 24 mmol)
after purification by column chromatography (CH2Cl2/MeOH/25%
aqueous NH4OH 2:8:0.2). Rf ) 0.80 (CH2Cl2/MeOH 9:1); IR (neat)
1737, 1698, 1661, 1610, 1504, 1263, 1207 cm-1; 1H NMR (CDCl3)
9-(2-Dimethylaminoethoxy)-6-(2-dimethylaminoethyl)-6H-in-
deno[1,2-c]isoquinolin-5,11-dione (6c). Compound 6c was ob-
tained as a red solid (185 mg, 45% yield from cis isomer) from
carboxylic acid 23c (1.27 g, 3 mmol) and aluminum chloride (2.0
g, 15 mmol) after purification by column chromatography (CH2Cl2/

Quinolin-7-one and Isoquinolin-5,11-dione DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3627
δ 8.59 (d, J ) 7.7 Hz, 1H, Ar), 8.26 (d, J ) 8.2 Hz, 1H, Ar), 8.18
(d, J ) 7.2 Hz, 2H, Ar), 7.74–7.64 (m, 3H, Ar), 7.55–7.50 (t, J )
7.7 Hz, 2H, Ar), 7.44–7.39 (m, 2H, Ar), 7.28 (dd, J ) 8.2 Hz, J )
2.3 Hz, 1H, Ar), 4.60 (t, J ) 7.9 Hz, 2H, CH2), 2.76 (t, J ) 7.9
Hz, 2H, CH2), 2.41 (s, 6H, CH3).
6-(3-Dimethylaminopropyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione (8b). Compound 8b was obtained as an orange solid (0.24
g, 60% yield) from N,N-dimethylpropane-1,3-diamine (0.15 g, 1.44
mmol) after concentration of the solution (20 mL), filtration, and
successive washings of the precipitate with ethyl ether/chloroform
2:1 (40 mL) and ethyl ether (10 mL). Rf ) 0.45 (CH2Cl2/MeOH
8:2); mp 166–168 °C (lit.26 168–171 °C); 1H NMR (CDCl3) δ 8.71
(d, J ) 8.0 Hz, 1H, Ar), 8.34 (d, J ) 7.6 Hz, 1H, Ar), 7.78–7.70
(m, 2H, Ar), 7.64 (dd, J ) 6.9 Hz, J ) 1.2 Hz, 1H, Ar), 7.50–7.40
(m, 3H, Ar), 4.60 (t, J ) 8.0 Hz, 2H, CH2), 2.53 (t, J ) 6.7 Hz,
2H, CH2), 2.31 (s, 6H, CH3), 2.12–2.00 (m, 2H, CH2); 13C NMR
(CDCl3) δ 190.60 (Cquat), 163.47 (Cquat), 155.70 (Cquat), 137.15
(Cquat), 135.21 (Cquat), 133.84 (CH), 133.30 (CH), 132.32 (Cquat),
130.96 (CH), 128.38 (CH), 127.12 (CH), 123.50 (CH), 123.43
(Cquat), 123.18 (CH), 122.93 (CH), 104.51 (Cquat), 56.87 (CH2),
45.77 (CH3), 43.57 (CH2), 27.38 (CH2); LC-MS.
DNA and Drugs Solutions. Calf thymus DNA (CT DNA,
Pharmacia) was deproteinized with sodium dodecyl sulfate (SDS,
protein content less than 0.2%) and extensively dialyzed against
the required experimental buffer. An extinction coefficient of 6600
M-1 cm-1 was used to measure the nucleotide concentration of
DNA solutions.34 All synthesized compounds, as well as camp-
tothecin and etoposide (Sigma), were dissolved as 10 mM solutions
in DMSO. Further dilutions were made in the appropriate aqueous
buffer.
Absorption Spectrometry and Melting Temperature Stud-
ies. Absorption spectra and melting curves were obtained using an
Uvikon 943 spectrophotometer coupled to a Neslab RTE111
cryostat. Typically, 20 µM of the various drugs 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 µM of CT DNA
and transferred into a quartz cuvette of 10 mm path length. The
spectra were recorded from 230 to 500 nm and are referenced
against a cuvette containing the same DNA concentration in the
same buffer. For the absorption titration, CT DNA was added
gradually from 1 to 20 µM with a spectrum recorded after each
addition. To perform the melting temperature measurement, CT
DNA (20 µM) was incubated alone (control Tm) or with increasing
concentrations of the tested compound in 1 mL of BPE buffer, thus
resulting in a drug/base pair ratio of 0.05, 0.1, 0.25, 0.5. The sample
was transferred into a quartz cell, and the absorbance at 260 nm
was measured every min over the range 20-100 °C with an
increment of 1 °C per min. The Tm values were obtained from first-
derived plots.
Fluorescence Measurements. Method 1. The intrinsic fluores-
cence of analogues of indenoquinolinone 1a was exploited to
determine their apparent binding constants. Fluorescence titration
data were recorded at room temperature using a SPEX Fluorolog
fluorometer. Excitation was set at 490 nm, and fluorescence
emission was monitored over the range 500–700 nm. Samples used
for titration experiments were prepared separately at a constant drug
concentration of 1 µM and DNA concentration ranging from 0.01
to 10 µM bp. Fluorescence titration data were fitted directly to
obtain apparent binding constants using a fitting function incorpo-
rated into Prism 3.0 software.
6-(3-Dimethylaminopropyl)-5,11-dioxo-5,11-dihydro-6H-in-
deno[1,2-c]isoquinolin-9-yl Benzoate (27b). Compound 27b was
obtained as a red oil (210 mg, 15% yield from cis-trans mixture)
from carboxylic acid 26b (0.85 g, 3 mmol) and aluminum chloride
(3.2 g, 24 mmol) after purification by column chromatography
(CH2Cl2/MeOH/25% aqueous NH4OH 2:8:0.2). Rf ) 0.80 (CH2Cl2/
MeOH 9:1); IR (neat) 1734, 1697, 1661, 1610, 1507, 1261, 1204
1
cm-1; H NMR (CDCl3) δ 8.69 (d, J ) 7.3 Hz, 1H, Ar), 8.33 (d,
J ) 8.1 Hz, 1H, Ar), 8.22 (d, J ) 7.3 Hz, 2H, Ar), 7.86 (d, J ) 8.8
Hz, 1H, Ar), 7.75–7.66 (m, 2H, Ar), 7.57–7.45 (m, 4H, Ar), 7.30
(dd, J ) 8.1 Hz, J ) 2.5 Hz, 1H, Ar), 4.59 (t, J ) 7.7 Hz, 2H,
CH2), 2.56 (t, J ) 6.6 Hz, 2H, CH2), 2.34 (s, 6H, CH3), 2.10–2.02
(m, 2H, CH2).
General Procedure for Synthesis of Compounds 7a,b. LiOH
(33 mg, 1.36 mmol, 2 equiv) was added to a solution of ester 27a,b
(0.68 mmol, 1 equiv) in CH2Cl2/MeOH 1:1, and the reaction mixture
was stirred for 1 h. Then 1 N HCl was added until pH 1 and the
aqueous layer was washed with CH2Cl2 (3 × 20 mL), alkalinized
with NH4OH until pH 8, and subsequently extracted with ethyl
acetate (3 × 40 mL). The organic layer was dried over MgSO4
and concentrated under reduced pressure. The desired products were
obtained after crystallization from methanol.
9-Hydroxy-6-(2-dimethylaminoethyl)-6H-indeno[1,2-c]iso-
quinolin-5,11-dione (7a). Compound 7a was obtained as a red solid
(0.14 g, 62% yield) from ester 27a (0.30 g, 0.68 mmol). Rf ) 0.50
(CH2Cl2/MeOH 9:1); mp 250 °C (dec); IR (neat) 1697, 1660, 1610,
1
1547, 1505, 1438 cm-1; H NMR (DMSO-d6) δ 10.58 (bs, 1H,
OH), 8.59 (d, J ) 8.1 Hz, 1H, Ar), 8.17 (d, J ) 8.2 Hz, 1H, Ar),
7.78 (t, J ) 7.1 Hz, 1H, Ar), 7.56 (d, J ) 8.2 Hz, 1H, Ar), 7.46 (t,
J ) 7.7 Hz, 1H, Ar), 6.98 (d, J ) 2.7 Hz, 1H, Ar), 6.86 (dd, J )
8.2 Hz, J ) 2.2 Hz, 1H, Ar), 4.54 (t, J ) 7.1 Hz, 2H, CH2),
2.66–2.63 (m, 2H, CH2), 2.06 (s, 6H, CH3); HPLC; LC-MS.
9-Hydroxy-6-(3-dimethylaminopropyl)-6H-indeno[1,2-c]iso-
quinolin-5,11-dione (7b). Compound 7b was obtained as a red solid
(0.16 g, 67% yield) from ester 27b (0.31 g, 0.68 mmol). Rf ) 0.50
(CH2Cl2/MeOH 9:1); mp 243 °C (dec); IR (neat) 1691, 1652, 1613,
1
1551, 1506, 1475, 1455 cm-1; H NMR (DMSO-d6) δ 8.49 (d, J
) 8.0 Hz, 1H, Ar), 8.16 (d, J ) 8.0 Hz, 1H, Ar), 7.76 (t, J ) 7.3
Hz, 1H, Ar), 7.64 (d, J ) 8.3 Hz, 1H, Ar), 7.44 (t, J ) 8.0 Hz, 1H,
Ar), 6.96 (d, J ) 2.4 Hz, 1H, Ar), 6.83 (dd, J ) 8.3 Hz, J ) 2.4
Hz, 1H, Ar), 4.43 (t, J ) 7.6 Hz, 2H, CH2), 2.41 (t, J ) 6.6 Hz,
2H, CH2), 2.16 (s, 6H, CH3), 1.89–1.84 (m, 2H, CH2); HPLC;
LC-MS.
General Procedure for Synthesis of Compounds 8a,b. Ap-
propriate dimethylaminoalkylamine (1.44 mmol, 1.2 equiv) was
added to a stirred solution of indenopyranedione 2829 (0.30 g, 1.20
mmol, 1 equiv) in chloroform (40 mL), and the reaction mixture
was stirred at room temperature for 18 h.
6-(2-Dimethylaminoethyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione (8a). Compound 8a was obtained as an orange solid (0.15
g, 39% yield) from N,N-dimethylethane-1,2-diamine (0.13 g, 1.44
mmol) after concentration of the solution (10 mL), filtration, and
successive washings of the precipitate with ethyl ether/chloroform
3:1 (20 mL) and ethyl ether (10 mL). Rf ) 0.60 (CH2Cl2/MeOH
Method 2. Since indeno[1,2-c]isoquinolin-5,11-dione derivatives
show weak fluorescence variation with DNA titration, the binding
studies were carried out through a competitive displacement
fluorometry assay using DNA-bound ethidium bromide.35,36 Excita-
tion was set at 515 nm, and the fluorescence emission was
monitored over the range 550–700 nm. Experiments were performed
with an ethidium bromide/DNA molar ratio of 12.6:10 and a drug
concentration range of 0.01–100 µM in a BPE buffer, pH 7.1. C50
values for ethidium bromide displacement were calculated using a
fitting function incorporated into Prism 3.0, and the apparent binding
constant was calculated as follows: Kapp ) (1.26/C50)Kethidium, with
1
8:2); mp 184–187 °C; H NMR (CDCl3) δ 8.71 (d, J ) 7.9 Hz,
1H, Ar), 8.34 (d, J ) 7.6 Hz, 1H, Ar), 7.76–7.67 (m, 2H, Ar), 7.64
(dd, J ) 7.0 Hz, J ) 1.0 Hz, 1H, Ar), 7.51–7.41 (m, 3H, Ar), 4.66
(t, J ) 8.0 Hz, 2H, CH2), 2.76 (t, J ) 8.0 Hz, 2H, CH2), 2.41 (s,
6H, CH3); 13C NMR (CDCl3) δ 190.48 (Cquat), 163.30 (Cquat),
155.69 (Cquat), 137.05 (Cquat), 135.09 (Cquat), 133.91 (CH), 133.46
(CH), 132.28 (Cquat), 131.03 (CH), 128.43 (CH), 127.22 (CH),
123.52 (CH), 123.37 (Cquat), 123.26 (CH), 122.54 (CH), 108.62
(Cquat), 57.32 (CH2), 45.90 (CH3), 43.23 (CH2); LC-MS; Anal.
(C20H18N2O2) C, H, N.
Kethidium ) 107 M-1
.
Topoisomerase Inhibition. The experimental procedure has been
previously detailed.37 Supercoiled pLAZ plasmid DNA (130 ng)
was incubated with 4 units of human topoisomerase I or II
(TopoGen) at 37 °C for 45 min in 20 µL of relaxation buffer (50

3628 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Ryckebusch et al.
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mM tris(hydroxymethyl)aminomethane, pH 7.8, 50 mM KCl, 10
mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, and 1 mM ATP)
in the presence of graded concentrations (from 1.0 to 50 µM) of
the tested compound. Reactions were terminated by adding of SDS
to 0.25% and proteinase K to 250 µg/mL and incubating at 50 °C
for a further 30 min. An amount of 3 µL of the electrophoresis dye
mixture was then added to DNA samples, which were then
separated by electrophoresis in a 1% agarose gel containing
ethidium bromide (1 µg/mL, topoisomerase DNA cleavage gel) or
not (inhibition of the relaxation of DNA) at room temperature for
2 h at 120 V. Gels run without ethidium bromide were then stained
using a bath containing ethidium bromide. Both gels were finally
washed and photographed under UV light.
Cell Cultures and Antiproliferative Assay. Human HL60 and
HL60/MX2 leukemia cells were obtained from the American Tissue
Culture Collection. Cells were grown at 37 °C in a humidified
atmosphere containing 5% CO2 in RPMI 1640 medium, supple-
mented with 10% fetal bovine serum, 2 mM L-glutamine, 1.5 g/L
sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium
pyruvate, penicillin (100 IU/mL), and streptomycin (100 µg/mL).
The cytotoxicity of the tested compounds was assessed using a cell
proliferation assay developed by Promega (CellTiter 96 AQueous
one solution cell proliferation assay). Briefly, 2 × 104 exponentially
growing cells were seeded in 96-well microculture plates with
various drug concentrations in a volume of 100 µL. After 72 h
incubation at 37 °C, 20 µL of the tetrazolium dye was added to
each well and the samples were incubated for a further 2 h at 37
°C. Plates were analyzed on a Labsystems Multiskan MS (type 352)
reader at 492 nm.
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paradigm for ternary trapping of the topoisomerase I-DNA covalent
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I inhibitors. J. Med. Chem. 2000, 43, 3688–3698.
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Acknowledgment. This work was supported by a postdoc-
toral grant (to A.R.) from the Ligue Nationale Contre le Cancer
and by grants (to A.R.) from the Ligue Nationale Contre le
Cancer, Comité du Nord. The authors thank Christine Mahieu
and William Laine for expert technical assistance and Nathalie
Dias for her contribution in performing biological assays.
Supporting Information Available: Analytical data and purity
assessment for key compounds; elemental analysis data for
compound 8a. This material is available free of charge via the
Internet at http://pubs.acs.org.
(22) Staker, B. L.; Feese, M. D.; Cushman, M.; Pommier, Y.; Zembower,
D.; Stewart, L.; Burgin, A. B. Structures of three classes of anticancer
agents bound to the human topoisomerase I-DNA covalent complex.
J. Med. Chem. 2005, 48, 2336–2345.
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of norindenoisoquinoline topoisomerase I poisons reveal an inhibitor
with a flipped orientation in the ternary DNA-enzyme-inhibitor
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activity of simplified indenoisoquinoline topoisomerase I inhibitors
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