Indeno[1,2-c]isoquinolin-5,11-diones conjugated to amino acids: Synthesis, cytotoxicity, DNA interaction, and topoisomerase II inhibition properties

Article abstract of DOI:10.1016/j.bmc.2010.08.025

Three series of indeno[1,2-c]isoquinolin-5,11-dione-amino acid conjugates were designed and synthesized. Amino acids were connected to the tetracycle through linkers with lengths of n = 2 and 3 atoms using ester (series I), amide (series II), and secondary amine (series III) functions. DNA binding was evaluated by thermal denaturation and fluorescence measurements. Lysine and arginine substituted derivatives with n = 3 provided the highest DNA binding. Arginine derivative 32 (n = 2, series II) and glycine derivative 34 (n = 2, series III) displayed high topoisomerase II inhibition. Incrementing the length of the N-6 side chain from two to three methylene units provided a significant increase in DNA affinity but a substantial loss in topoisomerase II inhibition. The most cytotoxic compounds toward HL60 leukemia cells were 19, 33, and 34 displaying micromolar IC₅₀ values. When tested with the topoisomerase II-mutated HL60/MX2 cell line, little variation of IC₅₀ values was found, suggesting that topoisomerase II might not be the main target of these compounds and that additional targets could be involved.

Full text of DOI:10.1016/j.bmc.2010.08.025

Bioorganic & Medicinal Chemistry 18 (2010) 8119–8133
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Indeno[1,2-c]isoquinolin-5,11-diones conjugated to amino acids: Synthesis,
cytotoxicity, DNA interaction, and topoisomerase II inhibition properties
Gang Ahn a, Amélie Lansiaux c, Jean-François Goossens e, Christian Bailly d, Brigitte Baldeyrou c,
Nadège Schifano-Faux e, Pierre Grandclaudon a,b, Axel Couture a, Adina Ryckebusch a,b,
⇑
a EA CMF 4478, Université Lille Nord de France-Université Lille 1, Bât C3(2), Cité Scientifique, 59655 Villeneuve d’Ascq Cedex, France
b Ecole Nationale Supérieure de Chimie de Lille, Avenue Dimitri Mendeleïev, Cité Scientifique, BP 90108, 59652 Villeneuve d’Ascq Cedex, France
c Inserm U837, Centre Oscar Lambret, Université Lille Nord de France-Université Lille 2, IRCL et IMPRT, 1 Place de Verdun 59045 Lille Cedex, France
d Centre de Recherche en Oncologie Expérimentale, Institut de Recherche Pierre Fabre, 3 rue des Satellites, 31140 Toulouse, France
e Laboratoire de Chimie Analytique, EA 4481, Université Lille Nord de France-Université Lille 2, 3 rue du Professeur Laguesse, BP 83, 59006 Lille Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Three series of indeno[1,2-c]isoquinolin-5,11-dione-amino acid conjugates were designed and synthe-
sized. Amino acids were connected to the tetracycle through linkers with lengths of n = 2 and 3 atoms
using ester (series I), amide (series II), and secondary amine (series III) functions. DNA binding was eval-
uated by thermal denaturation and fluorescence measurements. Lysine and arginine substituted deriva-
tives with n = 3 provided the highest DNA binding. Arginine derivative 32 (n = 2, series II) and glycine
derivative 34 (n = 2, series III) displayed high topoisomerase II inhibition. Incrementing the length of
the N-6 side chain from two to three methylene units provided a significant increase in DNA affinity
but a substantial loss in topoisomerase II inhibition. The most cytotoxic compounds toward HL60 leuke-
mia cells were 19, 33, and 34 displaying micromolar IC50 values. When tested with the topoisomerase II-
mutated HL60/MX2 cell line, little variation of IC50 values was found, suggesting that topoisomerase II
might not be the main target of these compounds and that additional targets could be involved.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 11 April 2010
Revised 9 August 2010
Accepted 11 August 2010
Available online 16 August 2010
Keywords:
Cancer
DNA ligand
Topoisomerases
Cytotoxicity
Amino acid conjugate
Indenoisoquinolin-5,11-dione
1. Introduction
Eukaryotic type II topoisomerases are homodimers that require
divalent metal ions and ATP for complete catalytic activity. Two
Topoisomerases are key enzymes required for the survival of all
organisms. They regulate the topological state of the DNA in vital
cellular processes of replication, transcription, recombination, re-
pair, chromatin assembly, and chromosome segregation. Topoi-
somerases act by generating transient breaks in the double helix.
There are two major classes of topoisomerases, type I and type II,
that are distinguished by the number of DNA strands that they
cleave, generating either single- or double-stranded breaks, respec-
tively, and the mechanism by which they alter the topological
properties of the genetic material.
Eukaryotic type I topoisomerases are monomeric enzymes that
require no high-energy co-factor. They have been further classi-
fied1,2 into three different sub-families: types IA, IB, and IC. Type
IB topoisomerases are the targets of important anticancer com-
pounds such as camptothecins,3,4 indolocarbazoles,5 and
indenoisoquinolines.6
isoforms are expressed by vertebrate species: topoisomerases II
and IIb displaying a high degree of amino acid sequence identity
is essential for the survival of actively
growing cells and its concentrations are dramatically upregulated
during periods of cell growth. It is generally believed to be the iso-
a
(ꢀ70%). Topoisomerase II
a
form
a that functions in growth-dependent processes, such as DNA
replication and chromosome segregation.7
Topoisomerases I and II control tightly DNA topology by cleav-
ing transiently DNA to form a complex known as the ‘cleavage
complex’ changing its topology by a controlled rotation of the
cleaved strand around the intact strand, and religating it to give
an intact DNA duplex. DNA topoisomerases are targets for several
clinically prescribed anticancer agents. Camptothecins, potent
topoisomerase I poisons, reversibly trap the topo I in the covalent
complex with DNA, inducing an accumulation of DNA damage that
triggers cell death. Among topoisomerase I poisons, the only FDA/
EMEA-approved compounds are the camptothecin derivatives iri-
notecan and topotecan used to treat colorectal, ovarian, and lung
cancers.
Topoisomerase II poisons represent some of the most important
and widely prescribed anticancer drugs. Among them, etoposide,
⇑
Corresponding author. Tel.: +33 3 2043 4440; fax: +33 3 2033 6309.
E-mail address: Adina.Ryckebusch@ensc-lille.fr (A. Ryckebusch).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.025

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G. Ahn et al. / Bioorg. Med. Chem. 18 (2010) 8119–8133
doxorubicin, mitoxantrone, and amsacrine are indicated for a vari-
ety of malignancies, including leukemias, lymphomas, sarcomas,
breast, and lung cancers.
Clinical uses of these molecules are limited by their toxicities
and development of multidrug resistance (MDR) phenomenon,
essentially due to the induction and action of the membrane
ATP-dependent multidrug efflux pump P-glycoprotein (MDR1
and ABCB1) which extrudes cytotoxic molecules, keeping intracel-
lular drug concentration below a cell-killing threshold.8
side-chains of the indenoisoquinolinedione framework. The cyto-
toxicity, DNA interaction, and topoisomerase I and II inhibition
properties of these molecules were evaluated. The goal was to in-
crease the overall basicity of the molecules in order to enhance
DNA binding as well as the aqueous solubility. Amino acids were
connected to the tetracycle through linkers with lengths of 2 and
3 atoms using ester (series I), amide (series II), and secondary
amine (series III) functions. In each of the three series, glycine ana-
logues were synthesized and evaluated as controls.
Over the past 20 years, new topoisomerases inhibitors have
been designed to overcome limitations cited above. Indenoisoquin-
olines are novel non-camptothecin topoisomerase I poisons en-
dowed with greater stability compared to camptothecins and
potentially lower toxicities. Topoisomerase I cleavage complexes
trapped by indenoisoquinolines display better stability, which
could increase the duration of action of the drugs. These com-
pounds are also weakly recognized by resistance efflux pumps
(MDR-1 and ABCG2). Among the over 400 indenoisoquinolines
evaluated, three have been selected as leads for clinical develop-
ment by the National Cancer Institute,6 particularly compound 1
(NSC 706744, Fig. 1).
Given the potential of this family, a great number of structural
modifications were introduced at both the N-6 position and at the
indenoisoquinoline skeleton on the aromatic A and D cycles9–12 to
provide structure–activity relationships.13,14 Modifications at the
N-6 position such as introduction of aminoalkyl chains provided
potent topoisomerase I inhibitors. Non-substituted molecules on
the A and D aromatic cycles could also display good topoisomerase
I inhibition and antiproliferative activity.15 A crystallographic
study performed with compound 2 (MJ-238, Fig. 1)16 bound to
the topoisomerase I-DNA cleavage complex revealed that the aro-
matic tetracycle interacts with both DNA and topoisomerase I,
whereas the side chain was placed in a hydrophilic binding pocket
of the enzyme.17 Introduction of dimethylaminoethoxy substituent
on D-cycle at 8-position and dimethylaminoethyl at N-6
position provided a good topoisomerase II inhibitor18 (compound
3, Fig. 1).
2. Chemistry
The synthesis of the targeted compounds is depicted in
Schemes 1 and 2. The indenopyrandione 38, obtained by a de-
scribed procedure9 slightly modified, was used as a precursor for
the synthesis of compounds 4–37. Esters 4–17 were prepared by
the condensation of lactone 38 with commercially available ami-
noethanol or aminopropanol followed by the reaction of resulting
alcohols 39 and 40 with the corresponding Boc-protected amino
acids using EDCI as an activator to provide esters 4a–17a. This
operation spared the stereogenic centers and subsequent removal
of tert-butoxycarbonyl groups by acidolysis (HCl in isopropanol)
yielded the desired products as hydrochloride salts.
Condensation of precursor 38 with N-Boc-protected N,N-ethy-
lenediamine 41 or N,N-propanediamine 42 followed by acidolysis
afforded the NH2 free intermediates 43 and 44. Amides 18a–33a
were obtained, respectively, by reaction with Boc-protected amino
acids using either EDCI/HOBt or HBTU/HOBt as coupling agents.
Deprotection of the tert-butoxycarbonyl groups was performed
by acidolysis to provide the desired products 18–33 as hydrochlo-
ride salts. It is noteworthy that for deprotection of compounds 32a
and 33a, the usually employed conditions (HCl in isopropanol)
failed. The use of SnCl4 was required to secure the formation of
arginine-containing derivatives 32 and 33.
For elaboration of compounds from series III bearing amine
functionalities, N-Boc-protected glycine and lysine were initially
converted into corresponding Weinreb amides 45 and 46 which
were subsequently reduced with LiAlH4 to provide the correspond-
In recent years, various drug-amino acid conjugates were re-
ported, such as DNA-acting drugs anthraquinones,19–21 naphthali-
mides,22,23 indolocarbazoles24,25 and camptothecins.26 In some
cases, promising results were obtained with conjugates showing
increased water-solubility and cytotoxicity, reduced drug resis-
tance and toxicity. These considerations prompted us to consider
a similar approach in the indenoisoquinoline series. In the current
work, we report the synthesis (Fig. 2) of a novel series of molecules
bearing basic amino acids (lysine, histidine, arginine) on the N-6
ing
a-aminoaldehydes. A reductive amination process involving
the aminoalkylated derivatives 43 and 44 delivered the N-Boc-pro-
tected compounds 34a, 36a, and 37a. Subsequent acidolysis with
HCl 5 M in isopropanol afforded very satisfactory yields of the de-
sired compounds 34, 36, and 37. Amine 35 was readily assembled
through an alternative pathway involving nucleophilic substitu-
tion between the tosylate 47 and the N-Boc-protected-N,N-ethy-
lenediamine 41, followed by standard acidolysis.
Many attempts to introduce N-protected arginines in Series I
and in Series III were performed. Unfortunately, concerning the
Series I, although the esterification reaction was proven to be suc-
cessful, the harsh conditions used for the deprotection step led to
the exclusive formation of the transcription side products. Several
strategies to synthesize the expected esters were subsequently
envisaged but all attempts to assemble these compounds were
invariably doomed to failure.
O
O
D
C
H3CO
H3CO
H
N
A
B
N
N
COOH
OH
O
O
1 (NSC 706744)
2 (MJ-238, NSC 700079)
As for the Series III, taking into account the undesirable racemi-
zation observed for D- or L-lysine derivatives, only the syntheses
H3C
N
CH3
involving the racemic DL-lysine were then considered. Histidine
derivatives were also eliminated from the current study due to
the failure to synthesize the histidinal: to the best of our knowl-
edge, no description of histidinal synthesis has been found in the
literature. And finally, arginine derivatives were dismissed from
further studies since even under forcing conditions, the ultimate
reductive amination liable to give access to the targeted com-
pounds failed and an inextricable mixture of unidentified products
was obtained instead.
O
O
N
CH3
N
O
CH3
3
Figure 1. Indenoisoquinolin-5,11-dione inhibitors of topoisomerases I and II.

G. Ahn et al. / Bioorg. Med. Chem. 18 (2010) 8119–8133
8121
O
O
O
H
N
H
N
N
R
N
R
O
N
R
( )n
( )n
n =2,3
( )n
n =2,3
O
O
O
O
n =2,3
O
Series II
Series III
Series I
cpd
n
cpd
n
CH2R
COR
COR
cpd
n
H
H
.
HCl
.
HCl
18
19
2
3
Gly
Gly
4
5
2
3
Gly
Gly
34
35
2 Gly(H2)
3 Gly(H2)
NH2
NH2
O
NH2
2HCl
20
21
22
23
24
25
2
2
2
3
3
3
L-Lys
NH2
2HCl
.
6
7
2
2
2
3
3
3
L-Lys
.
DL-Lys
D-Lys
L-Lys
36
37
2
3
DL-Lys(H2)
DL-Lys(H2)
DL-Lys
D-Lys
L-Lys
NH2
8
NH2
N
9
10
11
O
DL-Lys
D-Lys
DL-Lys
D-Lys
.
N
H
HCl
26
27
28
29
30
31
2
2
2
3
3
3
L-His
12
13
14
15
16
17
2
2
2
3
3
3
L-His
DL-His
D-His
L-His
DL-His
D-His
L-His
DL-His
D-His
NH2
O
DL-His
D-His
H
N
NH2
NH
2HCl
NH2
32
33
2
3
L-Arg
L-Arg
.
O
Figure 2. Compounds considered in the present study.
R
O
O
O
n
Series I Series II
O
4a
5a
6a
7a
8a
18a
19a
20a
21a
22a
23a
24a
25a
26a
27a
28a
29a
30a
31a
32a
33a
2
3
2
2
2
3
3
3
2
2
2
3
3
3
2
3
N-Boc-Gly
N-Boc-Gly
N-Boc-L-Lys(Boc)
N-Boc-DL-Lys(Boc)
N-Boc-D-Lys(Boc)
N-Boc-L-Lys(Boc)
N-Boc-DL-Lys(Boc)
N-Boc-D-Lys(Boc)
N-Boc-L-His(Boc)
N-Boc-DL-His(Boc)
N-Boc-D-His(Boc)
N-Boc-L-His(Boc)
N-Boc-DL-His(Boc)
N-Boc-D-His(Boc)
N-Boc-L-Arg(Boc)2
N-Boc-L-Arg(Boc)2
a
b
c
4-17
N
OH
( )n
O
N
O
R
( )n
O
39
O
38
O
O
(n = 2)
4a-17a
40 (n = 3)
9a
10a
11a
12a
13a
14a
15a
16a
O
O
O
c or f
c
e
d
H
N
18-33
38
N
R
N
NHBoc
( )n
N
NH2
( )n
( )n
O
O
O
O
17a
18a-33a
43a
44a
43
44
(n = 2)
(n = 3)
(n = 2)
(n = 3)
Scheme 1. Synthesis of compounds from series I and II. Reagents and conditions: (a) NH2(CH2)nOH, n = 2 (39) or 3 (40), CHCl3, rt; (b) corresponding N-Boc protected amino
acid, EDCI, DMAP, CH2Cl2, rt; (c) HCl, isopropanol, CHCl3, rt; (d) NH2(CH2)2NHBoc (41) or NH2(CH2)3NHBoc (42), CHCl3, rt; (e) corresponding N-Boc protected amino acid,
EDCI/HOBt or HBTU/HOBt, DMAP, CH2Cl2, rt; (f) SnCl4, AcOEt, rt.
significantly duplex stabilization as shown by comparison of series
III derivatives (34–37) with their respective amide counterparts
(18, 19, 21, and 24). Incrementing the length of the N-6 side chain
3. Results and discussion
3.1. DNA interactions
from two to three methylene units enhanced
DTm values for all
tested compounds from the three series, except for histidine deriv-
atives (12-17 and 26-31). As expected, among all amino acids used
in this study, lysine provided the strongest duplex stabilization. In
series II and III lysine derivatives were twofold more potent than
their reference glycine counterparts (20–22 vs 18, 23–25 vs 19,
36 vs 34, and 37 vs 35) and in series I they display three to four
times more potent stabilization (6–8 vs 4, 9–11 vs 5). In series II,
arginine derivatives (32 and 33) were equipotent to lysine (20
and 23). These values were similar to daunorubicin values
3.1.1. DNA thermal denaturation
The ability of the drugs to protect calf thymus DNA (CT DNA,
42% GC bp) against thermal denaturation was used as an indicator
of their relative capacity to bind and to stabilize the DNA double
helix. Variations of Tm values
D
Tm ¼ Tmdrug —DNA complex ꢁ TmDNA alone
are presented in Table 1. They ranged from 2.1 °C (compound 13)
to 26.7 °C (compound 37). Amides from series II (18–31) display
better
DTm values than their ester counterparts (4–17). Reduction
of amide (series II) to amine function (series III) increased

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G. Ahn et al. / Bioorg. Med. Chem. 18 (2010) 8119–8133
O
O
O
a
H
N
b
c
X
Y
N-Boc-Gly-OH
or
CH3
H
N
H
N
Boc
N
N
N
( )n
( )n
NHBoc
NH2
X
OCH3
N-Boc-Lys(Boc)-OH
O
O
34a n = 2, X = H
45 X = H
34 n = 2, Y = H
36a n = 2, X = (CH2)4NHBoc,
37a n = 3, X = (CH2)4NHBoc
46 X = (CH2)4NHBoc
36 n = 2, Y = (CH2)4NH2
37 n = 3, Y = (CH2)4NH2
O
O
O
O
c
d
e
N
N
( )3
OH
NH
N
NH
N
OTs
( )3
NHBoc
( )3
NH2
( )3
O
40
O
35a
O
35
O
47
Scheme 2. Synthesis of compounds from series III. Reagents and conditions: (a) N,O-dimethylhydroxylamine, EDCI, DMAP, CH2Cl2, rt; (b) (i) LiAlH4, THF, 0 °C, (ii) compound
43 or 44, NaHB(OAc)3, triethylamine, CH2Cl2, rt; (c) HCl, isopropanol, CHCl3, rt; (d) TsCl, triethylamine, CH2Cl2, rt; (e) compound 41, CH3CN, 80 °C.
reference intercalative compound. Finally, no significant differ-
and fluorescence experiments suggest an interacting mode, a
DNA unwinding assay was used (Fig. 3) to confirm it.
The experiments were based on the relaxation of supercoiled
plasmid DNA in the presence or not of topoisomerase I and increas-
ing concentration of the tested compounds. We selected com-
ences were found between
and II.
L- and D-stereoisomers from series I
The high
DTm values obtained with the lysine and arginine
derivatives are most likely attributable to additional electrostatic
interactions or hydrogen bonding between protonated basic side-
chains of the amino acids and the negatively charged DNA backbone.
pounds with the highest
DTm and Kapp in each series. We also
selected compounds with three carbon lactam substituent chain
length (n = 3) as they displayed more pronounced effect than their
shorter side-chain counterparts (n = 2). As shown in Figure 3, we
compared DNA relaxation of racemate and enantiomer compounds
in series I (Fig. 3A) and II (Fig. 3B). We also compared series II and
III with the lysine derivatives (Fig. 3A) and the lysine and arginine
derivatives (Fig. 3B). As shown in Figure 3, the DNA (lane DNA) re-
laxed by topoisomerase I alone (lane Topo) generated a family of
DNA topoisomers with a slow electrophoretic mobility. When the
drug was added while topoisomerase I was maintained in the
reaction mixture, the closed circular duplex DNA was progres-
sively supercoiled, indicating that the drug intercalates into
DNA.29,30
3.1.2. Fluorescence measurements
DNA binding affinities were quantified by means of fluores-
cence (Table 1). Since a weak fluorescence was observed upon
DNA titration with all compounds, an indirect method was privi-
leged. We used the conventional fluorescence quenching assay
based on DNA binding competition between the intercalating drug
ethidium bromide and the tested molecules.27,28 As reported in
Table 1, Kapp values ranged from 0.22 ꢂ 106 (compounds 13 and
14) to 16.97 ꢂ 106 (compound 37). Replacing ester (series I) by
amide (series II) connection had no significant effect on histidine
and glycine derivatives DNA affinity but provided a net increase
for lysine analogues displaying Kapp values (20–22 vs 6–8, 23–25
vs 9–11). Compounds from series III display higher Kapp values than
their amide and ester counterparts as noted for lysine derivatives
(36 vs 21 and 7, 37 vs 24 and 10). For glycine derivatives the effects
obtained are more pronounced as Kapp values are 10-fold superior
to their amide and ester counterparts (34 vs 18 and 4, 35 vs 19 and
5) which suggest the key role of the secondary amino group as
H-donor for the DNA interaction.
When considering the influence of the amino acid in DNA inter-
action, the best Kapp values in the three series were obtained for ly-
sine derivatives. The results obtained by the fluorescence assay
were consistent. Incrementing the length of the N-6 side chain
from two to three methylene units leads to a significant increase
in DNA affinity for lysine derivatives: 10-fold in series I (9–11 vs
6–8), twofold in series II (23–25 vs 20–22), 1.2-fold in series III
(37 vs 36).
All tested compounds in series I, II, and III modify the relaxation
of the DNA with a passage from negative to positive in the helical
superturn with 1 or 2 lM of compound. There are no differences
between compounds: any difference between racemate and enan-
tiomers in series I and II, between lysine derivatives in series I and
III, between lysine and arginine derivatives in series II.
Only compounds with space length of three methylene units
were reported on Figure 3. (n = 3) as they displayed more pro-
nounced effect than their shorter side-chain counterparts (n = 2).
In series I, compound 10 (Lys) intercalated more efficiently than
16 (His), as judged from the appearance of the supercoiled DNA
band, respectively, for 5 and 10 lM (data not shown). In series II,
for selected compounds, the ranking was 24 (Lys) > 19 (Gly) > 30
(His), 33 (Arg) (data not shown). Amides displayed better interca-
lation than ester counterparts as noted for lysine (24 vs 10) and
histidine (30 vs 16) analogues (data not shown). When comparing
the nature of the amino acid, the best intercalating profile was ob-
tained for lysine derivative 24 which achieved the same effect at
From both experiments (DTm and Kapp), DNA binding rank in the
order amine > amide > ester which follows the variation of per-
centages of ionization in the three series (Table 1), suggesting a
relationship between the two parameters as protonated forms
are more likely to provide DNA binding and stabilization. Com-
lower drug concentration (compare the 2
lM lanes). They also dis-
played the highest Tm and Kapp values in series II. In series III,
D
compound 37 displayed good intercalating profile comparable to
the best molecule in the amide series, compound 24.
pound 37 displayed the highest
DTm and Kapp values, indicating
both good affinity for DNA and duplex stabilization.
At this point, it is worth to mention that none of the compounds
was found to function as a topoisomerase I poison. Molecules were
tested in a topoisomerase I assay using ethidium bromide contain-
ing gels. In contrast to camptothecin, no topoisomerase I-induced
strand breaks was observed with any of the compounds.
3.1.3. Mode of binding to DNA
Different binding modes (i.e. intercalation and/or groove bind-
ing) can account for the melting temperature evaluation. As
DTm

G. Ahn et al. / Bioorg. Med. Chem. 18 (2010) 8119–8133
8123
Table 1
As shown in Figure 4A, in series I, lysine ester 6 (n = 2) displayed
the highest activity. Increasing the spacing arm length totally sup-
pressed the activity (6 vs 9, 7 vs 10). We did not detect a DNA topo-
isomerase II inhibition with the D-enantiomer 8 compared to the 6
L-enantiomers and the 7 DL-racemic mixture at tested concentra-
a
b
Compd
n
Aminoacyl
D
Tm
Kapp
% ionizationc
DNd
21.9
0
nde
Etopf
-
g
Series I
tions. Interestingly, for compounds 6 and 7 inhibition at 50
was lower than at 20 M indicating possibly inhibition of topoiso-
merase II mediated DNA cleavage at 50 M. They act as topoiso-
merase II poisons at low concentrations (<20 M) and as
topoisomerase II suppressors at high concentrations (>50 M). This
trend has already been reported for indenoisoquinoline deriva-
tives.31 Compounds with a three methylene chain of the N-6 side
chain in series I (9, 10, and 11) had no effect on the inhibition of
topoisomerase II. All other compounds in series I had no effect
on the inhibition of topoisomerase II (data not shown).
In series II, no inhibition was detected for lysine and histidine
derivatives(data not shown). In contrast, as shown in Figure4B, argi-
nine derivative 32 (n = 2) displayed high inhibition, comparable to
etoposide. Increasing the spacing arm length suppressed the activity
when compared to its counterpart 33 (n = 3). In series III, glycine
derivative 34 (n = 2, Fig. 4B) displayed high inhibition comparable
to 32. Increasing the spacing arm length decreased the inhibition
when compared to derivative 35 (n = 2). Replacing glycine by lysine
totally suppressed the activity (34–35 vs 36–37) (data not shown).
lM
4
5
6
2
3
2
Gly
Gly
3
5.5
9.1
0.68 0.05
0.66 0.04
0.89 0.07
62.4
66.0
100
l
l
L
-Lys
DL-Lys
-Lys
-Lys
DL-Lys
-Lys
-His
DL-His
-His
-His
DL-His
-His
l
7
2
2
3
3
3
2
2
2
3
3
3
9
0.87 0.06
0.90 0.09
8.36 0.49
8.09 0.39
8.27 0.52
0.58 0.07
0.25 0.03
0.22 0.03
0.60 0.09
0.69 0.07
0.72 0.06
100
100
100
100
100
61.3
61.3
61.3
65.1
65.1
65.1
l
8
10.4
15.2
12.6
15.2
3.0
D
9
L
10
11
12
13
14
15
16
17
D
L
2.1
2.2
D
2.3
L
3.5
4.7
D
Series II
18
19
2
3
2
Gly
Gly
5
9.8
13.2
0.71 0.05
0.95 0.08
5.86 0.18
85.5
86.8
100
20
L
-Lys
DL-Lys
-Lys
-Lys
DL-Lys
-Lys
-His
DL-His
-His
-His
DL-His
-His
21
22
23
24
25
26
27
28
29
30
31
32
33
2
2
3
3
3
2
2
2
3
3
3
2
3
13.1
12.6
23.8
20.9
18.4
3.4
6.03 0.29
5.98 0.22
10.24 0.32
10.59 0.41
11.09 0.45
0.65 0.06
0.62 0.03
0.67 0.05
0.72 0.08
0.73 0.05
0.69 0.08
7.29 0.44
10.23 0.55
100
100
100
100
100
86.7
86.7
86.7
88
D
L
3.3. In vitro antiproliferative activity
D
The antiproliferative activities of these 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 an insight into the involvement of topoisomerase II inhi-
bition in the cytotoxicity of the molecules, their antiproliferative
activity was assessed using the HL60/MX2 cell line resistant to
mitoxantrone, which displays altered catalytic activity and re-
duced levels of topoisomerase II32 (the values for mitoxantrone
and etoposide are given as references). All compounds were tested
L
4.5
7.3
D
7.9
L
4.9
88
5.5
88
D
13.6
21.0
100
100
L
-Arg
-Arg
L
Series III
34
35
2
3
2
Gly
Gly
11.4
14.9
23.8
6.99 0.38
12.35 0.58
13.88 0.59
99.8
99.9
100
except for
line, all compounds displayed IC50 values ranging from
1.2 0.145 (19 and 34) to 21.3 0.4 (10).
Compounds from series I displayed moderate to weak cytotox-
icities (IC50 values between 4.95 0.05 M and 21.3 0.4 M).
D-lysine derivatives. When evaluated on the HL60 cell
36
DL-Lys
DL-Lys
l
M
lM
37
3
26.7
16.97 0.74
100
Tm(Tmdrug DNA complex ꢁ TDmNA alone). Drug/CT
a
Variation in melting temperature
DNA ratio = 0.25; mean values correspond to n = 3 with deviation standard 10%.
D
l
l
Glycine derivative which exert a topoisomerase II inhibition (4,
n = 2) displayed the best activity. For intercalative compounds (9
and 10), the IC50 values were higher. Lysine derivatives which exert
an inhibition of topoisomerase II or a DNA intercalation were less
active. We hypothesize that low value obtained could be attribut-
able at least partially to the instability of the ester function.
In contrast, in series II, compounds displayed better cytotoxici-
b
Apparent binding constant measured by fluorescence using [BET]/[DNA] = 1.26,
Kapp in 106 Mꢁ1
.
c
Calculated percentage of ionized form at pH 7.4.
Daunorubicin (DTm at ratio 1).
Interferences of fluorescence spectra of DNA and BET were observed.
Etoposide.
28% of displacement of BET for etoposide at 10 lM.
d
e
f
g
ties, IC50 values ranging from 1.2 0.145 lM to 5.8 0.15 lM. Dif-
ferences in IC50 values between racemates and enantiomers were
not significative at micromolar range. Lysine amides 20–24 dis-
played IC50 values fourfold lower than for their ester counterparts
6–10. One of the highest cytotoxicity was obtained for arginine
derivative 33. This compound also displayed the strongest DNA
3.2. Topoisomerase II inhibition
A conventional DNA relaxation assay was used to assess the ef-
fects of the compounds from the three series on the catalytic activ-
ity of human topoisomerase II. In these experiments supercoiled
plasmid DNA was treated with topoisomerase II in the presence
interaction (DTm and Kapp) in this series and this could account,
at least partially for the good cytotoxicity obtained. In contrast, ly-
sine derivative 23 which displayed similar DNA interaction param-
eters to compound 33 was four times less cytotoxic, indicating that
the aminoacyl plays a role in the cytotoxicity and other parameters
could be involved such as membrane uptake or additional cellular
targets. DNA interaction was found to correlate neither with topo-
isomerase poisoning nor with the antiproliferative activity. The
highest cytotoxicities were obtained for compounds 19, 33 and
34. For compound 33 strong DNA interaction could account for
the high cytotoxicity obtained. Compound 34 displayed lower
of two concentrations of the tested drug (20 lM and 50 lM) and
the DNA relaxation products were then resolved by gel electropho-
resis on agarose gels. The reference drug etoposide produces a
marked level of DNA double stranded breaks, corresponding to lin-
ear DNA (lin, Fig. 4). We essentially compared results in function of
the length of the N-6 side chain (two or three) methylene chains in
series I (6–8 vs 9–11), II (32 vs 33), and III (34 vs 35). We also com-
pared results for lysine (6–8 vs 9–11) or arginine (32 vs 33) or gly-
cine (34 vs 35) derivatives.

8124
G. Ahn et al. / Bioorg. Med. Chem. 18 (2010) 8119–8133
Figure 3. (A) Effects of lysine derivatives from series I (9, 10, and 11), and series III (37) with n = 3 on the relaxation of plasmid DNA by human topoisomerase I. (B) Effect of
lysine (23, 24, 25) and arginine (33) compounds from series II. Native supercoiled pUC19 (130 ng, lane DNA) was incubated with 4 units of topoisomerase I in the absence
(lane Topo) or presence of tested compound at the indicated concentration (from 1 to 20 lM). 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.
Figure 4. (A) Effect of lysine derivatives from series I (6–11) compared with reference compounds (POM02 and POM03). (B) Effect of arginine derivatives from series II (32,
33) and glycine derivatives from series III (34, 35) on the relaxation of plasmid DNA by human topoisomerase II. Native supercoiled pUC19 (130 ng, lane DNA) was incubated
with 4 units topoisomerase II in the absence (lane Topo) or presence of tested compound at the indicated concentration (A: 20 and 50
lM, B: from 10 to 50
lM and 20 lM for
32, 33). Etoposide was used at the same concentrations. DNA samples were separated by electrophoresis on a 1% agarose gel containing 1
lg/mL ethidium bromide. Gels were
photographed under UV light. Nck: nicked; Sc: supercoiled; Lin: linear; Rel: relaxed.
DNA interaction but it is a good topoisomerase II inhibitor. Distri-
bution coefficient for compound 19 (c log D = 0.1) was higher
than for compounds 34 (c log D = ꢁ1.0) and 33 (c log D = ꢁ2.6)
indicating better membrane uptake which could at least partially
account for the good cytotoxicity obtained despite weaker DNA
interaction and lack of topoisomerase II inhibition.

G. Ahn et al. / Bioorg. Med. Chem. 18 (2010) 8119–8133
8125
Table 2
intercalant profiles at concentrations above 5
l
M, and two of them
(32 and 34) are good topoisomerase II poisons.
As noted in our previous study,18 compounds bearing a spacing
linker with three methylene units (n = 3) were more favorable to
DNA interaction than the ones with a spacing linker with two
methylene units (n = 2). Strong affinity to DNA is detrimental to
topoisomerase II inhibition as the best topoisomerase II poison
(32) displayed only moderate interaction to DNA and the best
DNA ligand (37) displayed no topoisomerase II inhibition. No sig-
nificant differences were observed for enantioselectivity of these
compounds in all of the experiments.
Compd
n
Aminoacyl
c log Db
IC50
(l
M)a
RRIc
HL60
HL60/MX2
MXd
0.063 0.020
0.486 0.190
1.51 0.08
48.5 1.5
24
100
Etope
Series I
4
5
6
2
3
2
Gly
Gly
1.6
1.6
1.2
4.95 0.05
ndf
12.3 0.2
27.5 0.85
nd
5.56
nd
2.60
32
3
L
-Lys
DL-Lys
-Lys
-Lys
DL-Lys
-Lys
-His
DL-His
-His
-His
DL-His
-His
7
2
2
3
3
3
2
2
2
3
3
3
1.2
1.2
1.1
1.1
1.1
1.2
1.2
1.2
1.4
1.4
1.4
14 0.5
nd
28 0.01
2.00
nd
8
nd
D
This present study revealed two new topoisomerase II poison,
arginine derivative 32 and glycine derivative 34 displaying potency
comparable to the reference topoisomerase II poison etoposide and
cytotoxicity in the micromolar range. Further studies about the role
of guanidine group in topoisomerase II inhibition are in progress.
No obvious relationship was found between cytotoxicity, DNA
interaction and topoisomerase II inhibition. This could be the result
of several contributions such as differences in cellular uptake and/
or involvement of targets and mechanisms different from topoiso-
merase II-mediated DNA cleavage. Uptake studies as well as stabil-
ity of the cleavage complex will be conducted on our compounds in
attempt to rationalize the results obtained.
9
14.55 0.5
21.3 0.4
nd
24.5 1.05
22.5 0.5
1.68
1.06
nd
L
10
11
12
13
14
15
16
17
nd
D
7.8 0.2
9.2 0.4
9.2 0.75
15.7 0.2
8.9 0.85
10.5 0.85
23
21.0
2
1
2.95
2.28
1.79
1.44
1.80
1.45
L
16.5 0.95
22.6 1.05
16.0 0.95
15.25 1.05
D
L
D
Series II
18
19
2
3
2
Gly
Gly
0.0
0.1
2.7
2.5 0.3
1.2 0.145
1.85 0.05
3.0 0.4
1.6 0.25
2.55 0.15
1.20
1.33
1.38
20
L
-Lys
DL-Lys
-Lys
-Lys
DL-Lys
-Lys
-His
DL-His
21
22
23
24
25
26
27
28
29
30
31
32
33
2
2
3
3
3
2
2
2
3
3
3
2
3
2.7
3
0.1
3.8 0.2
nd
1.27
nd
5. Experimental
5.1. General
2.7
nd
3.2 0.2
5.25 0.35
nd
D
2.6
4.95 0.15
7.25 0.55
nd
1.55
1.38
nd
L
2.6
2.6
D
All starting materials were purchased from Acros Organics, Alfa
Aesar, Sigma–Aldrich and Bachem. Common organic solvents such
as petroleum spirit, ethyl acetate, dichloromethane, chloroform,
acetone, and toluene were purchased from VWR. Tetrahydrofuran
(THF) and diethyl ether (Et2O) were pre-dried with anhydrous
Na2SO4, then distilled over sodium benzophenone ketyl under Ar
and stored over sodium wire before use. When required, an inert
atmosphere was obtained by using dry Ar. 1H NMR spectra were
recorded at 300 MHz and 13C NMR spectra at 75 MHz on a BRUKER
AM300WB spectrometer. Chemical shifts (ppm) were reported
with tetramethylsilane (SiMe4) as internal reference. Analytical
thin layer chromatography (TLC) was carried out on aluminum
plates pre-coated with Merck Kieselgel 60 GF254 and products visu-
alized by UV light. For flash column chromatography, Merck silica
0.05
0.05
0.05
0.07
0.07
0.07
2.7
3.6 0.4
5.8 0.15
4.6 0.05
3.0 0.15
2.45 0.35
2.8 0.05
2.0 0.15
1.3 0.15
4.0 0.5
14.7 0.7
17.5 0.75
2.8 0.05
4.65 0.55
4.9 0.15
3.1 0.3
2.3 0.25
1.11
2.53
3.80
0.93
1.90
1.75
1.55
1.77
L
L
-His
-His
L
DL-His
-His
D
L
-Arg
-Arg
2.6
L
Series
34
35
2
3
2
Gly
Gly
1.0
1.0
2.5
1.2 0.1
2.95 0.15
7.6 0.4
2
0.01
1.67
1.64
0.72
4.85 0.25
5.5 0.35
36
DL-Lys
DL-Lys
37
3
2.4
2.4 0.1
2.8 0.3
1.17
a
The cytotoxicity IC50 values are the concentrations corresponding to 50%
gel 60 (40–63 lm) was used. Melting points were determined by
growth inhibition.
Calculated log D.33
b
using an electrothermal REICHERT THERMOPAN and are uncor-
rected. Optical rotation values were given by Perkin Elmer Model
343 Polarimeter emitting lights with a Na/HaI lamp at a wave-
length of 589 nm, at 20.0 °C. High-resolution mass spectra (HRMS)
were obtained on an Orbitrap Exactive Thermo Scientific mass
spectrometer by positive mode electrospray ionization.
c
(MX-sensitive)
Relative resistance index: IC50(MX-resistant)/IC50
.
d
Mitoxantrone.
Etoposide.
e
f
Not determined.
The best in vitro topoisomerase II poisons 32 and 34 compara-
ble to etoposide displayed weak resistance index on MX2 which
could be explained by their binding to the enzyme at a different
site from mitoxantrone or specific kinetics.
5.2. Benz[d]indeno[1,2-b]pyran-5,11-dione (38)
To a suspension of 2-carboxybenzaldehyde (3.00 g, 20.0 mmol,
1.0 equiv) and phthalide (2.68 g, 20.0 mmol, 1.0 equiv) in EtOAc
(10 mL) a solution of sodium methoxide (25 mL of 3.2 M methano-
lic solution) was added at 65 °C. The resulting mixture was main-
tained at 65 °C for 18 h under stirring. The mixture was
subsequently diluted at 0 °C with distilled water (50 mL), washed
with Et2O (3 ꢂ 50 mL) and acidified with HCl 12 M until pH 1.
The resulting yellow precipitate was filtered, dried in vacuo and
was diluted with acetic anhydride (60 mL). The mixture was stirred
at 100 °C for 3 h and cooled to room temperature. The resulting so-
lid was filtrated and washed with Et2O to give the desired product
as bright orange solid. Yield: 2.75 g, 55%. mp: 257–259 °C. 1H NMR
(CDCl3, d ppm, J Hz): 8.40 (d, J = 7.9 Hz, 1H), 8.31 (d, J = 8.0 Hz, 1H),
4. Conclusion
In this study, amino-acid moieties especially with protonable
side-chains were introduced on indenoisoquinolin-5,11-dione
skeleton. Variations were introduced in the length of the spacing
arm, connecting function, amino-acid structure, and chirality to
provide structure–activity relationships.
Lysine and arginine derivatives displayed high DNA interaction
consistent with the establishment of additional electrostatic
and/or hydrogen interactions. The compounds displayed good

8126
G. Ahn et al. / Bioorg. Med. Chem. 18 (2010) 8119–8133
a
e
7.83 (dd, J = 8.9 Hz, J = 1.2 Hz, 1H), 7.62 (d, J = 6.9 Hz, 1H), 7.57–
7.49 (m, 4H).
5.4.3. 6-(N ,N -Di-Boc-lysyl-oxyethyl)-6H-indeno[1,2-c]iso-
quinolin-5,11-diones 6a–8a
Compounds 6a–8a were obtained from compound 39 (320 mg)
a
e
5.3. General procedure for the synthesis of 6-hydroxyalkyl-6H-
indeno[1,2-c]isoquinolin-5,11-diones 39 and 40
and appropriate N ,N -di-Boc-lysine (419 mg) as bright red amor-
phous solids. 1H NMR (CDCl3, d ppm, J Hz): 8.74 (d, J = 8.0 Hz, 1H),
8.35 (d, J = 7.5 Hz, 1H), 7.79–7.72 (m, 2H), 7.66 (dd, J = 7.0 Hz,
J = 1.3 Hz, 1H), 7.53–7.41 (m, 3H), 5.01 (d, J = 7.6 Hz, 1H), 4.92–
4.85 (m, 2H), 4.64–4.58 (m, 3H), 4.14 (q, J = 7.1 Hz, 1H), 3.00 (t,
J = 6.0 Hz, 2H), 1.65–1.58 (m, 2H), 1.35–1.21 (m, 4H), 1.51 (s, 9H),
1.41 (s, 9H).
2-Aminoethanol
or
3-aminopropan-1-ol
(40.0 mmol,
10.0 equiv) were added to a solution of 38 (1.00 g, 4.0 mmol,
1.0 equiv) in CHCl3 (12 mL). After stirring at room temperature
for 48 h, the reaction mixture was diluted in CHCl3 (100 mL) and
subsequently washed with distilled water (2 ꢂ 50 mL), HCl 0.1 M
(1 ꢂ 25 mL), and brine (1 ꢂ 50 mL), dried over Na2SO4, filtered
and concentrated in vacuo to afford dark-orange needles. The
crude product was used in the next step without further
purification.
a
e
5.4.3.1. 6-(N ,N -Di-Boc-
olin-5,11-dione (6a). Compound 6a was obtained from N ,N -di-Boc-
-lysine. Yield: 436 mg, 64%.
L-lysyl-oxyethyl)-6H-indeno[1,2-c]isoquin-
a
e
L
a
e
5.4.3.2. 6-(N ,N -Di-Boc-DL-lysyl-oxyethyl)-6H-indeno[1,2-c]iso-
5.3.1. 6-(2-Hydroxyethyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione (39)
quinolin-5,11-dione (7a). Compound 7a was obtained from
a
e
N ,N -di-Boc-DL-lysine. Yield: 266 mg, 39%.
Compound 39 was prepared from 2-aminoethanol (2.4 mL).
Yield: 1.04 g, 89%. mp: 210–212 °C. 1H NMR (CDCl3, d ppm, J Hz):
8.74 (d, J = 8.0 Hz, 1H), 8.36 (d, J = 8.2 Hz, 1H), 7.77 (dd, J = 6.0 Hz,
J = 1,3 Hz, 1H), 7.68–7.60 (m, 2H), 7.53–7.40 (m, 3H), 4.78 (t,
J = 5.9 Hz, 2H), 4.20 (q, J = 5.8 Hz, 2H), 2.45 (t, J = 5.3 Hz, 1H).
a
e
5.4.3.3. 6-(N ,N -Di-Boc-
D-lysyl-oxyethyl)-6H-indeno[1,2-c]iso-
quinolin-5,11-dione (8a). Compound 8a was obtained from
a
e
N ,N -di-Boc-
D-lysine. Yield: 559 mg, 82%.
a
e
5.3.2. 6-(3-Hydroxypropyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione (40)
5.4.4. 6-(N ,N -Di-Boc-lysyl-oxypropyl)-6H-indeno[1,2-c]iso-
quinolin-5,11-diones 9a–11a
Compound 40 was prepared from 3-aminopropan-1-ol (3.0 mL).
Yield: 1.16 g, 95%. mp: 191–192 °C. 1H NMR (CDCl3, d ppm, J Hz):
8.47 (d, J = 8.0 Hz, 1H), 8.15 (d, J = 7.5 Hz, 1H), 7.79–7.65 (m, 2H),
7.54–7.42 (m, 4H), 4.75 (t, J = 6.6 Hz, 2H), 3.72 (t, J = 5,0 Hz, 2H),
3.26 (t, J = 6.3 Hz, 1H), 2.19–2.15 (m, 2H).
Compounds 9a–11a were obtained from compound 40
a
e
(336 mg) and appropriate N ,N -di-Boc-lysine (419 mg) as bright
red amorphous solids. 1H NMR (CDCl3, d ppm, J Hz): 8.73 (d,
J = 7.9 Hz, 1H), 8.35 (d, J = 7.4 Hz, 1H), 7.76 (td, J = 7.5 Hz,
J = 1.3 Hz, 1H), 7.68–7.62 (m, 2H), 7.56–7.41 (m, 3H), 5.19 (d,
J = 7.4 Hz, 1H), 4.76–4.58 (m, 3H), 4.47–4.32 (m, 3H), 3.17–3.11
(m, 2H), 2.36–2.27 (m, 2H), 1.91–1.73 (m, 2H), 1.56–1.50 (m,
2H), 1.48–1.43 (m, 20H).
5.4. General procedure for the synthesis of 6-(N-Boc-aminoacyl-
oxyalkyl)-6H-indeno[1,2-c]isoquinolin-5,11-diones 4a–17a
To a suspension of 39 or 40 (1.1 mmol, 1.0 equiv) in CH2Cl2
(8 mL) DMAP (1.1 mmol, 1.0 equiv) and the N-Boc-protected amino
acid (1.2 mmol, 1.1 equiv) were added. The resulting mixture was
cooled to 0 °C and EDCI (1.3 mmol, 1.2 equiv) was subsequently
added. The mixture was stirred at 0 °C for 2 h; then at room tem-
perature for 18 h. The solvent was evaporated under reduced pres-
sure and the residue was taken up in EtOAc-distilled water mixture
(2:1, 60 mL). The organic layer was separated, washed with satu-
rated NaHCO3 (2 ꢂ 15 mL) and distilled water (2 ꢂ 15 mL), dried
on Na2SO4, filtered and concentrated in vacuo. Purification by flash
column chromatography on silica gel (CH2Cl2/EtOAc, 8:2, as eluent)
provided the desired products.
a
e
5.4.4.1. 6-(N ,N -Di-Boc-
L-lysyl-oxypropyl)-6H-indeno[1,2-c]iso-
quinolin-5,11-dione (9a). Compound 9a was obtained from
N ,N -di-Boc-
a
e
L
-lysine. Yield: 502 mg, 72%.
a
e
5.4.4.2. 6-(N ,N -Di-Boc-DL-lysyl-oxypropyl)-6H-indeno[1,2-c]-iso-
quinoline-5,11-dione (10a). Compound 10a was provided from
N ,N -di-Boc-DL-lysine. Yield: 411 mg, 59%.
a
e
a
e
5.4.4.3. 6-(N ,N -Di-Boc-
D-lysyl-oxypropyl)-6H-indeno[1,2-c]-iso-
quinolin-5,11-dione (11a). Compound 11a was obtained from
N ,N -di-Boc-D-lysine. Yield: 181 mg, 26%.
a
e
a
p
5.4.5. 6-(N ,N -Di-Boc-histidyl-oxyethyl)-6H-indeno[1,2-c]iso-
quinolin-5,11-diones 12a–14a
5.4.1. 6-(N-Boc-glycyl-oxyethyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione (4a)
Compounds 12a–14a were obtained from compound 39
Compound 4a was obtained as a red amorphous solid from 39
a
p
a
(320 mg) and N -Boc-glycine (212 mg). Yield: 301 mg, 61%. 1H
(320 mg) and appropriate N ,N -di-Boc-histidine (430 mg) as
bright red amorphous solids. 1H NMR (CDCl3, d ppm, J Hz): 8.70
(d, J = 8.0 Hz, 1H), 8.34 (d, J = 7.4 Hz, 1H), 7.80 (d, J = 6.7 Hz, 1H),
7.79 (s, 1H), 7.74 (td, J = 7.7 Hz, J = 1.3 Hz, 1H), 7.64 (dd,
J = 6.5 Hz, J = 1.2 Hz, 1H), 7.50–7.38 (m, 3H), 7.04 (s, 1H), 5.59 (d,
J = 8.0 Hz, 1H), 4.85 (t, J = 4.2 Hz, 2H), 4.77–4.45 (m, 3H), 2.99–
2.90 (m, 2H), 1.60 (s, 9H), 1.42 (s, 9H).
NMR (CDCl3, d ppm, J Hz): 8.75 (d, J = 8.0 Hz, 1H), 8.35 (d,
J = 7.6 Hz, 1H), 7.76 (dd, J = 7.7 Hz, J = 1.2 Hz, 1H), 7.69–7.66 (m,
2H), 7.53–7.40 (m, 3H), 4.93 (s, 1H), 4.85 (t, J = 6.0 Hz, 2H), 4.65
(t, J = 6.0 Hz, 2H), 3.81 (d, J = 5.7 Hz, 2H), 1.42 (s, 9H).
5.4.2. 6-(N-Boc-glycyl-oxypropyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione (5a)
a
p
5.4.5.1. 6-(N ,N -Di-Boc-
L-histidyl-oxyethyl)-6H-indeno[1,2-c]-iso-
Compound 5a was obtained as red amorphous solid from 40
a
(336 mg) and N -Boc-glycine (212 mg). Yield: 295 mg, 58%. 1H
quinolin-5,11-dione (12a). Compound 12a was obtained from
a
p
N ,N -di-Boc-L-histidine. Yield: 567 mg, 82%.
NMR (CDCl3, d ppm, J Hz): 8.73 (d, J = 8.2 Hz, 1H), 8.35 (d,
J = 8.0 Hz, 1H), 7.75 (dd, J = 7.5 Hz, J = 1.2 Hz, 1H), 7.67–7.61 (m,
2H), 7.52–7.35 (m, 3H), 5.01 (s, 1H), 4.66 (t, J = 7.6 Hz, 2H), 4.41
(t, J = 6.0 Hz, 2H), 4.00 (d, J = 5.6 Hz, 2H), 2.36–2.19 (m, 2H), 1.43
(s, 9H).
a
p
5.4.5.2. 6-(N ,N -Di-Boc-DL-histidyl-oxyethyl)-6H-indeno[1,2-c]-
isoquinolin-5,11-dione (13a). Compound 13a was obtained from
a
p
N ,N -di-Boc-DL-histidine. Yield: 436 mg, 63%.

G. Ahn et al. / Bioorg. Med. Chem. 18 (2010) 8119–8133
8127
a
p
5.4.5.3. 6-(N ,N -Di-Boc-
D
-histidyl-oxyethyl)-6H-indeno[1,2-c]iso-
2H), 1.05–1.59 (m, 6H). 13C NMR (DMSO-d6, d ppm): 190.35,
169.77, 162.96, 156.70, 136.88, 134.58, 134.53, 134.30, 132.13,
131.81, 128.47, 127.72, 124.47 (br, 2C), 123.06, 123.01, 107.65,
63.56, 52.23, 43.14, 38.48, 29.33, 26.60, 21.71.
quinolin-5,11-dione (14a). Compound 14a was obtained from
N ,N -di-Boc-D-histidine. Yield: 553 mg, 80%.
a
p
a
p
5.4.6. 6-(N ,N -Di-Boc-histidyl-oxypropyl)-6H-indeno[1,2-
c]isoquinolin-5,11-diones 15a–17a
5.5.3.1. 6-(L-Lysyl-oxyethyl)-6H-indeno[1,2-c]isoquinolin-5,11-
Compounds 15a–17a were obtained from compound 40
dione dihydrochloride (6). Yield: 51 mg, 52%. ½a D20
ꢃ
: +10.1 (c 0.2,
a
p
(336 mg) and appropriate N ,N -di-Boc-histidine (430 mg) as
bright red amorphous solids. 1H NMR (CDCl3, d ppm, J Hz): 8.71
(d, J = 7.9 Hz, 1H), 8.33 (d, J = 7.4 Hz, 1H), 7.93 (s, 1H), 7.73 (td,
J = 7.1 Hz, J = 1.3 Hz, 1H), 7.67–7.61 (m, 2H), 7.55–7.38 (m, 3H),
7.19 (s, 1H), 5.87 (d, J = 8.0 Hz, 1H), 4.68–4.58 (m, 3H), 4.45–4.30
(m, 2H), 3.18–3.05 (m, 2H), 2.33–2.24 (m, 2H), 1.56 (s, 9H), 1.42
(s, 9H).
MeOH). HRMS calcd for C24H26N3O4 [M+H]+ 420.1918, found
420.1916.
5.5.3.2. 6-(DL-Lysyl-oxyethyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione dihydrochloride (7). Yield: 70 mg, 71%. HRMS calcd for
C
24H26N3O4 [M+H]+ 420.1918, found 420.1920.
5.5.3.3. 6-(D-Lysyl-oxyethyl)-6H-indeno[1,2-c]isoquinoline-5,11-
dione dihydrochloride (8). Yield: 69 mg, 70%. ½a D20
ꢃ : ꢁ6.7 (c 0.2,
a
p
5.4.6.1. 6-(N ,N -Di-Boc-
quinolin-5,11-dione (15a). Compound 15a was obtained from
L
-histidyl-oxypropyl)-6H-indeno[1,2-c]iso-
MeOH). HRMS calcd for C24H26N3O4 [M+H]+ 420.1918, found
420.1917.
a
p
N ,N -di-Boc-L-histidine. Yield: 502 mg, 71%.
a
p
5.4.6.2. 6-(N ,N -Di-Boc-DL-histidyl-oxypropyl)-6H-indeno[1,2-
5.5.4. 6-(Lysyl-oxypropyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione dihydrochlorides 9–11
c]isoquinolin-5,11-dione (16a). Compound 16a was obtained
from N ,N -di-Boc-DL-histidine. Yield: 198 mg, 28%.
a
p
Compounds 9–11 were obtained from compounds 9a–11a
(127 mg) as red amorphous solids. 1H NMR (DMSO-d6, d ppm, J
Hz): 8.69 (br s, 3H), 8.52 (d, J = 8.0 Hz, 1H), 8.19–8.13 (m, 4H),
7.82–7.77 (m, 2H), 7.62–7.47 (m, 4H), 4.59 (br s, 2H), 4.38–4.32
(m, 2H), 4.00 (br s, 1H), 2.76 (br s, 2H), 2.19 (t, J = 6.1 Hz, 2H), 1.88
(d, J = 6.7 Hz, 2H), 1.60–1.50 (m, 4H). 13C NMR (DMSO-d6, d ppm):
190.32, 169.90, 162.98, 156.04, 136.22, 134.24, 134.00 (br, 2C),
131.68, 131.32, 128.05, 127.11, 123.69, 122.68, 122.63, 122.50,
107.04, 63.48, 51.64, 41.19, 38.10, 29.23, 27.96, 26.25, 21.21.
a
p
5.4.6.3. 6-(N ,N -Di-Boc-
D-histidyl-oxypropyl)-6H-indeno[1,2-
c]isoquinolin-5,11-dione (17a). Compound 17a was obtained
from N ,N -di-Boc-D-histidine. Yield: 318 mg, 45%.
a
p
5.5. General procedure for the syntheses of compounds 4–17
To a solution of 4a–17a (0.2 mmol) in CHCl3 (5 mL), HCl 5 M in
2-propanol (1 mL) was slowly added at 0 °C. The resulting mixture
was stirred at room temperature for 18 h. The precipitated product
was filtered, washed with CHCl3 and Et2O and dried in vacuo to af-
ford the desired products.
5.5.4.1. 6-(L-Lysyl-oxypropyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione dihydrochloride (9). Yield: 63 mg, 79%. ½a D20
ꢃ
: +11.1 (c 0.2,
MeOH). HRMS calcd for
433.2075.
C
25H28N3O4 [M+H]+ 433.2074, found
5.5.1. 6-(Glycyl-oxyethyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione hydrochloride (4)
5.5.4.2. 6-(DL-Lysyl-oxypropyl)-6H-indeno[1,2-c]isoquinolin-5,11-
Compound 4 was obtained from 4a (90 mg) as a red amorphous
solid. Yield: 48 mg, 62%. 1H NMR (DMSO-d6, d ppm, J Hz): 8.61–
8.58 (m, 1H), 8.37 (br s, 2H), 8.24–8.22 (m, 1H), 8.13 (br s, 1H),
7.91–7.83 (m, 2H), 7.61–7.52 (m, 4H), 4.81 (d, J = 5.3 Hz, 2H),
4.63 (br s, 2H), 3.59 (br s, 2H). 13C NMR (DMSO-d6, d ppm):
190.25, 168.00, 162.96, 156.56, 136.80, 134.56, 134.46, 134.15,
132.10, 131.68, 128.38, 127.54, 124.13, 123.02, 122.95 (broad,
2C), 107.67, 63.08, 43.24, 40.30. HRMS calcd for C20H17N2O4
[M+H]+ 349.1183, found 349.1180.
dione dihydrochloride (10). Yield: 64 mg, 80%. HRMS calcd for
C
25H28N3O4 [M+H]+ 433.2074, found 433.2070.
5.5.4.3. 6-(D-Lysyl-oxypropyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione dihydrochloride (11). Yield: 63 mg, 79%. ½a D20
ꢃ : ꢁ8.1 (c 0.2,
MeOH). HRMS calcd for C25H28N3O4 [M+H]+ 433.2074, found
433.2067.
5.5.5. 6-(Histidyl-oxyethyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione hydrochlorides 12–14
5.5.2. 6-(Glycyl-oxypropyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione hydrochloride (5)
Compounds 12–14 were obtained from 12a–14a (126 mg) as
dark red amorphous solids. 1H NMR (DMSO-d6, d ppm, J Hz):
9.06 (s, 1H), 8.73 (br s, 2H), 8.59 (d, J = 7.9 Hz, 1H), 8.22 (d,
J = 7.4 Hz, 1H), 7.92 (d, J = 7.5 Hz, 1H), 7.83 (td, J = 7.7 Hz,
J = 1.3 Hz, 1H), 7.60–7.47 (m, 5H), 7.43 (s, 1H), 4.83 (t, J = 5.4 Hz,
2H), 4.72–4.57 (m, 2H), 4.34 (t, J = 6.9 Hz, 1H), 3.25–3.07 (m, 2H).
13C NMR (DMSO-d6, d ppm): 190.48, 168.53, 163.17, 156.81,
136.96, 134.75, 134.69, 134.51, 134.37, 132.28, 131.77, 128.58,
127.73, 126.89, 124.33, 123.20, 123.16, 123.09, 118.56, 107.76,
63.81, 51.39, 43.04, 24.56.
Compound 5 was obtained from compound 5a (92 mg) as a red
amorphous solid. Yield: 41 mg, 52%. 1H NMR (DMSO-d6, d ppm, J
Hz): 8.54 (d, J = 8.2 Hz, 1H), 8.45 (br s, 2H), 8.19 (d, J = 7.9 Hz,
1H), 7.83–7.79 (m, 2H), 7.63–7.47 (m, 4H), 4.59 (t, J = 6.9 Hz, 2H),
4.35 (t, J = 6.2 Hz, 2H), 3.81 (d, J = 4.7 Hz, 2H), 2.19 (m, 2H). 13C
NMR (DMSO-d6, d ppm): 189.89, 167.63, 162.53, 156.12, 136.31,
134.31, 134.06, 133.98, 131.76, 131.29, 128.04, 127.13, 123.70,
122.74, 122.67, 122.55, 107.08, 63.29, 41.21, 39.73, 27.96. HRMS
calcd for C21H19N2O4 [M+H]+ 363.1339, found 363.1337.
5.5.5.1. 6-(L-Histidyl-oxyethyl)-6H-indeno[1,2-c]isoquinolin-5,11-
5.5.3. 6-(Lysyl-oxyethyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione dihydrochlorides 6–8
dione hydrochloride (12). Yield: 68 mg, 73%. ½a D20
ꢃ : ꢁ7.0 (c 0.2,
MeOH). HRMS calcd for
429.1558.
C
24H21N4O4 [M+H]+ 429.1557, found
Compounds 6–8 were obtained from compounds 6a–8a
(124 mg) as red amorphous solids. 1H NMR (DMSO-d6, d ppm, J
Hz): 8.57 (br d, J = 8.0 Hz, 4H), 8.22 (d, J = 8.0 Hz, 1H), 8.00 (br s,
3H), 7.95 (d, J = 7.5 Hz, 1H), 7.83 (t, J = 7.0 Hz, 1H), 7.62–7.50 (m,
4H), 4.85 (br s, 2H), 4.70–4.54 (m, 2H), 3.77 (br s, 1H), 2.62 (br s,
5.5.5.2. 6-(DL-Histidyl-oxyethyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione hydrochloride (13). Yield: 81 mg, 87%. HRMS calcd
for C24H21N4O4 [M+H]+ 429.1557, found 429.1556.

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G. Ahn et al. / Bioorg. Med. Chem. 18 (2010) 8119–8133
5.5.5.3. 6-(
D
-Histidyl-oxyethyl)-6H-indeno[1,2-c]isoquinolin-5,
reaction mixture was diluted with CHCl3 (125 mL), washed with
11-dione hydrochloride (14). Yield: 66 mg, 71%. ½a D20
ꢃ
: +7.6 (c
distilled water (3 ꢂ 50 mL) and brine (1 ꢂ 50 mL), dried over
MgSO4, filtered and concentrated under reduced pressure to afford
the desired product as an orange-red solid. The crude product was
used in the next step without further purification.
0.2, MeOH). HRMS calcd for C24H21N4O4 [M+H]+ 429.1557, found
429.1551.
5.5.6. 6-(Histidyl-oxypropyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione hydrochlorides 15–17
5.8.1. 6-(2-Boc-aminoethyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione (43a)
Compounds 15–17 were obtained from 15a–17a (129 mg) as
red amorphous solids. 1H NMR (DMSO-d6, d ppm, J Hz): 9.09 (s,
1H), 8.92 (br s, 2H), 8.50 (d, J = 7.9 Hz, 1H), 8.17 (d, J = 7.6 Hz,
1H), 7.76 (m, 2H), 7.57–7.48 (m, 4H), 4.60–4.48 (m, 3H), 4.34 (t,
J = 6.2 Hz, 2H), 3.50–3.36 (m, 2H), 2.19–2.10 (br t, J = 6.1 Hz, 2H).
13C NMR (DMSO-d6, d ppm): 189.71, 168.12, 162.38, 155.85,
136.06, 134.13, 134.06, 133.98, 133.88, 131.59, 131.20, 127.95,
126.99, 126.59, 123.62, 122.59, 122.53, 122.42, 118.16, 107.00,
64.28, 51.56, 41.58, 28.37, 25.59.
Compound 43a was prepared from compound 41 (1.35 g). mp:
243–245 °C. Yield: 2.07 g, 95%. 1H NMR (CDCl3, d ppm, J Hz): 8.75
(d, J = 8.0 Hz, 1H), 8.35 (d, J = 8.0 Hz, 1H), 7.96 (d, J = 7.4 Hz, 1H),
7.76 (td, J = 7.0 Hz, J = 1.3 Hz, 1H), 7.67–7.63 (m, 1H), 7.54–7.35
(m, 3H), 5.05 (br s, 1H), 4.69 (t, J = 6.5 Hz, 2H), 3.67 (t, J = 6.5 Hz,
2H), 1.41 (s, 9H).
5.8.2. 6-(3-Boc-aminopropyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione (44a)
5.5.6.1. 6-(
L
-Histidyl-oxypropyl)-6H-indeno[1,2-c]isoquinolin-
Compound 44a was prepared from compound 42(1.46 g). mp:
269–270 °C. Yield: 2.06 g, 91%. 1H NMR (CDCl3, d ppm, J Hz): 8.69
(d, J = 7.9 Hz, 1H), 8.33 (d, J = 8.0 Hz, 1H), 7.74 (t, J = 7.3 Hz, 1H),
7.63 (d, J = 6.7 Hz, 1H), 7.57–7.38 (m, 4H), 5.39 (s, 1H), 4.63 (t,
J = 6.9 Hz, 2H), 3.28 (t, J = 6.1 Hz, 2H), 2.10 (m, 2H), 1.46 (s, 9H).
5,11-dione hydrochloride (15). Yield: 57 mg, 60%. ½a D20
ꢃ
: +4.5 (c
0.2, MeOH). HRMS calcd for C25H23N4O4 [M+H]+ 443.1714, found
443.1713.
5.5.6.2. 6-(DL-Histidyl-oxypropyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione hydrochloride (16). Yield: 58 mg, 61%. HRMS calcd
for C25H23N4O4 [M+H]+ 443.1714, found 443.1712.
5.9. General procedure for the synthesis of 6-aminoalkyl-6H-
indeno[1,2-c]isoquinolin-5,11-dione hydrochlorides 43 and 44
5.5.6.3. 6-(
D
-Histidyl-oxypropyl)-6H-indeno[1,2-c]isoquinolin-
To a solution of compound 43a or 44a (2.5 mmol) in CHCl3
(125 mL) a solution of HCl 5 M in 2-propanol (10 mL) was added
dropwise. The resulting mixture was stirred at room temperature
for 18 h. The precipitated product was filtered, washed with CHCl3
and Et2O and dried in vacuo to afford the desired product as an or-
ange-red solid. The crude product was used in the next step with-
out further purification.
5,11-dione hydrochloride (17). Yield: 0.10 g, 70%. ½a D20
ꢃ : ꢁ4.0 (c
0.2, MeOH). HRMS calcd for C25H23N4O4 [M+H]+ 443.1714, found
443.1701.
5.6. 2-(Boc-amino)-ethylamine (41)
A solution of di-tert-butyl dicarbonate (7.27 g, 33.3 mmol,
1.0 equiv) in THF (30 mL) was added dropwise at 0 °C, over
30 min to a stirred solution of ethylenediamine (6.7 mL, 100 mmol,
3.0 equiv) dissolved in THF at room temperature for 18 h. The sol-
vent was removed under reduced pressure, and the residue taken
up in EtOAc-brine mixture (1:1, 100 mL). The organic phase was
washed with brine (2 ꢂ 15 mL), dried over Na2SO4, filtered, and
concentrated in vacuo to afford a colorless oil. The crude desired
product was used in the next step without further purification.
Yield: 4.62 g, 87%. 1H NMR (CDCl3, d ppm, J Hz): 5.01 (br s, 1H),
3.20–3.12 (m, 2H), 2.78 (t, J = 6.0 Hz, 2H), 1.43 (s, 9H), 1.31 (s, 2H).
5.9.1. 6-(2-Aminoethyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione hydrochloride (43)
Compound 43 was prepared from compound 43a (0.98 g).
Yield: 0.62 g, 76%. 1H NMR (DMSO-d6, d ppm, J Hz): 8.53 (d,
J = 7.9 Hz, 1H), 8.44 (br s, 3H), 8.19 (d, J = 7.6 Hz, 1H), 8.03 (d,
J = 7.3 Hz, 1H), 7.81 (td, J = 7.0 Hz, J = 1.2 Hz, 1H), 7.57–7.47 (m,
4H), 4.75 (t, J = 6.7 Hz, 2H), 3.24 (t, J = 6.0 Hz, 2H).
5.9.2. 6-(3-Aminopropyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione hydrochloride (44)
Compound 44 was prepared from compound 44a (1.00 g). Yield:
0.74 g, 87%. 1H NMR (DMSO-d6, d ppm, J Hz): 8.53 (d, J = 8.0 Hz,
1H), 8.19 (d, J = 8.0 Hz, 1H), 8.08 (br s, 3H), 7.83–7.78 (m, 2H),
7.60–7.47 (m, 4H), 4.54 (t, J = 6.9 Hz, 2H), 3.01–2.86 (m, 2H), 2.13
(m, 2H).
5.7. 3-(Boc-amino)-propylamine (42)
A solution of di-tert-butyl dicarbonate (2.18 g, 10.0 mmol,
1.0 equiv) in CHCl3 (20 mL) was added dropwise over 3 h to a stir-
red solution of 1,3-diaminepropane (4.1 mL, 50.0 mmol, 5.0 equiv)
dissolved in CHCl3 (200 mL). The reaction mixture was stirred at
room temperature for 18 h and filtered. The filtrate was subse-
quently concentrated in vacuo and the resulting oil was dissolved
in EtOAc (100 mL), washed with brine (3 ꢂ 30 mL), dried over
Na2SO4, filtered and concentrated in vacuo to afford a colorless
oil. The crude product was used in the next step without further
purification. Yield: 1.39 g, 80%. 1H NMR (CDCl3, d ppm, J Hz): 4.96
(br s, 1H), 3.29–3.18 (m, 2H), 2.78–2.74 (t, J = 6.6 Hz, 2H), 1.65–
1.56 (m, 2H), 1.44 (s, 9H).
5.10. General procedure for the synthesis of 6-(N-Boc-amino-
acyl-aminoalkyl)-6H-indeno[1,2-c]isoquinolin-5,11-diones
18a–33a
5.10.1. Procedure A
EDCI (0.86 mmol, 2.0 equiv), HOBt (0.86 mmol. 2.0 equiv) and
triethylamine (1.1 mmol, 2.5 equiv) were added successively to a
solution of the appropriate N-Boc-protected amino acid
(0.86 mmol, 2.0 equiv) in dry THF (5 mL) at room temperature.
The mixture was stirred for 15 min, then a solution 43 or 44
(0.43 mmol, 1.0 equiv) and TEA (0.64 mmol, 1.5 equiv) in dry THF
(6 mL) were added. The reaction mixture was stirred at room tem-
perature for 18 h. The solvent was evaporated and the residue was
diluted with CHCl3 (50 mL). The organic layer was washed with HCl
0.1 M (2 ꢂ 15 mL), distilled water (2 ꢂ 15 mL), NaHCO3 0.5 M
(2 ꢂ 15 mL), brine (2 ꢂ 15 mL), dried over Na2SO4 and concentrated
5.8. General procedure for the synthesis of 6-(Boc-aminoalkyl)-
6H-indeno[1,2-c]isoquinolin-5,11-diones 43a, 44a
To a suspension of 38 (5.6 mmol, 1.0 equiv, 1.39 g) in CHCl3
(28 mL), appropriate Boc-aminoalkylamine (8.4 mmol, 1.5 equiv)
was added. After stirring at room temperature for 72 h, the

G. Ahn et al. / Bioorg. Med. Chem. 18 (2010) 8119–8133
8129
in vacuo. Purification by flash column chromatography on silica gel
(CH2Cl2/EtOAc, 8:2, as eluent) furnished the desired products.
ppm, J Hz): 8.73 (d, J = 8.0 Hz, 1H), 8.35 (d, J = 7.4 Hz, 1H), 7.76
(td, J = 7.8 Hz, J = 1.2 Hz, 1H), 7.65 (dd, J = 6.7 Hz, J = 1.3 Hz, 1H),
7.55–7.40 (m, 4H), 7.21 (s, 1H), 5.25 (s, 1H), 4.72–4.53 (m, 3H),
4.17–4.10 (m, 1H), 3.49–3.21 (m, 2H), 3.14 (t, J = 6.3 Hz, 2H),
2.17–2.10 (m, 2H), 1.76–1.65 (m, 2H), 1.57–1.50 (m, 2H), 1.47–
1.44 (m, 20H).
5.10.2. Procedure B
Appropriate N-Boc-protected amino acid (0.64 mmol,
1.5 equiv), HBTU (0.86 mmol, 2.0 equiv), HOBt (0.86 mmol
2.0 equiv), and N,N-diisopropylethylamine (2.15 mmol, 5.0 equiv)
were added successively to a solution of compound 43 or 44
(0.43 mmol, 1.0 equiv) in CH2Cl2 (3 mL). The resulting mixture
was stirred at room temperature for 4 h, diluted with CH2Cl2
(50 mL) and washed with aq NaHCO3 1.0 M solution (2 ꢂ 15 mL).
The organic layer was separated and dried over Na2SO4 and con-
centrated in vacuo. Purification by flash column chromatography
on silica gel (CH2Cl2/EtOAc, 8:2, as eluent) yielded the desired
products.
a
e
5.10.6.1. 6-(N ,N -Di-Boc-
L-lysyl-aminopropyl)-6H-indeno[1,2-
c]isoquinolin-5,11-dione (23a). Compound 23a was obtained
a
e
from N ,N -di-Boc-L-lysine. Yield: 117 mg, 43%.
a
e
5.10.6.2. 6-(N ,N -Di-Boc-DL-lysyl-aminopropyl)-6H-indeno[1,2-
c]isoquinolin-5,11-dione (24a). Compound 24a was obtained
a
e
from N ,N -di-Boc-DL-lysine.Yield: 162 mg, 60%.
a
e
5.10.6.3. 6-(N ,N -Di-Boc-
D-lysyl-aminopropyl)-6H-indeno[1,2-
5.10.3. 6-(N-Boc-glycyl-aminoethyl)-6H-indeno[1,2-
c]isoquinolin-5,11-dione (18a)
c]isoquinolin-5,11-dione (25a). Compound 25a was obtained
from N ,N -di-Boc-D-lysine. Yield: 202 mg, 75%.
a
e
a
Compound 18a was obtained from 43 (141 mg) and N -Boc-gly-
a
p
cine (151 mg) according to procedure A as a red amorphous solid.
Yield: 129 mg, 67%. 1H NMR (CDCl3, d ppm, J Hz): 8.74 (d, J = 7.9 Hz,
1H), 8.35 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 7.6 Hz, 1H), 7.77 (td,
J = 7.7 Hz, J = 1.3 Hz, 1H), 7.67–7.63 (m, 1H), 7.57–7.40 (m, 3H),
6.91 (t, J = 6.4 Hz, 1H), 4.71 (t, J = 6.7 Hz, 2H), 3.83–3.78 (m, 4H),
1.47 (s, 9H).
5.10.7. 6-(N ,N -Di-Boc-histidyl-aminoethyl)-6H-indeno[1,2-
c]isoquinolin-5,11-diones 26a–28a
Compounds 26a–28a were obtained from compound 43
a
p
(141 mg) and appropriate N ,N -di-Boc-histidine according to
either procedure A or B as red amorphous solids. 1H NMR (CDCl3,
d ppm, J Hz): 8.73 (d, J = 8.0 Hz, 1H), 8.33 (d, J = 7.4 Hz, 1H), 8.07
(d, J = 7.6 Hz, 1H), 7.83 (s, 1H), 7.76 (td, J = 7.6 Hz, J = 1.2 Hz, 1H),
7.63 (dd, J = 7.0 Hz, J = 1.0 Hz, 1H), 7.57–7.39 (m, 4H), 7.15 (s,
1H), 6.16 (m, 1H), 4.70–4.50 (m, 2H), 4.46–4.38 (m, 1H), 3.72 (td,
J = 6.9 Hz, J = 6.3 Hz, 2H), 3.13–2.86 (m, 2H), 1.58 (s, 9H), 1.47 (s,
9H).
5.10.4. 6-(N-Boc-glycyl-aminopropyl)-6H-indeno[1,2-
c]isoquinolin-5,11-dione (19a)
a
Compound 19a was obtained from 44 (147 mg) and N -Boc-gly-
cine (151 mg) according to procedure A as a bright red amorphous
solid. Yield: 161 mg, 81%. 1H NMR (CDCl3, d ppm, J Hz); 8.61 (d,
J = 8.0 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.68 (t, J = 7.3 Hz, 1H), 7.55
(d, J = 7.0 Hz, 1H), 7.47–7.32 (m, 5H), 5.43 (t, J = 5.7 Hz, 1H), 4.54
(t, J = 6.3 Hz, 2H), 3.89 (d, J = 5.7 Hz, 2H), 3.39 (q, J = 6.1 Hz, 2H),
2.09 (m, 2H), 1.44 (s, 9H).
a
p
5.10.7.1. 6-(N ,N -Di-Boc-
2-c]isoquinolin-5,11-dione (26a). Compound 26a was obtained
from N ,N -di-Boc-
Yield: 224 mg, 83%.
L-histidyl-aminoethyl)-6H-indeno[1,
a
p
L-histidine (306 mg) according to procedure A.
a
p
a
e
5.10.7.2. 6-(N ,N -Di-Boc-DL-histidyl-aminoethyl)-6H-indeno[1,
5.10.5. 6-(N ,N -Di-Boc-lysyl-aminoethyl)-6H-indeno[1,2-
c]isoquinolin-5,11-diones 20a–22a
2-c]isoquinolin-5,11-dione (27a). Compound 27a was obtained
from N ,N -di-Boc-DL-histidine (229 mg) according to procedure
B. Yield: 177 mg, 64%.
a
p
Compounds 20a–22a were obtained from 43 (141 mg) and
a
e
appropriate N ,N -di-Boc-lysine (298 mg) according to procedure
A as red amorphous solids. 1H NMR (CDCl3, d ppm, J Hz): 8.72 (d,
J = 8.1 Hz, 1H), 8.33 (d, J = 7.4 Hz, 1H), 7.99 (d, J = 7.5 Hz, 1H),
7.75 (td, J = 7.7 Hz, J = 1.3 Hz, 1H), 7.64 (d, J = 7.2 Hz, 1H), 7.53–
7.24 (m, 3H), 6.97 (t, J = 6.3 Hz, 1H), 5.12 (br s, 1H), 4.69 (td,
J = 6.1 Hz, J = 2.2 Hz, 2H), 4.61 (br s, 1H), 4.07 (br s, 1H), 3.78 (m,
2H), 3.05 (q, J = 6.1 Hz, 2H), 1.86–1.80 (m, 1H), 1.61–1.57 (m,
1H), 1.45 (br s, 18H), 1.44–1.27 (m, 4H).
a
p
5.10.7.3. 6-(N ,N -Di-Boc-
D-histidyl-aminoethyl)-6H-indeno[1,
2-c]isoquinolin-5,11-dione (28a). Compound 28a was obtained
from N ,N -di-Boc-
Yield: 237 mg, 86%.
a
p
D-histidine (229 mg) according to procedure B.
a
p
5.10.8. 6-(N ,N -Di-Boc-histidyl-aminopropyl)-6H-indeno[1,2-
c]isoquinolin-5,11-diones 29a–31a
a
e
Compounds 29a–31a were obtained from 44 (147 mg) and
5.10.5.1. 6-(N ,N -Di-Boc-
quinolin-5,11-dione (20a). Compound 20a was obtained from
N ,N -di-Boc-L-lysine. Yield: 210 mg, 79%.
L-lysyl-aminoethyl)-6H-indeno[1,2-c]iso-
a
p
appropriate N ,N -di-Boc-histidine according either procedure A
or B. 1H NMR (CDCl3, d ppm, J Hz): 8.72 (d, J = 8.2 Hz, 1H), 8.34
(d, J = 8.0 Hz, 1H), 8.05 (s, 1H), 7.75 (t, J = 7.6 Hz, 1H), 7.65 (d,
J = 6.9 Hz, 2H), 7.53–7.39 (m, 4H), 7.21 (s, 1H), 6.22 (br s, 1H),
4.68–4.44 (m, 2H), 3.49–3.47 (m, 2H), 3.19–2.99 (m, 2H), 2.10–
2.01 (m, 2H), 1.57 (s, 9H), 1.48 (s, 9H).
a
e
a
e
5.10.5.2. 6-(N ,N -Di-Boc-DL-lysyl-aminoethyl)-6H-indeno[1,2-
c]isoquinolin-5,11-dione (21a). Compound 21a was obtained
from N ,N -di-Boc-DL-lysine. Yield: 138 mg, 52%.
a
e
a
e
a
p
5.10.5.3. 6-(N ,N -Di-Boc-
D-lysyl-aminoethyl)-6H-indeno[1,2-
5.10.8.1. 6-(N ,N -Di-Boc-
L-histidyl-aminopropyl)-6H-indeno[1,
c]isoquinolin-5,11-dione (22a). Compound 22a was obtained
from N ,N -di-Boc-D-lysine. Yield: 176 mg, 66%.
2-c]isoquinolin-5,11-dione (29a). Compound 29a was obtained
a
e
a
p
from N ,N -di-Boc-L-histidine (306 mg) according to procedure A.
Yield: 190 mg, 69%.
a
e
5.10.6. 6-(N ,N -Di-Boc-lysyl-aminopropyl)-6H-indeno[1,2-
c]isoquinolin-5,11-diones 23a–25a
a
p
5.10.8.2. 6-(N ,N -Di-Boc-DL-histidyl-aminopropyl)-6H-indeno-
Compounds 23a–25a were obtained from compound 44
[1,2-c]isoquinolin-5,11-dione (30a). Compound 30a was ob-
tained from N ,N -di-Boc-DL-histidine (306 mg) according to
procedure A. Yield: 185 mg, 67%.
a
e
a
p
(147 mg) and appropriate N ,N -di-Boc-lysine (298 mg) according
to procedure A as bright red amorphous solids. 1H NMR (CDCl3, d

8130
G. Ahn et al. / Bioorg. Med. Chem. 18 (2010) 8119–8133
a
p
5.10.8.3. 6-(N ,N -Di-Boc-
D
-histidyl-aminopropyl)-6H-indeno[1,
(t, J = 5.4 Hz, 1H), 8.56 (dd, J = 8.0 Hz, J = 3.6 Hz, 1H), 8.30 (br s,
3H), 8.21 (d, J = 8.0 Hz, 1H), 8.19–8.08 (m, 4H), 7.83 (t, J = 7.3 Hz,
1H), 7.57–7.49 (m, 4H), 4.56 (br s, 2H), 3.70 (br s, 2H), 3.51–3.45
(m, 1H), 2.70 (br s, 2H), 1.62–1.52 (m, 4H), 1.31 (br d, J = 7.0 Hz,
2H). 13C NMR (DMSO-d6, d ppm): 190.32, 169.78, 162.92, 156.68,
136.82, 134.63, 134.42, 134.34, 132.15, 131.76, 128.44, 127.55,
124.38, 123.14, 122.99, 122.93, 107.44, 52.35, 43.74, 38.58, 37.69,
30.41, 26.67, 21.68.
2-c]isoquinolin-5,11-dione (31a). Compound 31a was obtained
from N ,N -di-Boc-
Yield: 240 mg, 87%.
a
p
D-histidine (229 mg) according to procedure B.
0
5.10.9. 6-(N ,N ,Nx -Tri-Boc-
L-arginyl-aminoethyl)-6H-
indeno[1,2-c]isoquinolin-5,11-dione (32a)
a
x
Compound 32a was prepared from 43 (141 mg) and commer-
0
cially available N ,N ,Nx -tri-Boc-
L
-arginine (408 mg, 1.1 mmol)
a
x
according to procedure A. The crude product was purified by flash
column chromatography on silica gel (CHCl3/acetone, 8:2, as elu-
ent). Yield: 222 mg, 69%. 1H NMR (CDCl3, d ppm, J Hz): 9.14 (br s,
2H), 8.67 (d, J = 8.0 Hz, 1H), 8.29 (J = 8.0 Hz, 1H), 8.07 (d,
J = 7.6 Hz, 1H), 7.70 (td, J = 7.7 Hz, J = 1.3 Hz, 1H), 7.59–7.34 (m,
5H), 6.05 (br s, 1H), 4.73–4.58 (m, 2H), 4.21 (br s, 1H), 3.90–3.52
(m, 6H), 3.20 (q, J = 7.5 Hz, 2H), 1.43–1.41 (m, 27H).
5.11.3.1. 6-(L-Lysyl-aminoethyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione dihydrochloride (20). Compound 20 was obtained
from 20a. Yield: 87 mg, 89%. ½a D20
ꢃ
: +26.6 (c 0.2, MeOH). HRMS
calcd for C24H27N4O3 [M+H]+ 419.2078, found 419.2073.
5.11.3.2. 6-(DL-Lysyl-aminoethyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione dihydrochloride (21). Compound 21 was afforded
from 21a. Yield: 54 mg, 55%. HRMS calcd for C24H27N4O3 [M+H]+
419.2078, found 419.2075.
0
5.10.10. 6-(N ,N ,Nx -Tri-Boc-
L
-arginyl-aminopropyl)-6H-
a
x
indeno[1,2-c]isoquinolin-5,11-dione (33a)
5.11.3.3. 6-(D-Lysyl-aminoethyl)-6H-indeno[1,2-c]isoquinolin-
Compound 33a was prepared from 44 (0.15 g) and commer-
0
cially available N ,N ,Nx -tri-Boc-
L-arginine (0.41 g, 1.1 mmol)
a
x
5,11-dione dihydrochloride (22). Compound 22 was afforded
from 22a. Yield: 89 mg, 91%. ½a D20
ꢃ : ꢁ23.3 (c 0.2, MeOH). HRMS
according to procedure A. The crude product was purified by flash
column chromatography on silica gel (CHCl3/acetone, 8:2, as elu-
ent). Yield: 291 mg, 89%. 1H NMR (CDCl3, d ppm, J Hz): 9.31 (br s,
2H), 8.65 (d, J = 7.9 Hz, 1H), 8.28 (d, J = 7.5 Hz, 1H), 7.70 (td,
J = 7.7 Hz, J = 1.3 Hz, 1H), 7.58 (d, J = 6.9 Hz, 1H), 7.51–7.41 (m,
4H), 7.36 (t, J = 7.2 Hz, 1H), 5.93 (d, J = 8.3 Hz, 1H), 4.54 (t,
J = 7.0 Hz, 2H), 4.36–4.28 (m, 1H), 3.98–3.73 (m, 2H), 3.51–3.31
(m, 2H), 2.13–2.01 (m, 2H), 1.85–1.81 (m, 2H), 1.75–1.61 (m,
2H), 1.51 (s, 9H), 1.46 (s, 9H), 1.45 (s, 9H).
calcd for C24H27N4O3 [M+H]+ 419.2078, found 419.2074.
5.11.4. 6-(Lysyl-aminopropyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione dihydrochlorides 23–25
Compounds 23–25 were obtained from 23a–25a (127 mg) as
red amorphous solids. 1H NMR (DMSO-d6, d ppm, J Hz): 8.94 (br
s, 1H), 8.56 (d, J = 8.0 Hz, 1H), 8.33 (br s, 3H), 8.21 (d, J = 8.0 Hz,
1H), 8.04 (br s, 3H), 7.85–7.78 (m, 2H), 7.63–7.49 (m, 4H), 4.51
(m, 2H), 3.79 (br s, 1H), 3.44–3.25 (m, 2H), 2.76 (br s, 2H), 1.95
(br t, J = 6.1 Hz, 2H), 1.77 (m, 2H), 1.59 (m, 2H), 1.45–1.38 (m,
2H). 13C NMR (DMSO-d6, d ppm): 189.68, 168.55, 162.27, 155.85,
136.10, 134.18, 134.00, 133.87, 131.57, 131.22, 127.97, 127.01,
123.63, 122.59, 122.53, 122.43, 106.93, 51.88, 42.24, 38.12, 36.33,
30.01, 28.89, 26.25, 21.16.
5.11. General procedure for the synthesis of compounds 18–33
To a solution of 18a–33a (0.2 mmol) in CHCl3 (5 mL), a solution
of HCl 5 M in 2-propanol (1 mL) was slowly added at 0 °C. The
resulting mixture was stirred at room temperature for 18 h. The
precipitated product was filtered, washed with CHCl3 and Et2O
and dried in vacuo to provide the desired product.
5.11.4.1. 6-(L-Lysyl-aminopropyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione dihydrochloride (23). Yield: 83 mg, 82%. ½a D20
ꢃ
: +19.2
(c 0.2, MeOH). HRMS calcd for C25H29N4O3 [M+H]+ 433.2234, found
5.11.1. 6-(Glycyl-aminoethyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione hydrochloride (18)
433.2232.
Compound 18 was obtained as an orange amorphous solid from
18a (89 mg). Yield: 29 mg, 38%. 1H NMR (DMSO-d6, d ppm, J Hz):
8.93 (t, J = 5.7 Hz, 1H), 8.57 (d, J = 8.0 Hz, 1H), 8.21 (d, J = 7.9 Hz,
1H), 8.16 (br s, 3H), 8.09 (d, J = 7.0 Hz, 1H), 7.83 (t, J = 7.7 Hz, 1H),
7.60–7.49 (m, 4H), 4.56 (t, J = 6.6 Hz, 2H), 3.60–3.54 (m, 2H), 3.46
(br s, 2H). 13C NMR (DMSO-d6, d ppm): 190.43, 167.23, 163.04,
156.82, 136.99, 134.75, 134.54, 134.29, 132.29, 131.81, 128.51,
127.58, 124.17, 123.27, 122.01, 107.52, 43.86, 40.14, 37.73. HRMS
calcd for C20H18N3O3 [M+H]+ 348.1343, found 348.1342.
5.11.4.2. 6-(DL-Lysyl-aminopropyl)-6H-indeno[1,2-c]isoquino-
lin-5,11-dione dihydrochlorides (24). Yield: 48 mg, 47%. HRMS
calcd for C25H29N4O3 [M+H]+ 433.2234, found 433.2231.
5.11.4.3. 6-(D-Lysyl-aminopropyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione dihydrochlorides 25. Yield: 70 mg, 69%. ½a D20
ꢃ : ꢁ15.5
(c 0.2, MeOH). HRMS calcd for C25H29N4O3 [M+H]+ 433.2234, found
433.2237.
5.11.5. 6-(Histidyl-aminoethyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione hydrochlorides 26–28
5.11.2. 6-(Glycyl-aminopropyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione hydrochloride (19)
Compounds 26–28 were obtained as red amorphous solids from
26a–28a (126 mg). 1H NMR (DMSO-d6, d ppm, J Hz): 9.29 (br s, 1H),
8.99 (br s, 1H), 8.57 (br d, J = 7.9 Hz, 3H), 8.20 (d, J = 7.9 Hz, 1H),
8.10 (d, J = 7.0 Hz, 1H), 7.83 (t, J = 7.7 Hz, 1H), 7.60–7.47 (m, 5H),
4.55 (t, J = 6.5 Hz), 4.21–4.15 (m, 1H), 3.68–3.44 (m, 2H), 3.25–
Compound 19 was obtained as an orange amorphous solid from
19a (92 mg). Yield: 43 mg (54%). 1H NMR (DMSO-d6, d ppm, J Hz):
8.70 (br s, 1H), 8.55 (d, J = 8.0 Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 8.15
(br s, 3H), 7.83–7.74 (m, 2H), 7.63–7.47 (m, 4H), 4.51 (t, J = 7.0 Hz,
2H), 3.57 (br s, 2H), 3.37–3.31 (m, 2H), 1.97 (br t, J = 7.0 Hz, 2H). 13
C
3.07 (m, 2H). 13C NMR (DMSO-d6,
d ppm): 189.80, 167.90,
NMR (DMSO-d6, d ppm): 189.80, 165.95, 162.36, 155.98, 136.22,
134.27, 134.00 (br, 2C), 131.68, 131.24, 127.99, 127.07, 123.65,
122.68, 122.61, 122.51, 106.98, 42.12, 40.05, 36.31, 29.03. HRMS
calcd for C21H20N3O3 [M+H]+ 362.1499, found 362.1498.
162.40, 156.08, 136.24, 134.11, 133.91 (br, 2C), 131.65, 131.23,
127.90, 127.00, 126.80, 123.78, 122.65, 122.44 (br, 3C), 117.74,
106.98, 51.27, 43.08, 37.30, 25.95.
5.11.5.1. 6-(L-Histidyl-aminoethyl)-6H-indeno[1,2-c]isoquino-
5.11.3. 6-(Lysyl-aminoethyl)-6H-indeno[1,2-c]isoquinolin-5,11-
dione dihydrochlorides 20–22
lin-5,11-dione hydrochloride (26). Yield: 47 mg, 51%. ½a D20
ꢃ
:
+13.1 (c 0.2, MeOH). HRMS calcd for
C
24H23N5O3 [M+H]+
Compounds 20–22 were obtained from 20a–22a (124 mg) as
orange amorphous solids. 1H NMR (DMSO-d6, d ppm, J Hz): 9.14
428.1717, found 428.1714.

G. Ahn et al. / Bioorg. Med. Chem. 18 (2010) 8119–8133
8131
5.11.5.2. 6-(DL-Histidyl-aminoethyl)-6H-indeno[1,2-c]isoquino-
lin-5,11-dione hydrochloride (27). Yield: 42 mg, 45%. HRMS
calcd for C24H23N5O3 [M+H]+ 428.1717, found 428.1720.
ppm): 189.88, 168.58, 163.53, 155.56, 136.53, 134.29, 133.68,
133.62, 132.09, 130.92, 127.76, 126.91, 123.05, 122.96, 122.87,
122.59, 108.78, 52.79, 42.16, 40.50, 36.59, 33.50, 28.99, 28.38,
24.14. ½a 2D0
ꢃ
: +18.1 (c 0.4, MeOH). HRMS calcd for C25H29N6O3
5.11.5.3. 6-(
D
-Histidyl-aminoethyl)-6H-indeno[1,2-c]isoquino-
[M+H]+ 461.2296, found 461.2294.
lin-5,11-dione hydrochloride (28). Yield: 65 mg, 70%. ½a D20
ꢃ
:
ꢁ12.1 (c 0.2, MeOH). HRMS calcd for
C
24H23N5O3 [M+H]+
5.13. General procedure for the synthesis of Weinreb amides of
N-Boc-protected a-amino acids 45 and 46
428.1717, found 428.1719.
5.11.6. 6-(Histidyl-aminopropyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione hydrochlorides 29–31
N,O-Dimethylhydroxylamine-HCl (371 mg, 3.8 mmol, 1.3
equiv), and DMAP (35 mg, 1 mol %) were added to a solution of
the appropriate N-Boc-protected amino acid (2.9 mmol, 1.0 equiv)
in CH2Cl2 (14.5 mL). The resulting mixture was cooled to 0 °C.
Triethylamine (0.7 mL, 4.9 mmol, 1.7 equiv) was added slowly over
5 min then EDCI (657 mg, 3.4 mmol, 1.2 equiv) was introduced.
The solution was stirred for 1 h at 0 °C and for 18 h at room tem-
perature. The reaction mixture was subsequently diluted with
50 mL of CH2Cl2 (50 mL) and washed with HCl 1 M (2 ꢂ 20 mL),
saturated NaHCO3 (2 ꢂ 20 mL), and brine (2 ꢂ 20 mL). The organic
layer was dried over Na2SO4, concentrated, and dried in vacuo to
provide the crude desired product which was used in the next step
without further purification.
Compounds 29–31 were obtained as red amorphous solids from
29a–31a (128 mg). 1H NMR (DMSO-d6, d ppm, J Hz): 9.04 (s, 1H),
8.97 (t, J = 5.4 Hz, 1H), 8.57 (d, J = 7.9 Hz, 1H), 8.54 (br s, 2H),
8.22 (d, J = 7.6 Hz, 1H), 7.86–7.77 (m, 2H), 7.65–7.49 (m, 5H),
4.65–4.44 (m, 2H), 4.29–4.23 (br s, 1H), 3.17–3.74 (m, 4H), 1.96
(m, 2H). 13C NMR (DMSO-d6, d ppm): 189.59, 167.26, 162.19,
155.68, 135.98, 134.08, 134.00, 133.76, 131.52, 131.12, 127.87,
126.96, 126.89, 123.55, 122.52, 122.44, 122.41, 122.39, 117.72,
106.90, 51.36, 42.14, 36.56, 28.69, 26.30.
5.11.6.1. 6-(
L-Histidyl-aminopropyl)-6H-indeno[1,2-c]isoquino-
lin-5,11-dione hydrochloride (29). Yield: 74 mg, 77%.
+27.3 (c 0.2, MeOH). HRMS calcd for
½
a 2D0
:
ꢃ
C
25H24N5O3 [M+H]+
a
442.1874, found 442.1869.
5.13.1. N -Boc-glycyl-N-methoxy-N-methylamine (45)
a
Compound 45 was obtained from N -Boc-glycine (508 mg) as a
yellow amorphous solid. Yield: 563 mg, 89%. 1H NMR (CDCl3, d
ppm, J Hz): 5.29 (br s, 1H), 4.10 (d, J = 4.5 Hz, 2H), 3.73 (s, 3H),
3.22 (s, 3H), 1.47 (s, 9H).
5.11.6.2. 6-(DL-Histidyl-aminopropyl)-6H-indeno[1,2-c]isoquin-
olin-5,11-dione hydrochloride (30). Yield: 76 mg, 80%. HRMS
calcd for C25H24N5O3 [M+H]+ 442.1874, found 442.1866.
5.11.6.3. 6-(
D-Histidyl-aminopropyl)-6H-indeno[1,2-c]isoquino-
a
e
5.13.2. N ,N -Di-Boc-(DL)-lysyl-N-methoxy-N-methylamine (46)
lin-5,11-dione hydrochloride (31). Yield: 67 mg, 70%.
½
a 2D0
:
ꢃ
a
e
Compound 46 was obtained from N ,N -di-Boc-DL-lysine
(1.00 g) as a yellow oil. Yield: 1.03 g, 91%. NMR (CDCl3, d ppm, J
Hz): 5.24 (d, J = 8.5 Hz, 1H), 4.66 (br s, 2H), 3.78 (s, 3H), 3.22 (s,
3H), 3.15–3.06 (m, 2H), 1.58–1.50 (m, 6H), 1.45 (s, 18H).
ꢁ25.6 (c 0.2, MeOH). HRMS calcd for
C
25H24N5O3 [M+H]+
442.1874, found 442.1865.
5.12. General procedure for synthesis of compounds 32, 33
5.14. General procedure for the synthesis of 6-(2-Boc-
aminoalkylamino)alkyl-6H-indeno[1,2-c]isoquinolin-5,11-
diones 34a, 36a, 37a
To a stirred solution of 32a or 33a (0.3 mmol) in EtOAc (4 mL)
under Ar atmosphere, SnCl4 (0.2 mL, 1.3 mmol) was added. The
resulting mixture was stirred at room temperature for 4 h under
Ar atmosphere. The solvent and the excess of SnCl4 were evapo-
rated in vacuo. The residue was subsequently dissolved in MeOH
(10 mL) and Et2O was then added until an orange precipitate was
formed. The precipitate was filtered, washed with Et2O and dried
in vacuo to afford the desired product as orange amorphous solids.
Appropriate Weinreb amide 45 or 46 (2.4 mmol, 1.0 equiv) was
dissolved in dry THF (20 mL) and the solution was cooled to 0 °C.
LiAlH4 (110 mg, 2.9 mmol, 1.2 equiv) was added in small portions
and the resulting mixture was stirred for 40 min at 0 °C. The reac-
tion mixture was quenched at 0 °C by a slow addition of a 0 °C
precooled KHSO4 0.4 M solution (10 mL). The resulting mixture
was stirred for 12 min at room temperature. Distilled water
(20 mL) was added to the mixture and the aqueous layer was ex-
tracted with EtOAc (4 ꢂ 25 mL). The combined organic layers
were washed with HCl 2 M (3 ꢂ 25 mL), saturated NaHCO3
(2 ꢂ 25 mL), and brine (2 ꢂ 25 mL), dried over Na2SO4, concen-
trated on a rotary evaporator, and dried under vacuum (30 min).
The resulting clear oil was immediately dissolved in CH2Cl2
(15 mL) then 43 or 44 (2.4 mmol, 1.0 equiv) and triethylamine
(0.34 mL, 2.4 mmol, 1.0 equiv) were carefully added to the reac-
tion mixture and the solution was stirred for 5 min. NaHB(OAc)3
(615 mg, 2.9 mmol, 1.2 equiv) was subsequently added to the
reaction mixture and the solution was stirred for 90 min. The
resulting solution was diluted with a mixture of saturated NaH-
CO3 (17 mL) and saturated K2CO3 (25 mL) then stirred for
10 min. The resulting mixture was extracted with CH2Cl2
(4 ꢂ 15 mL). The combined organic layers were dried over Na2SO4,
concentrated and dried in vacuo. The residue was purified by flash
column chromatography on silica gel (CH2Cl2/MeOH, 95:5, as elu-
ent) to give the desired product.
5.12.1. 6-(L-Arginyl-aminoethyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione dihydrochloride (32)
Compound 32 was obtained from 32a (224 mg). Yield: 62 mg,
40%. 1H NMR (CD3OD, d ppm, J Hz): 8.46 (d, J = 7.9 Hz, 1H), 8.12
(d, J = 7.7 Hz, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.64 (td, J = 7.6 Hz,
J = 1.2 Hz, 1H), 7.52–7.36 (m, 5H), 4.71–4.57 (m, 2H), 3.94–3.82
(m, 2H), 3.67–3.58 (m, 1H), 3.20 (t, J = 6.7 Hz, 2H), 1.92–1.65 (m,
4H). 13C NMR (CD3OD, d ppm): 190.36, 169.02, 163.63, 157.18,
155.68, 136.57, 134.48, 133.68, 133.62, 132.08, 130.87, 127.73,
126.91, 123.41, 122.98, 122.85, 122.49, 108.19, 52.68, 43.34,
40.48, 38.13, 28.09, 23.98. ½a D20
ꢃ
: +18.6 (c 0.4, MeOH). HRMS calcd
for C24H27N6O3 [M+H]+ 447.2139, found 447.2137.
5.12.2. 6-(L-Arginyl-aminopropyl)-6H-indeno[1,2-c]isoquinolin-
5,11-dione dihydrochloride (33)
Compound 33 was obtained from 33a (228 mg). Yield: 24 mg,
15%. 1H NMR (CD3OD, d ppm, J Hz): 8.53 (d, J = 8.0 Hz, 1H), 8.16
(d, J = 8.0 Hz, 1H), 7.72–7.65 (m, 2H), 7.57–7.39 (m, 5H), 4.56 (br
s, 2H), 4.01 (t, J = 6.3 Hz, 1H), 3.66–3.40 (m, 2H), 3.36–3.31 (m,
2H), 2.15–1.97 (m, 4H), 1.85–1.77 (m, 2H). 13C NMR (CD3OD, d

8132
G. Ahn et al. / Bioorg. Med. Chem. 18 (2010) 8119–8133
5.14.1. 6-(2-(2-Boc-aminoethylamino)ethyl)-6H-indeno[1,2-
c]isoquinolin-5,11-dione (34a)
was stirred at room temperature for 18 h. The precipitated product
was filtered, washed with CHCl3 and Et2O then dried in vacuo to
provide the desired products.
Compound 34a was obtained from 45 (524 mg) and 43
(784 mg) as a dark red amorphous solid. Yield: 385 mg, 37%. 1H
NMR (CDCl3, d ppm, J Hz): 8.73 (d, J = 8.0 Hz, 1H), 8.37 (d,
J = 7.4 Hz, 1H), 7.76 (td, J = 7.7 Hz, J = 1.3 Hz, 1H), 7.64 (dd,
J = 7.2 Hz, J = 7.0 Hz, 2H), 7.52–7.40 (m, 3H), 5.00 (br s, 1H), 4.67
(t, J = 7.0 Hz, 2H), 3.25 (td, J = 6.0 Hz, J = 5.6 Hz, 2H), 3.15 (t,
J = 7.02 Hz, 2H), 2.86 (t, J = 5.8 Hz, 2H), 1.46 (s, 9H).
5.15.1. 6-(2-(2-Aminoethylamino)ethyl)-6H-indeno[1,2-c]iso-
quinolin-5,11-dione dihydrochloride (34)
Compound 34 was obtained from 34a (173 mg) as an orange
amorphous solid. Yield: 111 mg, 68%. 1H NMR (DMSO-d6, d ppm,
J Hz): 10.01 (br s, 2H), 8.54 (br s, 3H), 8.43 (d, J = 8.0 Hz, 1H),
8.12 (d, J = 8.0 Hz, 1H), 8.05 (d, J = 7.2 Hz, 1H), 7.74 (t, J = 7.7 Hz,
1H), 7.52–7.38 (m, 4H), 4.76 (t, J = 6.2 Hz, 2H), 3.46 (br s, 2H),
3.33 (br s, 2H), 3.24 (br s, 2H). 13C NMR (DMSO-d6, d ppm):
189.86, 162.79, 155.90, 135.90, 134.05, 133.95, 133.88, 131.63,
131.25, 127.99, 127.07, 123.88, 122.78, 122.57, 122.42, 107.30,
45.11, 44.39, 40.37, 35.02. HRMS calcd for C20H20N3O2 [M+H]+
334.1550, found 334.1543.
5.14.2. 6-(2-(2,6-Di-Boc-diaminohexylamino)ethyl)-6H-
indeno[1,2-c]isoquinolin-5,11-dione (36a)
Compound 36a was obtained from 46 (934 mg) and 43
(784 mg) as a dark red amorphous solid. Yield: 421 mg, 29%. 1H
NMR (CDCl3, d ppm, J Hz): 8.72 (d, J = 8.0 Hz, 1H), 8.35 (d,
J = 8.2 Hz, 1H), 7.75 (t, J = 7.6 Hz, 2H), 7.64 (t, J = 6.2 Hz, 2H),
7.51–7.39 (m, 3H), 4.66 (br t, J = 6.8 Hz, 5H), 3.66 (br s, 1H),
3.17–3.10 (m, 5H), 2.74 (d, J = 5.6 Hz, 2H), 1.56–1.45 (m, 26H).
5.15.2. 6-(3-(2-Aminoethylamino)propyl)-6H-indeno[1,2-c]iso-
quinolin-5,11-dione dihydrochloride (35)
5.14.3. 6-(3-(2,6-Di-Boc-diaminohexylamino)propyl)-6H-
indeno[1,2-c]isoquinolin-5,11-dione (37a)
Compound 35 was afforded from 35a (179 mg) as an orange
amorphous solid. Yield: 155 mg, 92%. 1H NMR (DMSO-d6, d ppm):
9.43 (br s, 2H), 8.59 (d, J = 7.9 Hz, 1H), 8.30 (br s, 3H), 8.23 (d,
J = 7.5 Hz, 1H), 7.87–7.82 (m, 2H), 7.63–7.50 (m, 4H), 4.59 (t,
J = 6.7 Hz, 2H), 3.18 (br s, 6H), 2.22 (m, 2H). HRMS calcd for
Compound 37a was obtained from 46 (934 mg) and 44
(818 mg) as a dark red amorphous solid. Yield: 0.33 g, 52%. 1H
NMR (CDCl3, d ppm, J Hz): 8.73 (d, J = 8.0 Hz, 1H), 8.36 (d,
J = 8.2 Hz, 1H), 7.74 (t, J = 6.7 Hz, 2H), 7.66 (d, J = 7.0 Hz, 1H),
7.54–7.36 (m, 3H), 4.77–4.66 (m, 4H), 3.74–3.67 (m, 1H), 3.14 (br
d, J = 6.0 Hz, 3H), 2.83 (t, J = 6.2 Hz, 2H), 2.69 (br d, J = 5.3 Hz, 2H),
2.13–1.98 (m, 2H), 1.55–1.41 (m, 26H).
C
21H22N3O2 [M+H]+ 348.1706, found 348.1703.
5.15.3. 6-(2-(2,6-Diaminohexylamino)ethyl)-6H-indeno[1,2-c]-
isoquinolin-5,11-dione trihydrochloride (36)
5.14.4. 3-(5,11-Diketo-6H-indeno[1,2-c]isoquinolin-6-yl)propyl
4-methylbenzenesulfonate (47)
Compound 36 was afforded from 36a (242 mg) as a red amor-
phous solid. Yield: 117 mg, 57%. 1H NMR (DMSO-d6, d ppm, J Hz):
10.14 (br s, 2H), 8.76 (br s, 3H), 8.39 (d, J = 8.0 Hz, 1H), 8.17 (br s,
3H), 8.10 (d, J = 7.9 Hz, 2H), 7.72 (t, J = 7.6 Hz, 1H), 7.51–7.42 (m,
4H), 4.78 (br s, 2H), 3.60 (br s, 2H), 3.45–3.33 (m, 3H), 2.77 (br s,
2H), 1.73–1.48 (m, 6H). 13C NMR (DMSO-d6, d ppm): 189.85,
162.71, 155.87, 135.82, 133.98 (br, 2C), 133.83, 131.60, 131.24,
127.96, 127.07, 123.90, 122.76, 122.55, 122.39, 107.31, 48.70,
47.74, 45.32, 40.24, 38.15, 29.59, 26.15, 21.16. HRMS calcd for
TsCl (159 mg, 0.8 mmol, 1.1 equiv) was added to a solution of
40 (212 mg, 0.7 mmol, 1.0 equiv) in CH2Cl2 (8 mL). Triethylamine
(0.3 mL, 2.1 mmol, 3.0 equiv) was added and the mixture was stir-
red at room temperature for 18 h. The resulting mixture was di-
luted with CH2Cl2 (50 mL) and washed with distilled water
(2 ꢂ 20 mL) and then brine (20 mL). The organic layer was dried
over Na2SO4 and concentrated. Purification by flash column chro-
matography on silica gel (CH2Cl2/MeOH, 99:1, as eluent) afforded
the desired products as a dark red solid. Yield: 180 mg, 56%. mp:
180–182 °C. 1H NMR (DMSO-d6, d ppm, J Hz): 8.64 (d, J = 7.9 Hz,
1H), 8.27 (d, J = 8.0 Hz, 1H), 7.91 (t, J = 7.6 Hz, 1H), 7.83 (d,
J = 8.2 Hz, 3H), 7.66–7.51 (m, 4H), 7.50 (d, J = 8.2 Hz, 2H), 4.60 (t,
J = 7.2 Hz, 2H), 4.35 (t, J = 5.9 Hz, 2H), 2.47 (s, 3H), 2.23 (m, 2H).
C
24H29N4O2 [M+H]+ 405.2285, found 405.2283.
5.15.4. 6-(3-(2,6-Diaminohexylamino)propyl)-6H-indeno[1,2-c]-
isoquinolin-5,11-dione trihydrochloride (37)
Compound 37 was afforded from 37a (248 mg) as a red amor-
phous solid. Yield: 116 mg, 55%. 1H NMR (DMSO-d6, d ppm): 9.80
(br s, 2H), 8.75 (br s, 3H), 8.29 (d, J = 8.0 Hz, 1H), 8.17 (br s, 3H),
8.02 (d, J = 7.9 Hz, 1H), 7.70–7.60 (m, 2H), 7.49 (t, J = 6.5 Hz, 1H),
7.43–7.33 (m, 3H), 4.40 (br s, 2H), 3.57 (br s, 1H), 3.39–2.98 (m,
4H), 2.75 (br s, 2H), 2.21 (br s, 2H), 1.87–1.31 (m, 6H). 13C NMR
5.14.5. 6-(3-(2-Boc-aminoethylamino)propyl)-6H-indeno[1,2-
c]isoquinolin-5,11-dione (35a)
Compound 41 (370 mg, 2.3 mmol, 10 equiv) was added to a
solution of compound 47 (106 mg, 0.2 mmol) in acetonitrile
(10 mL). The resulting mixture was heated at 80 °C for 18 h. The sol-
vent was evaporated in vacuo. The residue was taken up in CH2Cl2
(50 mL), washed with brine (2 ꢂ 25 mL), dried over Na2SO4 and
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (CH2Cl2/EtOAc, 7:3, as eluent) to yield
the desired product as a red amorphous solid. Yield: 83 mg, 80%. 1H
NMR (CDCl3, d ppm, J Hz): 8.73 (d, J = 8.2 Hz, 1H), 8.36 (d, J = 8.0 Hz,
1H), 7.76 (td, J = 7.7 Hz, J = 1.3 Hz, 1H), 7.67 (td, J = 7.6 Hz, J = 1.0 Hz,
2H), 7.52–7.39 (m, 3H), 5.25 (br s, 1H), 4.67 (t, J = 7.2 Hz, 2H), 3.35
(q, J = 5.2 Hz, 4H), 2.88–2.84 (m, 4H), 2.18 (m, 2H), 1.48 (s, 9H).
(DMSO-d6,
d ppm): 189.57, 162.36, 155.56, 135.76, 134.03,
133.93, 133.79, 131.46, 131.12, 127.82, 126.91, 123.68, 122.47,
122.40, 122.34, 107.01, 48.29, 47.80, 44.89, 41.56, 38.13, 29.46,
26.10, 25.56, 21.16. HRMS calcd for
419.2441, found 419.2438.
C
25H31N4O2 [M+H]+
5.16. DNA and drugs solutions
Calf thymus DNA (CT DNA, Pharmacia) was deproteinized with
sodium dodecyl sulfate (SDS, protein content <0.2%) and exten-
sively dialyzed against the required experimental buffer. An
extinction coefficient of 6600 Mꢁ1 cmꢁ1 was used to measure the
nucleotide-concentration of DNA solutions.34 All synthesized com-
pounds, as well as camptothecin and etoposide (Sigma), were dis-
solved as 10 mM solutions in DMSO. Further dilutions were made
in the appropriate aqueous buffer.
5.15. General procedures for the syntheses of 6-(aminoalkyl-
amino)alkyl)-6H-indeno[1,2-c]isoquinolin-5,11-dione hydro-
chlorides 34–37
To a solution of 34a–37a (0.4 mmol) in CHCl3 (10 mL) a solution
of HCl 5 M in 2-propanol (2 mL) was added. The resulting mixture

G. Ahn et al. / Bioorg. Med. Chem. 18 (2010) 8119–8133
8133
5.17. Melting temperature studies
assessed using a cell proliferation assay developed by Promega
(CellTiter 96 AQueous one solution cell proliferation assay). Briefly,
2 ꢂ 104 exponentially growing cells were seeded in 96-well micro-
culture plates with various drug concentrations in a volume of
Melting curves were obtained using an Uvikon 943 spectropho-
tometer coupled to a Neslab RTE111 cryostat. Typically, 20 lM of
the various drugs were prepared in 1 mL of BPE buffer (6 mM
100 lL. After 72 h incubation at 37 °C, 20 lL of the tetrazolium
Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA, pH 7.1) in the presence or
absence of 20 lM of CT DNA and transferred into a quartz cuvette
of 10 mm path length. The spectra were recorded from 230 nm to
500 nm and are referenced against a cuvette containing the same
DNA concentration in the same buffer. For the absorption titration,
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.
Acknowledgements
CT DNA was added gradually from 1 to 20
corded after each addition. To perform the melting temperature
measurement, CT DNA (20 M) 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 of 20–100 °C with an increment of 1 °C per min. The Tm val-
ues were obtained from first-derived plots.
lM with a spectrum re-
This research work was supported by the Ligue Contre le Cancer
Comité du Nord, the Centre National de la Recherche Scientifique,
MENESR (grant to G.A.) and by the Programme PRIM (Région Nord-
Pas-de-Calais). The authors thank Christine Mahieu for expert
technical assistance.
l
References and notes
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weak fluorescence variation with DNA titration, the binding stud-
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etry assay using DNA-bound ethidium bromide.27,28 Excitation was
set at 546 nm and the fluorescence emission was monitored over
the range 560–700 nm. Experiments were performed with an ethi-
dium bromide/DNA molar ratio of 12.6:10 and a drug concentra-
6. Pommier, Y.; Cushman, M. Mol. Cancer Ther. 2009, 8, 1008.
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stant was calculated as follows: Kapp = (1.26/C50)Kethidium, with
12. Nagarajan, M.; Morrell, A.; Ioanoviciu, A.; Antony, S.; Kohlhagen, G.; Agama, K.;
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Kethidium = 107 Mꢁ1
.
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5.19. Topoisomerase inhibition
16. Strumberg, D.; Pommier, Y.; Paull, K.; Jayaraman, M.; Nagafuji, P.; Cushman, M.
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The experimental procedure has been previously detailed.35
Supercoiled pUC19 plasmid DNA (130 ng) was incubated with
4 units of human topoisomerase I or II (TopoGen) at 37 °C for
45 min in 20 lL of relaxation buffer (50 mM tris(hydroxy-
methyl)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
pound. Reactions were terminated by adding of SDS to 0.25% and
proteinase K to 250 g/mL and incubating at 50 °C for a further
30 min. 3 L 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 g/mL; topoiso-
lM) of the tested com-
l
l
22. Yang, Q.; Yang, P.; Qian, X.; Tong, L. Bioorg. Med. Chem. Lett. 2008, 18, 6210.
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l
merase DNA cleavage gel) or not (inhibition of the relaxation of
DNA) at room temperature for 2 h at 120 V. Gels run without ethi-
dium bromide were then stained using a bath containing ethidium
bromide. Both gels were finally washed and photographed under
UV light.
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33. pKa and log D values were calculated using ACD/pKa DB software from
Advanced Chemistry Development Inc.: Toronto, Canada.
34. Wells, R. D.; Larson, J. E.; Grant, R. C.; Shortle, B. E.; Cantor, C. R. J. Mol. Biol.
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35. Bailly, C. Methods Enzymol. 2001, 340, 610.
5.20. 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 med-
ium, supplemented with 10% fetal bovine serum, 2 mM L-gluta-
mine, 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 lg/mL). The cytotoxicity of the tested compounds was