Synthesis, cytotoxicity and topoisomerase inhibition properties of multifarious aminoalkylated indeno[1,2-c]isoquinolin-5,11-diones

Article abstract of DOI:10.1016/j.bmcl.2011.02.106

A number of mono- or diaminoalkylated indeno[1,2-c]isoquinolin-5,11-diones analogs of 1 were synthesized and evaluated for their DNA binding affinities, topoisomerase inhibition properties and antiproliferative activities against human cancer cell lines (

Full text of DOI:10.1016/j.bmcl.2011.02.106

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2259–2263
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, cytotoxicity and topoisomerase inhibition properties of multifarious
aminoalkylated indeno[1,2-c]isoquinolin-5,11-diones
Gang Ahn a, , Nadège Schifano-Faux b, Jean-François Goossens b, Brigitte Baldeyrou c, Axel Couture a,
⇑
Pierre Grandclaudon a,d, Amélie Lansiaux c, Adina Ryckebusch a,d
a EA CMF 4478, Université Lille 1-Lille Nord de France, Bat C3(2), Cité Scientifique, 59655 Villeneuve d’Ascq Cedex, France
b Laboratoire de Chimie Analytique, EA 4481, Université Lille 2-Lille Nord de France, 3 rue du Professeur Laguesse, BP 83, 59006 Lille Cedex, France
c Inserm U837, Centre Oscar Lambret, Université Lille 2- Lille Nord de France, IRCL et IMPRT, 1 Place de Verdun, 59045 Lille Cedex, France
d Ecole Nationale Supérieure de Chimie de Lille, Avenue Dimitri Mendeleïev, Cité Scientifique, BP 90108, 59652 Villeneuve d’Ascq Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of mono- or diaminoalkylated indeno[1,2-c]isoquinolin-5,11-diones analogs of 1 were synthe-
sized and evaluated for their DNA binding affinities, topoisomerase inhibition properties and antiprolif-
erative activities against human cancer cell lines (HL60). Impact of the side chain connected to the
aromatic D ring and to the N6 lactam position on the biological profile will be discussed.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 17 January 2011
Revised 24 February 2011
Accepted 25 February 2011
Available online 1 March 2011
Keywords:
Cancer
DNA intercalators
Topoisomerases
Indenoisoquinolines
Topoisomerase inhibitors occupy a place of choice in the arsenal
of current chemotherapy strategies involved in the four-decade-
long war against cancer,1 a disease that is estimated to have affected
12.7 million people and to have taken away 7.6 million lives world-
wide in 2008.2 The identification of this ubiquitous enzyme in the
early 1970s3,4 led to the development of an array of DNA-intercalat-
ing and/or cytotoxic molecules capable of inhibiting the topoisome-
rases. FDA-approved topotecan5 (HYCAMTINÒ) falls into this class of
compounds. It targets the single DNA strand regulating-topoisomer-
ase type I (Topo I), whereas constitutionally diverse models such as
Consequently a variety of new compounds overcoming such
limitations and drawbacks were extensively developed during
the late 1980s and 1990s, leading to the discovery by the National
Cancer Institute of NSC 314622, a representative example of the
class of indenoisoquinoline derivatives (Fig. 2).11 Several struc-
ture–activity relationships and pharmacomodulations on the lead
compound then led to the development of structurally diverse ana-
logs with improved pharmacological activities on Topo I,12 as
exemplified by indimitecan or indotecan.13–17 Recently an array
of new indenoisoquinoline derivatives including 118 and 219 were
shown to display good inhibition capacities on Topo II. Addition-
ally, structurally related analogs such as indeno[1,2-c]quinolines
3 have recently proved to be potent dual inhibitors of Topo I and
Topo II (Fig. 2).20–22
daunorubicin/daunomycin6
(CERUBEDINEÒ),
mitoxantrone7
(NOVANTRONEÒ) and etoposide8 (VEPESIDÒ) interfere with the
duplex regulating-topoisomerase type II (Topo II) (Fig. 1).
Despite their efficiency, the therapeutic activity of these com-
mercialized drugs is fraught with pharmacokinetic flaws, undesir-
able side effects and resistance from tumor cells, most widely by
action of membrane ATP-dependent multidrug efflux pump
mediated by P-glycoprotein (MDR1, ABCB1).9 Besides, the poten-
cies of Topo I inhibitors are seriously compromised by their chem-
ical instabilities whilst, in case of Topo II inhibitors, the efficiencies
of the drugs are lowered by the reduction of the expression of Topo
II and by mutations of the targeted enzyme.10
In a previous study from our group,18 we reported that the most
potent compound on Topo II in this series was a tetracyclic model
equipped with two basic tertiary amino groups appended to the C8
and N6 lactam positions and linked to the tetracyclic intercalant
moiety through ethoxy and ethyl group spacers respectively. We
then surmised that a more thorough investigation of influence of
multifarious aromatic D-ring substitution patterns coupled with
varied lactam side chain at N6 could help to get better insight
about Topo II inhibition activity. For this purpose, an array of mod-
els tailed with constitutionally diverse dimethylaminoalkyl chains
at C7, C8 and C9 positions coupled with suitable spacing linkers at
the N6 lactam position of the tetracyclic template were initially de-
⇑
Corresponding author.
E-mail address: gangahn3@gmail.com (G. Ahn).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.106

2260
G. Ahn et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2259–2263
R4
R3
CH3
N
O
4
O
OH
CH3
HO
a
b
H
CH3
H
3
OH
2
HO
R2
O
O
O
N
4: R2 = O-(CH2)2-N(CH3)2; R3, R4 = H
2-, 3-, or 4-
hydroxybenzaldehyde
OCH3O
OH
O
O
CH3
OH
N
5: R2 = O-(CH2)3-N(CH3)2; R3, R4 = H
O
6: R2, R4 = H; R3 = O-(CH2)2-N(CH3)2
: R , R4 = H; R3 = O-(CH2)3-N(CH3)2
2
C2H5
7
8
: R , R3 = H; R4 = O-(CH2)3-N(CH3)2
2
OH O
NH2
R4
R3
topotecan
daunorubicin/daunomycin
O
c
+
O
R2
N
OH
R1
O
HO
O
O
CH3
11: R1 = (CH2)2-N(CH3)2; R2 = O-(CH2)2-N(CH3)2; R3, R4 = H
H
N
O
12: R1 = (CH2)3-N(CH3)2; R2 = O-(CH2)2-N(CH3)2; R3, R4 = H
OH
OH
O
O
HN
HN
OH
OH
O
: R = (CH2)2-N(CH3)2; R2 = O-(CH2)3-N(CH3)2; R3, R4 = H
1
13
14
15
16
17
18:
19
20
: R = (CH2)3-N(CH3)2; R2 = O-(CH2)3-N(CH3)2; R3, R4 = H
1
O
: R = (CH2)4-N(CH3)2; R2, R4 = H; R3 = O-(CH2)2-N(CH3)2
1
: R = (CH2)2-N(CH3)2; R2, R4 = H; R3 = O-(CH2)3-N(CH3)2
1
O
: R = (CH2)3-N(CH3)2; R2, R4 = H; R3 = O-(CH2)3-N(CH3)2
1
N
H
R
1 = (CH2)4-N(CH3)2; R2, R4 = H; R3 = O-(CH2)3-N(CH3)2
: R = (CH2)2-N(CH3)2; R2, R3 = H; R4 = O-(CH2)3-N(CH3)2
1
H3CO
OCH3
: R = (CH2)3-N(CH3)2; R2, R3 = H; R4 = O-(CH2)3-N(CH3)2
1
OH
R4
R3
mitoxantrone
etoposide
HO
O
N
d, e
Figure 1. Structures of FDA-approved Topo I and Topo II inhibitors.
R2
R1
O
O
21: R1 = (CH2)2-N(CH3)2; R2 = O-(CH2)2-N(CH3)2; R3, R4 = H
22: R1 = (CH2)3-N(CH3)2; R2 = O-(CH2)2-N(CH3)2; R3, R4 = H
23: R1 = (CH2)2-N(CH3)2; R2 = O-(CH2)3-N(CH3)2; R3, R4 = H
24: R1 = (CH2)3-N(CH3)2; R2 = O-(CH2)3-N(CH3)2; R3, R4 = H
25: R1 = (CH2)4-N(CH3)2; R2, R4 = H; R3 = O-(CH2)2-N(CH3)2
26: R1 = (CH2)2-N(CH3)2; R2, R4 = H; R3 = O-(CH2)3-N(CH3)2
O
O
O
O
D
O
C
N
H3CO
H3CO
H3CO
H3CO
A
B
N
R1
CH3
: R1 = (CH2)3-N(CH3)2; R2, R4 = H; R3 = O-(CH2)3-N(CH3)2
27
O
O
28:
29
R
1 = (CH2)4-N(CH3)2; R2, R4 = H; R3 = O-(CH2)3-N(CH3)2
NSC 314622
: R1 = (CH2)2-N(CH3)2; R2, R3 = H; R4 = O-(CH2)3-N(CH3)2
: R1 = (CH2)3-N(CH3)2; R2, R3 = H; R4 = O-(CH2)3-N(CH3)2
(CH2)3
(CH2)3
N
indimitecan:
indotecan:
N
30
R4
N
O
9
O
8
R3
R3
7 R2
9
D
7
O
C
N
R1
8
N
R2
O
31: R1 = (CH2)2-N(CH3)2; R2 = O-(CH2)2-N(CH3)2; R3, R4 = H
32: R1 = (CH2)3-N(CH3)2; R2 = O-(CH2)2-N(CH3)2; R3, R4 = H
33: R1 = (CH2)2-N(CH3)2; R2 = O-(CH2)3-N(CH3)2; R3, R4 = H
34: R1 = (CH2)3-N(CH3)2; R2 = O-(CH2)3-N(CH3)2; R3, R4 = H
35: R1 = (CH2)4-N(CH3)2; R2, R4 = H; R3 = O-(CH2)2-N(CH3)2
36: R1 = (CH2)2-N(CH3)2; R2, R4 = H; R3 = O-(CH2)3-N(CH3)2
A
B
O
N
OCH3
N
R1
O
: R1 = (CH2)2-N(CH3)2; R2 = O-(CH2)2-N(CH3)2; R3 = H
1
: R1 = (CH2)2-N(CH3)2; R2 = R3 = H
POM02
: R1 = (CH2)3-N(CH3)2; R2, R4 = H; R3 = O-(CH2)3-N(CH3)2
37
38:
39
40
POM03: R1 = (CH2)3-N(CH3)2; R2 = R3 = H
2: R1 = (CH2)2-NH-C(O)-Arg.2HCl, R2 = R3 = H
N
OH
1
R
= (CH2)4-N(CH3)2; R2, R4 = H; R3 = O-(CH2)3-N(CH3)2
3
: R1 = (CH2)2-N(CH3)2; R2, R3 = H; R4 = O-(CH2)3-N(CH3)2
: R1 = (CH2)3-N(CH3)2; R2, R3 = H; R4 = O-(CH2)3-N(CH3)2
Figure 2. Structures of potent indenoisoquinolines.
Scheme 1. Reagents and conditions: (a) appropriate dimethylamino alkyl chloride
Cl-(CH2)n-N(CH3)2 (n = 2 or 3), K2CO3,DMF, reflux, 6 h; (b) R1-NH2, MgSO4, CHCl3, rt,
18 h. For the synthesis of compounds 15 and 18, TEA was also added; (c) THF
(MeOH for the synthesis of compounds 25, 26 and 28), 0 °C, 1 h; (d) SOCl2, reflux,
18 h; (e)AlCl3, CH2Cl2, 0 °C, 3 h.
signed. These newly synthetized compounds were subsequently
evaluated for DNA interaction measurements, Topo I and Topo II
inhibition activities and for cytotoxicity against HL60 sensitive
and resistant leukemic cell lines.
The targeted indenoisoquinoline-dione derivatives 31–40 were
readily assembled as depicted in Scheme 1.18 The first fact of the
synthesis was the preliminary O-alkylation of 2-, 3- or 4-hydroxy-
benzaldehyde with 2-dimethylaminoethyl or 3-dimethylaminopro-
pyl chlorides afforded the corresponding hydroxybenzaldehyde
derivatives 4–8. Subsequent condensation of benzaldehyde analogs
4–8 with dimethylaminoethyl-, propyl- and butylamine provided
the imines 11–20, which were then allowed to react with homoph-
thalic anhydride to afford a diastereomeric mixture of 3-aryl-4-car-
boxyisoquinolones 21–30. The cis/trans ratio of these isoquinolones
ranged from 1:2 to 1:6.6 in favor of the trans-diastereoisomer. It is
likely that the formation of these lactamic compounds proceeds
via the mechanism proposed by Cushman and co-workers23 and
Gonzalez-Lopez et al.24 but it is worth mentioning that this mecha-
nism could not account for the favored formation of the trans-dia-
stereoisomer when increasing the side chain length.
It is also noteworthy that these diastereoisomers could not be
separated despite many attempts with multiple purification condi-
tions, as already previously mentioned.14,18 Subsequent treatment
of the mixture with thionyl chloride followed by intramolecular

G. Ahn et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2259–2263
2261
4
3
chain on the aromatic D ring or on the N6 lactam side chain re-
sulted in an upward shift in Tm values. This trend was also ob-
served for the N-substituted indenoisoquinoline derivatives (49
vs 50). However, further increase in the N6 lactam side chain
HO
a
H
D
H
R3
2
R2
O
O
41: R2 = O-C(O)-Ph; R3 = H
2- or 3-
hydroxybenzaldehyde
42: R2 = H; R3 = O-C(O)-Ph
length did not end up with the highest
DTm values (36, 37 vs
38), suggesting that the presence of three methylene units may
be the optimum for the duplex stabilization.
The competitive ethidium displacement method was applied to
our newly designed compounds.26–28 Apparent DNA binding affin-
ities Kapp using CT DNA were quantified by means of fluorescence.
The values of compounds 48–50 could not be obtained due to the
high insolubility of the molecules. The Kapp values ranged from
25.69 ꢁ 106 Mꢀ1 to 130.28 ꢁ 106 Mꢀ1 (Table 1). C7-aminoalkoxy-
substituted indenoisoquinolines 31–34 displayed the highest Kapp
values, followed by compounds bearing the aromatic D ring side
chain at C9 position (33 vs 39 and 34 vs 40). And similar to what
O
O
R3
R3
b
R2
R2
N
N
R1
R1
O
O
43: R1 = (CH2)2-N(CH3)2; R2 = O-C(O)-Ph; R3 = H
44: R1 = (CH2)3-N(CH3)2; R2 = O-C(O)-Ph; R3 = H
45: R1 = (CH2)2-N(CH3)2; R2 = H; R3 = O-C(O)-Ph
46: R1 = (CH2)3-N(CH3)2; R2 = H; R3 = O-C(O)-Ph
: R1 = (CH2)3-N(CH3)2; R2 = OH; R3 = H
: R1 = (CH2)2-N(CH3)2; R2 = H; R3 = OH
: R1 = (CH2)3-N(CH3)2; R2 = H; R3 = OH
48
49
50
was observed for
DTm values, C8-aminoalkoxy-substituted coun-
Scheme 2. Reagents and conditions: (a) benzoyl chloride, TEA, CH2Cl2; (b) R1-NH2,
MgSO4, CHCl3, rt, 18 h; (c)homophthalic anhydride, THF or MeOH, 0 °C, 1 h; (d)
SOCl2, reflux, 18 h; (e) AlCl3, CH2Cl2, 0 °C, 3 h; (f) LiOH, CH2Cl2/MeOH (1:1), rt, 1 h.
terparts exhibited the lowest affinities, with Kapp values 2.5- to
2.7-fold less than those of C9-aminoalkoxy counterparts (39 vs
36 and 40 vs 37). Incrementing one methylene unit either on D ring
side chain or on the N6-lactam side chain resulted, once again, in
the increase in Kapp values. But the amplitude of the variation de-
pended on the position of the side chain on which the addition
of a CH2 group was performed. In fact, increasing the length of
the N6-lactam side chain did not bring significant variations in Kapp
values (31 vs 32, 33 vs 34, 36 vs 37 and 38). The previous study
showed also this trend.18 On the other hand, increase in the D ring
side chain length brought about significant raise in the DNA affin-
ity constants (31 vs 33, 32 vs 34). These results show that, unlike
Table 1
Variations in
D
Tm and in apparent DNA affinity constants
a
b
Compound
D
Tm poly(dAT)2
Kapp 106 Mꢀ1
Daunorubicin
21.9c
30.5
42.7
44.7
41.3
33.9
31.4
40.6
37.1
36
—
31
32
33
34
35
36
37
38
39
40
48
49
50
55.79
52.67
130.28
107.67
25.69
28.71
33.38
41.63
71.99
89.31
—
D
Tm values, lengthening the spacer up to four methylene units
did produce highest Kapp values (36 and 37 vs 38).
A conventional DNA relaxation assay was used to assess the ef-
fects of the compounds in the three series on the catalytic activity
of recombinant Topo I (Fig. 3A).29 Negatively supercoiled plasmid
DNA was first treated with the enzyme in the presence of the
tested drug at different concentrations and the DNA relaxation
products were then resolved by electrophoresis. All tested com-
46.9
17.1
7.7
—
—
16.6
a
drug-DNA complex
alone
Variation in melting temperature
Drug/DNA ratio = 0.25.
D
Tm = Tm
ꢀ TmDNA
.
pounds modified the relaxation of the DNA at 1 lM or higher con-
b
Apparent binding constant measured by fluorescence using [BET]/[DNA] = 1.26,
centrations of the INDO. But the compounds were not able to
stabilize the cleavage complex, as opposed to CPT. Nevertheless,
these results were consistent with the binding (unwinding of
closed circular duplex DNA) of our compounds through intercala-
tion mode on DNA.
Kapp in 106 Mꢀ1
.
c
D
Tm of daunorubicin measured at ratio = 1. Kapp of this compound could not be
measured due to the interferences of fluorescence spectra of DNA and BET.
Friedel–Crafts acylation delivered the targeted N6- and D ring-
substituted indenoisoquinolines 31–40, albeit in moderate yield.25
The low overall yield may be attributable to the deleterious forma-
tion of diastereoisomeric isoquinolones not prone to cyclize,
namely those lacking electron-donating groups on the A ring, asso-
ciated with the generation of the regioisomers as byproducts upon
the ultimate intramolecular acylation reaction. The phenolic deriv-
atives 48–50 were readily constructed following a similar strategy
combined with an alkaline hydrolysis reaction at the final stage to
remove the benzoxylcarbonyl protecting group (Scheme 2).
The ability of the drugs to protect poly(dAT)2 DNA against ther-
mal denaturation was used as an indicator of the relative capacity
of the new molecules to bind and to stabilize the DNA double helix.
The same tests, with Topo I being replaced with Topo II, were
performed to evaluate effects on the catalytic activity of Topo II.
In these experiments supercoiled plasmid DNA was treated with
Topo II in the presence of graded concentrations of the tested drug
and the DNA relaxation products were then resolved by electro-
phoresis. The reference drug, etoposide, produced a marked level
of DNA double stranded breaks, corresponding to linear DNA
(Fig. 3B and C). The activity of the compounds was expressed semi-
quantitatively as follows on Table 2: 0: no specific inhibition activ-
ity, + and ++: weak activity, +++: good activity, ++++ as active as
etoposide. Inhibition of Topo II was clearly detected with our
new derivatives. Etoposide produced a marked level of DNA double
stranded breaks (linear DNA).
The agarose gel resulting from the assay revealed that some of
our C8-substituted analogs 36 and 37 showed the ability to stabi-
lize the cleavage complex DNA-Topo II (Fig. 3B). None of C7 and
C9-aminoalkoxy-substituted INDOs was able to specifically inhibit
Topo II. As illustrated in the Figure 3C, the previously reported
indenoisoquinoline 1 displayed the same potency as the etoposide
(++++), whereas compounds 36 and 37 exhibited good potencies
(+++) in Topo II inhibition among newly synthesized derivatives.
The length of the N6 lactam side chain had an impact on the Topo
II inhibition abilities, as compounds 35 (+) and 38 (+) were shown
These experiments were perfomed in poly(dAT)2 especially rich in
drug-DNA complex
AT. Variations of
D
Tm = Tm
ꢀ TmDNA alone ranged from
7.7 °C to 46.9 °C (Table 1). As expected, indenoisoquinolines 48–50,
bearing phenolic functions and tested as control molecules, dis-
played lower
DTm values (7.7–17.7 °C) than the analogs 31–40
bearing two cationic side chains (29–46.9 °C). C8-aminoalkoxy-
substituted products were found to less stabilize DNA from heat
denaturation compared to their C7-aminoalkoxy- and C9-amin-
oalkoxy counterparts (36 vs 33 vs 39 and 37 vs 34 vs 40). Insertion
of an additional methylene unit either on the aminoalkoxy side

2262
G. Ahn et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2259–2263
Figure 3. (A) Effects of the compounds 34, 37, 39 and 40 on the relaxation of plasmid DNA by Topo I. Native supercoiled pUC19 (130 ng, lane DNA) was incubated with 8 units
of Topo I in the absence (lane Topo I) or presence of tested compounds at the indicated concentration (1–20 M). Camptothecin (CPT) was used at 20 M. DNA samples were
l
l
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. B and C. Effects of novel indenoisoquinoline derivativeson the relaxation of plasmid DNA by Topo II. Native supercoiled
pUC19 (350 ng, lane DNA) was incubated with 8 units of Topo II in the absence (lane Topo II) or presence of tested compounds at the indicated concentration (B: 50
lM and C:
20–100 M). Etoposide was used at indicated concentrations. DNA samples were separated by electrophoresis on a 1% agarose gel containing 1 g/mL ethidium bromide. Gels
l
l
were photographed under UV light. Nck: nicked; Sc: supercoiled; Lin: linear; Rel: relaxed.
The antiproliferative activities of our compounds were tested
using two human leukemia30 cell lines, HL60 and HL60/MX2,
respectively sensitive and resistant to the antitumor drug mitoxan-
trone. The assays on HL60/MX2 cell line, displaying altered cata-
lytic activity and reduced levels of Topo II, were performed to
gain an insight into the involvement of Topo II inhibition in the
cytotoxicity of our derivatives. Table 2 shows that most of our
newly synthesized compounds 31–40, 48–50 displayed micromo-
lar IC50 values, ranging from 0.625 lM to 5.6 lM. The most cyto-
toxic compound 50 presented submicromolar IC50 value, close to
that of 1, which remained our most potent compound in HL60 cell
line (0.66 lM, ++++). No obvious relationship could be observed
Table 2
Cytotoxicities and Topo II inhibitory activity of the novel INDOs
Composé
IC50 (in
l
M)
RRIb
Topo II inhibition
HL60a
HL60/MX2
MTX
Eto
1
0.063 0.020
1.4 0.0
0.66 0.02
1.10 0.10
1.4 0.0
2.6 0.0
5.60 0.25
3.7 0.2
1.1 0.0
1.00 0.14
2.2 0.3
2.8 0.5
5.05 0.05
—
1.51 0.08
15.75 1.85
1.5 0.3
24
—
11.25
2.3
1.2
1.4
0.9
0.9
1.85
1.3
3.6
1.2
1.1
0.6
—
++++
++++
0
0
0
0
+
+++
+++
+
0
0
—
++
++
31
32
33
34
35
36
37
38
39
40
48
49
50
1.3 0.10
2.4 0.0
2.3 0.2
4.90 0.25
6.85 0.25
1.4 0.0
3.6 1.0
2.65 0.05
3.2 0.0
between the structures of the indenoisoquinoline derivatives and
cytotoxicities. The inhibition of cleavage complex is unlikely the
main contributor to the high cytotoxicity of compound 50
3.5 0.2
(0.625 lM), thus suggesting the possible involvement of additional
—
targets other than Topo II or differences in cellular uptake. The
weak resistance index RRI obtained for our derivatives may be ex-
plained by their binding to the enzyme at a site different from that
of mitoxantrone or by specific kinetics. As for cell cycle effects and
apoptosis, in light of the work on structurally related analogs from
other groups, one possibility could be the cell cycle arrest, by our
products, either in the S or the G2-M phase, depending on the con-
centration of the drug, whilst the cytotoxicity is p53-dependant.
Cell cycle arrest in sub-G1 phase, as shown by Tseng et al. with
the indeno[1,2-c]quinolines could be another possibility, subse-
quently followed by caspase-3 activation and PARP cleavage.22
Phosphorylation of the histone protein H2AX at Ser139 residue fol-
lowing DNA double-strand break event can also be expected from
our compounds.22,30
In conclusion, 13 structurally and constitutionally new analogs
of the indeno[1,2-c]isoquinoline 1 have been designed and evalu-
ated against DNA, Topo II and HL60 cell line. Despite the fact that
no lead compound could emerge from this study, the C8 position
has been confirmed as the sole position on the D-ring capable of
inhibiting the topoisomerase by stabilizing the ternary cleavage
complex. C7 and C9 positions, more favorable to the binding to
the DNA, are not active against Topo II. The most potent compound
remains the lead 1 whereas analogs 36 and 37 equipped with an
aminopropoxy side chain on the D ring displayed also good poten-
1.4 0.0
0.625 0.050
4.15 0.35
1.55 0.35
2.9
2.4
a
The cytotoxicity IC50 values are the concentrations corresponding to 50%
growth inhibition.
ðMX-resistantÞ
ðMX-sensitiveÞ
b
Relative resistance index: IC
=IC
.
50
50
to be poor Topo II inhibitors (data not shown). As for the 8-hydrox-
ylated indenoisoquinolines 49 and 50, they also displayed weak
Topo II inhibition (++), emphasizing that the aromatic D ring side
chain seems to play a major role in Topo II inhibition (data not
shown). These results undoubtedly illustrate the prominence of
the C8 position on the D ring in Topo II inhibition. C7 and C9 substi-
tuted derivatives did not show any inhibition activities. The side
chains lengths were proved to be key-points for the inhibition
activity. The optimal side chain length at both aromatic D ring
and N6-lactam positions seems to be two methylene units. Three
methylene units were well tolerated, except in the case of models
with an INDO bearing three methylene units at the N6 lactam po-
sition and two CH2 groups in the C8 position of the aromatic D ring,
as previously observed.18 On the other hand, spacers longer than
four CH2 groups were proved to be deleterious for the inhibition
activity, as evidenced by indenoisoquinolines 35 and 38. The tests
also revealed no connections between DNA affinities and Topo II
inhibition abilities.

G. Ahn et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2259–2263
2263
13. Nagarajan, M.; Morrell, A.; Ioanoviciu, A.; Antony, S.; Kohlhagen, G.; Agama, K.
K.; Hollingshead, M.; Pommier, Y.; Cushman, M. J. Med. Chem. 2006, 49, 6283.
14. Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. J. Med. Chem.
2006, 49, 7740.
15. Morrell, A.; Placzek, M.; Parmley, S.; Grella, B.; Antony, S.; Pommier, Y.;
Cushman, M. J. Med. Chem. 2007, 50, 4388.
cies against Topo II and HL60 cell line. These results suggested that
Topo II inhibition may be favored with shorter N6 lactam side
chain. Lengthening the N6 side chain beyond three methylene
units proved to be deleterious to the Topo II inhibition activity.
No correlation has been observed between DNA binding ability,
Topo II inhibition and cytotoxicity, suggesting Topo II is not the
only mechanism involved in the antiproliferative activity.
16. Morrell, A.; Placzek, M.; Parmley, S.; Antony, S.; Dexheimer, T. S.; Pommier, Y.;
Cushman, M. J. Med. Chem. 2007, 50, 4419.
17. Pommier, Y.; Cushman, M. Mol. Cancer Ther. 2009, 8, 1008.
18. Ryckebusch, A.; Garcin, D.; Lansiaux, A.; Goossens, J. F.; Baldeyrou, B.; Houssin,
R.; Bailly, C.; Hénichart, J. P. J. Med. Chem. 2008, 51, 3617.
19. Ahn, G.; Lansiaux, A.; Goossens, J. F.; Bailly, C.; Baldeyrou, B.; Schifano-Faux, N.;
Grandclaudon, P.; Couture, A.; Ryckebusch, A. Bioorg. Med. Chem. 2010, 18,
8119.
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