Synthesis and antitumor activity of some indeno[1,2-b]quinoline-based bis carboxamides

Article abstract of DOI:10.1016/S0968-0896(00)00039-0

A series of bis(11-oxo-11H-indeno[1,2-b]quinoline-6-carboxamides) linked through the 6-carboxamides were prepared by coupling the requisite acid imidazolides with various diamines. Compounds with mono-cationic linker chains were more potent cytotoxins than the corresponding monomer in a panel of rodent and human cell lines, while those with the dicationic linker chains (CH₂)₂NR(CH₂)₂NR(CH₂)₂ and (CH₂)₂NR(CH₂)₃NR(CH₂)₂ showed extraordinarily high potencies (for example, IC₅₀s of 0.18-1.4 nM against human Jurkat leukemia; up to 1000-fold more potent than the parent monomer). As seen previously in the monomeric series, small, lipophilic 4-substituents significantly increased potency in cell culture. The dimeric compounds were all slightly to significantly more potent in the mutant JL(A) and JL(D) cell lines that under-express topo II, suggesting that this enzyme is not their primary target. An 11-imino-linked dimer was much less active, and an asymmetric indeno[1,2-b]quinoline-6-carboxamide/naphthalimide dimer was less active than the comparable symmetric bis(indeno[1,2-b]quinoline-6- carboxamide). Selected analogues were active against sub-cutaneously implanted colon 38 tumors in mice, giving growth delays comparable to that of the clinical topo I inhibitor irinotecan at up to 10-fold lower doses. These compounds form an interesting new class of putative topo I inhibitors. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00039-0

Bioorganic & Medicinal Chemistry 8 (2000) 977±984
Synthesis and Antitumor Activity of Some
Indeno[1,2-b]quinoline-based bis Carboxamides
Leslie W. Deady, a,* Jose Desneves, a Anthony J. Kaye, a Graeme J. Finlay, b
Bruce C. Baguley b and William A. Denny b
aDepartment of Chemistry, La Trobe University, Bundoora, Victoria 3083, Australia
bAuckland Cancer Society Research Laboratory, Faculty of Medical and Health Sciences, The University of Auckland,
Private Bag 92019, Auckland 1000, New Zealand
Received 21 September 1999; accepted 7 December 1999
AbstractÐA series of bis(11-oxo-11H-indeno[1,2-b]quinoline-6-carboxamides) linked through the 6-carboxamides were prepared by
coupling the requisite acid imidazolides with various diamines. Compounds with mono-cationic linker chains were more potent
cytotoxins than the corresponding monomer in a panel of rodent and human cell lines, while those with the dicationic linker chains
(CH2)2NR(CH2)2NR(CH2)2 and (CH2)2NR(CH2)3NR(CH2)2 showed extraordinarily high potencies (for example, IC50s of 0.18±
1.4 nM against human Jurkat leukemia; up to 1000-fold more potent than the parent monomer). As seen previously in the mono-
meric series, small, lipophilic 4-substituents signi®cantly increased potency in cell culture. The dimeric compounds were all slightly
to signi®cantly more potent in the mutant JLA and JLD cell lines that under-express topo II, suggesting that this enzyme is not their
primary target. An 11-imino-linked dimer was much less active, and an asymmetric indeno[1,2-b]quinoline-6-carboxamide/naph-
thalimide dimer was less active than the comparable symmetric bis(indeno[1,2-b]quinoline-6-carboxamide). Selected analogues were
active against sub-cutaneously implanted colon 38 tumors in mice, giving growth delays comparable to that of the clinical topo I
inhibitor irinotecan at up to 10-fold lower doses. These compounds form an interesting new class of putative topo I inhibitors.
# 2000 Elsevier Science Ltd. All rights reserved.
Introduction
relationships for ring substitution broadly similar to
those for the monomers. Small lipophilic 5-substituents
provided compounds of the highest potency (e.g. 3; IC50
of 2 nM against the Lewis lung carcinoma).9
A number of recent reports have delineated a new class
of anti-cancer drugs, comprised of dimeric forms of
lipophilic, neutral, DNA-intercalating chromophores
joined by a ¯exible cationic or dicationic linker chain.
The chromophores employed include naphthalimides,1 4
benzonaphthalimides,5 imidazoacridinones,6,7 anthracy-
clines,8 acridines9 and phenazines.10 In all of the above
cases, while the corresponding monomeric compounds
are active cytotoxins and topoisomerase inhibitors, the
best dimeric compounds are considerably more cyto-
toxic, and many show excellent activity against a variety
of human solid tumor cell lines, both in culture and as
xenografts in nude mice. Two compounds of the bis-
(naphthalimide) series; DMP 840 and LU 79553 (2) are
reported to be in clinical trial.3,4 A series of substituted
We recently reported the development of a new class of
tetracyclic 11-oxo-11H-indeno[1,2-b]quinoline-6-carb-
oxamides (4) as potent cytotoxins and potential dual
topo I/II inhibitors.12,13 We now report the synthesis
and initial biological evaluation of a series of symme-
trical dimeric analogues of this chromophore (5a±5l),
together with three miscellaneous analogues (6±8).
Results
Preparation of the required precursor acids 9±11 has
been described.13 The isomeric acid 15 has been pre-
pared by an improvement of the original procedure12
(Scheme 1). The bis(amides) 5 and 8 were prepared by
activation of the appropriate acid to the corresponding
imidazolide, and coupling this with half an equivalent of
the required diamine14 (Scheme 2). The intermediate
imidazolide (12: RˆH) from acid 9 was isolated, but
bis(acridine-4-carboxamides linked by
(Me)(CH2)3 chain were signi®cantly more cytotoxic than
their monomeric analogues,11 with structure±activity
a (CH2)3N
*Corresponding author. Tel.: +61-3-9479-2561; fax: +61-3-9479-
1399; e-mail: l.deady@latrobe.edu.au
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00039-0

978
L. W. Deady et al. / Bioorg. Med. Chem. 8 (2000) 977±984
Scheme 2. (i) CDI/dioxan/re¯ux/3 h; (ii) diamine/CH2Cl2/20 ꢀC/24 h.
Scheme 3. (i) H2NO(CH2)6ONH2/5% HCl/re¯ux/3 h.
others were reacted in situ with the required diamine.
The bisoxime (6) was prepared from the known12 amide
4 and O,O0-1,6-hexanediylbishydroxylamine in acid
conditions (Scheme 3). Most of the polyamine linkers
were commercially available. N,N0-bis(2-Aminoethyl)-
N,N0-dimethyl-1,2-ethane- (and 1,3-propane) diamines,
though both known,5,15 were prepared by a common
procedure, slightly di€erent to the existing literature.
N,N0-dimethylethane- and propane-diamines were reacted
with chloroacetonitrile, then with borane by a literature
procedure16 for nitrile reduction. An unsymmetrical bis
compound 7, which combined both the above indeno-
quinoline subunit and the naphthalimide subunit of LU
79553 (2) was also prepared, by reacting N-[3-[4-(3-
aminopropyl)piperazin-1-yl]propyl]naphthalimide (pre-
pared from 1,8-naphthalic anhydride) with the imida-
zolide (12: RˆH).
leukemias. JLC is the wild-type (sensitive) line, while
JLA is resistant to the DNA intercalator amsacrine and
similar agents (85-fold resistant to amsacrine) by virtue
of a reduced level of topo II enzyme, and JLD is a dox-
orubicin-resistant line, primarily by virtue of altered
levels of topo II, but probably also by additional
mechanisms.17,18 IC50 values are given for the P388,
LLTC and JLC lines, together with ratios of IC50 values
against JLC and the other two Jurkat lines (JLA/JLC
and JLD/JLC). Values of these ratios of less than about
2-fold suggest a non-topo II mediated mechanism of
action.
Compounds 5a, 5b and 5l have mono-cationic linker
chains. The one (5a) with the shortest (5-atom) linker,
was less e€ective than the monomer 4, but those with
the longer linkers (5b; 7 atoms) and (5l; 8 atoms) were
signi®cantly more potent. The pairs of dicationic com-
pounds (5c/5d and 5e/5g) are of particular interest. In
the ®rst pair of compounds, linked by a (CH2)2NR
(CH2)2NR(CH2)2 chain, the RˆH analogue 5c showed
activity comparable to 5b, with the RˆMe compound
5d being about 2±20-fold less cytotoxic. The second pair
of compounds, with (CH2)2NR(CH2)3NR(CH2)2 linkers
(an extra atom in the chain), had signi®cantly higher
cytotoxicities, but in this case the R±Me compound 5g
was overall about 2-fold more cytotoxic than the RˆH
example. While the interpair di€erences are not large,
they show the similar trends to those seen earlier with
related pairs of bis(naphthalimides).4 In particular, 5e
and 5g exhibit very high potency (sub-nM) against the
human leukemia line JLC.
Discussion
Table 1 shows IC50 values for the compounds in a panel
of cell lines in culture. As discussed previously,11,12 P388
is a murine leukemia, LLTC is a late-passage murine
Lewis lung carcinoma, and the Jurkat lines are human
Compounds 5e/5f and 5g±5i explore the consequences
of substitutions in the 4-position of the chromophore.
Scheme 1. (i) KMnO4; (ii) heat ca. 300 ꢀC.

L. W. Deady et al. / Bioorg. Med. Chem. 8 (2000) 977±984
979
Table 1. Growth inhibitory data for the indeno[1,2-b]quinoline-based bis carboxamides
a
IC50
Ratiosb
P388c
LLd
JLC
JLAf/JLC
JLDg/JLC
e
No.
Fm
R
X
4h
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
6
130
1188
34
34
545
26
88
22
7.9
23
7200
40
55
91
240
12
14
33
180
165
23
2.5
21
0.75
1.4
0.35
0.18
0.36
155
2.3
84
1100
21
1.2
0.4
0.3
0.2
0.3
0.3
0.55
0.3
0.4
0.6
0.2
0.3
0.1
0.8
1.0
0.45
0.3
0.9
0.7
0.5
0.5
0.5
0.5
0.65
0.4
0.6
0.4
1.0
0.4
1.0
0.6
0.5
1.1
0.6
A
A
A
A
A
A
A
A
A
A
A
A
B
H
H
H
H
H
Me
H
Me
Cl
H
(CH2)2NH(CH2)2
(CH2)3NMe(CH2)3
(CH2)2NH(CH2)2NH(CH2)2
(CH2)2NMe(CH2)2NMe(CH2)2
(CH2)2NH(CH2)3NH(CH2)2
(CH2)2NH(CH2)3NH(CH2)2
(CH2)2NMe(CH2)3NMe(CH2)2
(CH2)2NMe(CH2)3NMe(CH2)2
(CH2)2NMe(CH2)3NMe(CH2)2
(CH2)3NH(CH2)2NH(CH2)3
(CH2)3NpipN(CH2)3
4.7
5.0
2.7
0.69
1.1
550
2.4
27
680
11
6.5
11
H
H
(CH2)3NH(CH2)4
925
130
23
7
C
D
8
2 (LU 79553)
0.8
0.7
57
aIC50, concentration of drug (nM) to reduce cell number to 50% of control cultures (see text). The value is the average of at least two independent
determinations; the coecient of variation was ca. 25%.
bRatios of IC50s in the cell lines shown.
cMurine P388 leukemia.
dMurine Lewis lung carcinoma.
eJLC, wild-type human Jurkat leukemia.
fJLA, amsacrine-resistant Jurkat.
gJLD, doxorubicin-resistant Jurkat.
hData from ref 12.
In previous structure±activity studies13 on the mono-
meric compounds, we showed that small, lipophilic 4-
substituents substantially increased cytotoxicity (up to
8-fold, with the 4-Me and 4-Cl analogues showing IC50s
of 35 and 55 nM, respectively, in the JLC line, compared
with 180 nM for 4 itself). In the dimeric series the 4-
substituents had lesser but still positive e€ects on abso-
lute potencies in the (CH2)2NR(CH2)3NR(CH2)2 series,
with 5h and 5i showing IC50s in JLC of 0.18 nM (an
increase of 1000-fold over 4). Extending the linker chain
further, in the (CH2)3NH(CH2)3NH(CH2)3 analogue 5j,
led to a substantial loss of cytotoxicity (e.g. IC50 in JLC
155 nM compared with 0.75 nM for the (CH2)2NH
(CH2)3NH(CH2)2 analogue 5e). As seen previously with
bis(DACA) derivatives,9 these dimeric compounds all
proved to be slightly to signi®cantly (up to 10-fold)
more potent in the mutant JLA and JLD cell lines that
under-express topo II. In contrast, the corresponding
monomeric compounds show essentially equivalent
activity in the wild-type and mutant cell lines.12 These
results suggest that the dimeric analogues do not act
through topo II, and are likely to be primarily topo I
inhibitors.
The 11-imino-linked dimer 6 was prepared to compare
the possible utility of linking the two chromophores in a
di€erent geometry, but proved much less active. The
asymmetric dimer 7 is a hybrid of the 11-oxo-11H-
indeno[1,2-b]quinoline-6-carboxamide and naphthali-
mide chromophores. While symmetric bis(naphthali-
mides) (e.g. 2) are potent cytotoxins, the hybrid dimer 7
was less active than the similarly-linked bis(11-oxo-11H-
indeno[1,2-b]quinoline-6-carboxamide) 5k. Finally, the
isomeric bis(6-oxo-6H-indeno[2,1-b]quinoline-4-carbox-
amide) (8) was 2±4-fold less potent in the cell line panel
than the corresponding 11-oxo analogue 5g. In the
initial study of the monomers,12 the 6-oxo isomer also
proved somewhat less active.
Four of the most potent compounds in cell culture (5e,
5f, 5g and 5h) were evaluated in vivo against trans-
planted sub-cutaneous colon 38 tumors in mice, and

980
L. W. Deady et al. / Bioorg. Med. Chem. 8 (2000) 977±984
Table 2. Comparative in vivo activity of selected bis(indeno[1,2-b]-
quinolinecarboxamides) in colon 38 tumors (optimal doses using a
q4DÂ3 schedule)
line-4,11-dicarboxylic acid (14) (63% yield from 2-inda-
none): mp >300 ꢀC; 1H NMR d 7.60 (t), 7.77±7.82 (m, 2
H), 7.86 (d), 7.91 (t), 8.14 (d), 8.32 (d). This was de-
carboxylated at reduced pressure,13 and the sublimate
was recrystallized from DMSO to give 15 (51%): mp
No.
Dose (mg/kg/day)
Growth delay (days)
ꢀ
ꢀ
1
5e
5f
5g
13.3
7.5
65
3.3
30
8.0
3.0
3.0
7.5
7.0
6.0
(DMSO) >300 C; H NMR (75 C) d 7.55 (t), 7.77±
7.90 (m, 3H), 8.03 (d), 8.29 (d), 8.40 (d), 8.87 (s).
5h
Irinotecan
LU 79553
Linker diamines
5.9
Diethylenetriamine, N,N0-bis(2-aminoethyl)-1,3-propane-
diamine,1,4-bis(3-aminopropyl)piperazine, 3,30-diamino-
N-methyldipropylamine and spermidine were used
without further puri®cation. The free base of triethyl-
enetetramine was prepared from its hydrochloride.19
O,O0-1,6-hexanediylbishydroxylamine dihydrochloride
was prepared as reported.20
compared to the clinical topo I inhibitor irinotecan
and the experimental bis(naphthalimide) LU 79533
(Table 2). For the bis(indeno[1,2-b]quinoline-6-carbox-
amides), there were little obvious structure±activity
relationships. There were large improvements in activity
and potency between the H and 4-Me substituted com-
pounds when the linker chain was methylated (5g and
5h), but the opposite when it was not (5e and 5f). There
was also no consistent correlation between in vivo
activity and methylated/unmethylated linker chains.
Overall, the best activities were shown by the least (5e)
and most (5h) lipophilic compounds. Both had activity
comparable to that of irinotecan and LU 79553, with 5h
in particular being more potent than both.
N,N0-bis(2-Aminoethyl)-N,N0-dimethyl-1,2-ethanediamine.
A mixture of chloroacetonitrile (4.4 g, 58.2 mmol), N,N0-
dimethylethylenediamine (2.5 g, 28.4 mmol) and anhy-
drous K2CO3 (14 g) in dry acetone (25 mL) was stirred
and re¯uxed for 24 h. The solid was ®ltered o€, washed
with CH2Cl2 and the solvent removed from the ®ltrate
under reduced pressure to give the intermediate bisace-
tonitrile (3.95 g, 84%) as a golden oil, which was used
1
without further puri®cation; H NMR (CDCl3) d 2.39
(s, 3H, NCH3), 2.61 (s, 2H, NCH2), 3.60 (s, 2H,
CH2CN).
Conclusions
The bis(indeno[1,2-b]quinoline-6-carboxamides) are an
interesting new class of putative topo I inhibitors, some
exhibiting very high cytotoxicity in cell culture, and
activity comparable to irinotecan against sub-cutaneous
colon 38 tumors in vivo.
Diborane was prepared in situ16 and, with a nitrogen
carrier gas, was bubbled through a solution of the above
bisacetonitrile (2.6 g) in THF at room temperature over
ca. 1 h (exothermic). This mixture was allowed to stir
for a further 1 h, then EtOH was added cautiously to
destroy the excess diborane, before HCl gas was passed
into the solution. The salt which formed was ®ltered o€,
dissolved in water, the pH was taken to 12 with 10%
NaOH, and the solvent was removed under reduced
pressure. The residue was extracted with hot toluene,
and the solvent was removed under reduced pressure to
give the title compound (1.4 g, 53%) (lit.5 bp 115 ꢀC/
0.3 mm Hg), which was suciently pure to be used in
amide formation. 1H NMR (CDCl3) d 2.18 (s, 3H,
NCH3), 2.36 (t, J=6 Hz, 2H, CH2), 2.42 (s, 2H, CH2),
2.68 (t, J=6 Hz, 2H, CH2). ESMS: m/z 175.1 (M+1).
Experimental
Chemistry
Microanalyses were performed at the Campbell Micro-
analytical Laboratory, University of Otago, New Zeal-
1
and. H NMR spectra were obtained at 300 MHz, in
CDCl3 unless stated otherwise, and are referenced to
Me4Si. In the listings, proton counts for aromatic pro-
tons (which have not been assigned) are given only for
unresolved multiplets; the other aromatic signals are
single proton doublets and triplets with J=6±8 Hz,
except the pyrido ring proton, a singlet. Electrospray
mass spectra were recorded on a VG Bio-Q triple
quadrupole mass spectrometer, with water:MeOH:
AcOH (50:50:1) as the mobile phase.
N,N0-bis(2-Aminoethyl)-N,N0-dimethyl-1,3-propanediamine.
The same two-step sequence with N,N0-dimethyl-1,3-
propanediamine gave the intermediate bisacetonitrile
(91%); (1H NMR (CDCl3) d 1.59 (m, 2H, C-CH2),
2.33 (s, 6H, NCH3), 2.48 (t, J=7 Hz, 4H, NCH2), 3.50
(s, 4H, CH2CN) and ®nal tetraamine (55%) (lit.15
bp 32±33 ꢀC/0.5 mmHg). H NMR (CDCl3) d 1.54 (m,
1
2H, C-CH2), 2.12 (s, 6H, NCH3), 2.25±2.35 (m, 8H,
CH2), 2.67 (t, J=6 Hz, 4H, CH2). ESMS: m/z 189.3
(M+1).
Acids
Known acids 9±11 were prepared as described.13
6-Oxo-6H-indeno[2,1-b]quinoline-4-carboxylic acid (15).
Isatin-7-carboxylic acid and 2-indanone were reacted
by the general P®tzinger reaction (Method A) reported
previously.13 The resulting crude 13 was oxidized13 with
alkaline KMnO4 to give 6-oxo-6H-indeno[2,1-b]quino-
Preparation of N,N0-(iminodi-2,1-ethanediyl)bis-[11-oxo-
11H-indeno[1,2-b]quinoline-6-carboxamide] (5a): exam-
ple of the amidation reaction. Oxo acid 9 (0.3 g) and
1,10-carbonyldiimidazole (0.5 g) in dry dioxan (20 mL)
were heated under re¯ux until dissolution was complete

L. W. Deady et al. / Bioorg. Med. Chem. 8 (2000) 977±984
981
(ca. 3 h). The solvent was removed under reduced pres-
sure and the residue was dissolved in CH2Cl2 (30 mL).
The organic layer was washed twice with warm water
(20 mL), and dried over MgSO4. The solvent was
removed to give 6-(1H-imidazol-1-ylcarbonyl)-11H-
indeno[1,2-b]quinolin-11-one (12; RˆH) as an orange/
red solid (0.31 g, 87%): mp 190±196 ꢀC (dec.); 1H NMR
d 7.11 (s, 1H, H-40), 7.48±7.53 (m, 2H, H-2,50), 7.60 (t,
1H, J=6.7 Hz, H-3), 7.66 (t, 1H, J=7.7 Hz, H-8), 7.75
(d, 1H, J=7.5 Hz, H-1), 7.80 (d, 1H, J=7.3 Hz, H-4),
7.88 (s, 1H, H-20), 7.98 (d, 1H, J=7.0 Hz, H-9), 8.14 (d,
1H, J=8.0 Hz, H-7), 8.42 (s, 1H, H-10).
N,N0-[(2-Aminoethyl)imino]di-3,1-propanediyl]bis-[11-oxo-
11H-indeno[1,2-b]quinoline-6-carboxamide] (5e). ꢀ As a
1
pale orange solid: mp (MeCN/CHCl3) 130±131 C; H
NMR (CDCl3) d 1.83 (m, 1H), 2.88 (t, 2H), 2.93 (t, 2H),
3.67 (q, 2H), 7.27 (t), 7.45 (d), 7.51 (t), 7.60 (t), 7.90 (d),
8.01 (d), 8.22 (s), 8.80 (d), 10.98 (s, 1H, NH). Anal.
.
calcd. for C42H34N6O4 H2O: C, 71.1; H, 5.2; N, 12.4.
Found: C, 71.2: H, 5.1: N, 12.1%.
N,N0-[(2-Aminoethyl)imino]di-3,1-propanediyl]bis-[4-meth-
yl-11-oxo-11H-indeno[1,2-b]quinoline-6-carboxamide] (5f).
The imidazolide (12: RˆMe) was prepared in situ from
acid 10, and was reacted with N,N0-bis(2-aminoethyl)-
1,3-propanediamine. The crude product (69%) was
subjected to column chromatography (alumina; CHCl3:
MeOH, 96:4) and the fraction with Rf=0.55 was col-
lected. The solvent was removed under reduced pressure
Diethylenetriamine (0.063 g, 0.61 mmol) was added to a
solution of 12 (RˆH) (0.4 g, 1.24 mmol) in dry CH2Cl2
(30 mL), and the mixture was stirred at room tempera-
ture for 24 h, then washed with 10% Na2CO3 solution
(2Â20 mL), warm water (2Â20 mL) and dried (MgSO4).
The solvent was removed to give 5a as a red solid
(0.26 g, 69%): mp (MeCN) 245±247 ꢀC; 1H NMR
[(CD3)2SO] d 3.06 (s, CH2NHCO), 3.69 (s, CH2NH),
7.13±7.15 (m, 2H), 7.36±7.46 (m, 2H), 7.82 (d), 7.95 (d),
8.16 (d), 8.22 (s), 10.72 (br s, NH). ESMS: m/z 618
to give 5f: mp (EtOH) 138±140 ꢀC; H NMR (CDCl3) d
1
1.60±1.8 (m, 1H, C-CH2), 2.7±2.8 (m, 5H, CH3+
NCH2), 2.85±2.95 (m, 2H, NCH2), 3.60±3.70 (m, 2H,
CH2NHCO), 7.25±7.40 (m, 2H), 7.50±7.55 (m, 2H),
7.85 (d), 8.27 (s), 8.75 (d), 10.69 (br s, 1H, NH). ESMS:
m/z 703 (M+1), 352 [(M+2)/2]. Anal. calcd. for
.
.
(M+1). Anal. calcd for C38H27N5O4 H2O: C, 71.8; H,
4.6; N, 11.0. Found: C, 72.0: H, 4.5: N, 11.2%.
C43H38N6O4 3H2O: C, 68.2; H, 5.9; N, 11.1. Found: C,
68.6; H, 5.9; N, 11.0%.
The following amides were prepared in this manner
(from imidazolide (12: RˆH) unless stated otherwise):
N,N0-[(2-Aminoethyl)methylimino]di-3,1-propanediyl]bis-
[11-oxo-11H-indeno[1,2-b]quinoline-6-carboxamide] (5g).
The ®rst obtained material was extracted with hot light
petroleum (bp 90±110 ꢀC). The insoluble material was
recrystallized twice from CHCl3/MeCN and had mp
N,N0 -(Methyliminodi-3,1-propanediyl)bis-[11-oxo-11H-
indeno[1,2-b]quinoline-6-carboxamide] (5b). As a pale
orange semi-solid (85%); H NMR (CDCl3) d 2.02 (m,
185±186 ꢀC; H NMR (CDCl3) d 1.80±2.0 (m, 2H, C-
1
1
4H, CH2), 2.39 (s, 3H, N-CH3), 2.76 (t, 4H, J=7.1 Hz,
CH2N), 3.62 (q, 4H, J=6.1 Hz, CH2NHCO), 7.08±7.18
(m, 4H), 7.42 (t, 2H), 7.51 (t, 2H), 7.59 (d, 2H), 7.81 (d,
2H), 8.08 (s, 2H), 8.63 (d, 2H), 10.58 (t, 2H, J=4.9 Hz,
NH). ESMS: m/z 660 (M+1), 330.7 [(M+2)/2]. A
hygroscopic perchlorate salt was prepared in 2-propanol
and had mp 173±176 ꢀC, but could not be freed of trace
impurity.
CH2), 2.34 (s, 6H, NCH3), 2.50±2.60 (m, 8H, CH2),
3.55±3.65 (m, 4H, CH2NHCO), 7.18±7.24 (m, 4H),
7.38±7.46 (m, 2H), 7.62 (t, 2H), 7.89 (dd, J=7.7, 1.5 Hz,
2H), 8.11 (s, 2H), 8.81 (dd, J=7.7, 1.5 Hz, 2H), 10.78
(br s, 2H, NH). ESMS: m/z 703.3 (M+1), 352.2
[(M+2)/2]. Anal. Calcd. for C43H38N6O4: C, 73.5; H,
5.5; N, 12.0. Found: C, 73.3: H, 5.3: N, 12.1%.
N,N0-[(2-Aminoethyl)methylimino]di-3,1-propanediyl]bis-
[4-methyl-11-oxo-11H-indeno[1,2-b]quinoline-6-carbox-
amide] (5h). The imidazolide from acid 10 was prepared
in situ and then reacted with N,N0bis(2-aminoethyl)-
N,N0-dimethyl-1,3-propanediamine. Column chromato-
graphy of the crude product (57%) (alumina; CHCl3:
MeOH, 99:1) gave 5h: mp 103±105 ꢀC; 1H NMR
(CDCl3) d 1.55±1.7 (m, 2H, CCH2), 2.24 (s, 6H, NCH3),
2.40±2.45 (m, 4H, NCH2), 2.54±2.60 (m, 4H, NCH2),
2.76 (s, 6H, CH3), 3.54±3.65 (m, 4H, CH2NHCO), 7.25±
7.35 (m, 4H), 7.48±7.60 (m, 4H), 7.86 (d, 2H), 8.24 (s,
2H), 8.75 (d, 2H), 10.58 (br s, 2H, NH). ESMS: m/z
731.2 (M+1), 366.4 [(M+2)/2]. The perchlorate salt
had mp (water) 204±206 ꢀC. Anal. calcd for C45H42
N,N0-[(2-Aminoethyl)imino]di-2,1-ethanediyl]bis-[11-oxo-
11H-indeno[1,2-b]quinoline-6-carboxamide] (5c). After
72 h reaction time, as an orange solid (73%): mp
(DMSO) 181±184 ꢀC; H NMR [(CD3)2SO] d 2.80±2.88
1
(m, 6H, CH2NH), 7.37±7.41 (m, 2H), 7.55±7.65 (m,
2H), 8.05±8.15 (m, 2H), 8.32 (s), 8.42 (d), 10.61 (br s,
NH). ESMS: m/z 661 (M+1). Anal. calcd for C40H32
.
N6O4 2.5H2O: C, 68.1; H, 5.3; N, 11.9. Found: C, 68.1:
H, 5.3: N, 12.1%.
N,N0 -[(2-Aminoethyl)methylimino]di-2,1-ethanediyl]bis-
[11-oxo-11H-indeno[1,2-b]quinoline-6-carboxamide] (5d).
The ®rst material obtained was dissolved in CH2Cl2
and addition of hexane gave the product in 70% yield.
Column chromatography (alumina/CHCl3) followed by
recrystallization from MeCN gave a sample with mp
.
N6O4 2HClO4.H2O: C, 56.9; H, 4.9; N, 8.8. Found: C,
56.9; H, 4.9; N, 8.8%.
192±193 ꢀC; H NMR (CDCl3) d 2.43 (s, 3H), 2.69 (t,
N,N0-[(2-Aminoethyl)methylimino]di-3,1-propanediyl]bis-
[4-chloro-11-oxo-11H-indeno[1,2-b]quinoline-6-carbox-
amide] (5i). The imidazolide from acid 11 was prepared
in situ and then reacted with N,N0-bis(2-aminoethyl)-
N,N0-dimethyl-1,3-propanediamine. Column chromato-
graphy of the crude product (23%) (alumina; CHCl3:
1
2H), 2.77 (s, 2H), 3.59 (q, 2H), 7.30 (t), 7.47±7.51 (m,
2H), 7.60 (t), 7.88 (d), 8.09 (d), 8.15 (s), 8.72 (d), 10.88
(s, 1H, NH). ESMS: m/z 689 (M+1). Anal. calcd. for
.
C42H36N6O4 0.5H2O: C, 72.3; H, 5.3; N, 12.0. Found:
C, 72.3: H, 5.3: N, 12.3%.

982
L. W. Deady et al. / Bioorg. Med. Chem. 8 (2000) 977±984
MeOH, 98:2) gave 5i, mp (MeCN) 193±195 ꢀC; 1H
NMR (CDCl3) d 1.6±1.8 (m, 2H, CH2), 2.32 (s, 6H,
NCH3), 2.46 (m, 4H, NCH2), 2.68 (m, 4H, NCH2), 3.64
(m, 4H, CH2NH), 7.19 (t, 2H), 7.40±7.50 (m, 4H), 7.64
(t, 2H), 7.91 (d, 2H), 8.29 (s, 2H), 8.85 (d, 2H), 10.9 (br
s, 2H, NH). ESMS: m/z 771 (100%), 772 (50), 773 (83),
774 (43), 775 (15), all (M+1) for C43H36Cl2N6O4. Anal.
lic anhydride (0.2 g, 1.0 mmol) in CH2Cl2 (20 mL) was
added dropwise with stirring over 1 h to a solution
of 1,4-bis(3-aminopropyl)piperazine (1 g, 5 mmol) in
CH2Cl2 (5 mL). The mixture was stirred at rt for 16 h,
then the solvent was removed under reduced pressure
and the excess of diamine was distilled at 150±152 ꢀC/2
mmHg to leave crude N-[3-[4-(3-aminopropyl)piper-
azin-1-yl]propylnaphthalimide as a golden oil. This
crude product was reacted directly with imidazolide (12:
RˆH) as above, to give 7 (0.16 g, 25%): mp (MeCN)
.
calcd for C43H36Cl2N6O4 1.5H2O: C, 64.7; H, 4.9; N,
10.5. Found: C, 64.9; H, 4.6; N, 10.8%.
N,N0 -[(3-Aminopropyl)imino]di-3,1-propanediyl]bis-[11-
oxo-11H-indeno[1,2-b]quinoline-6-carboxamide] (5j). The
crude solid was extracted with hot light petroleum (bp
90±110 ꢀC), then with hot MeCN to give the insoluble
pale orange product (78%), mp >300 ꢀC. This could
not be recrystallized and no suitable crystalline salt was
obtained. 1H NMR (DMSO/TFA) d aliphatic region
poorly resolved, 7.58 (t), 7.70±7.75 (m, 2H), 7.92 (d),
8.04 (d), 8.48 (d), 8.53 (s), 8.62 (d), 10.59 (br s, NH).
ESMS: 703 (M+1).
189±191 ꢀC; H NMR (CDCl3) d 1.85 (m, 4H), 2.40±
1
2.50 (m, 12H), 3.67 (q, J=6.2 Hz, 2H), 4.22 (t, J=
7.3 Hz, 2H), 7.60 (t, 1H), 7.65 (t, 1H), 7.73 (t, 2H), 7.86
(d, 1H), 7.93 (d, 1H), 7.99 (d, 1H), 8.18 (d, 2H), 8.45 (s,
1H), 8.57 (d, 2H), 8.87 (d, 1H), 11.0 (s, 1H, NH).
ESMS: m/z 638.3 (100%) (M+1); 319.7 (50%) [(M+2)/
. .
2]. Anal. calcd. for C39H35N5O4 1 5H2O: C, 70.5; H,
5.8; N, 10.5. Found: C, 70.2; H, 5.7; N, 10.8%.
N,N0-[(2-Aminoethyl)methylimino]di-2,1-propanediyl]bis-
[6-oxo-6H-indeno[2,1-b]quinoline-4-carboxamide] (8).
The imidazolide from acid 15 was prepared in situ and
then was reacted with N,N0-bis(2-aminoethyl)-N,N0-
dimethyl-1,3-propanediamine. The crude product was
extracted with hot light petroleum (bp 90±110 ꢀC) (2Â),
then with hot MeCN, to leave the insoluble yellow solid
N,N0 -(1,4-Piperazinediyldi-3,1-propanediyl)bis-[11-oxo-
11H-indeno[1,2-b]quinoline-6-carboxamide] (5k). As a
cream solid (47%): mp (DMSO) 280±282 ꢀC; H NMR
1
[(CD3)2SO] d 1.85±1.95 (m, 2H, CH2), 2.45±2.53 (m,
6H, pipCH2N), 3.59 (q, J=5.6 Hz, CONHCH2), 7.65
(t), 7.71 (t), 7.78±7.84 (m, 2H), 8.06 (d), 8.24 (d), 8.52
(d), 8.68 (s). ESMS: m/z 715 (M+1), 358 [(M+2)/2].
8 (33%): mp (EtOH/CHCl3) 207±209 ꢀC; H NMR d
1
1.79 (m, 2H, CH2), 2.35 (s, 6H, NCH3), 2.57 (t, 4H,
NCH2), 2.70 (t, 4H, NCH2), 3.64 (m, 4H, CH2NHCO),
7.24 (t, 2H), 7.44±7.58 (m, 8H), 7.73 (d, 2H), 7.97 (s,
2H), 8.58 (d, 2H), 10.60 (br s, 2H, NH). ESMS: 703
(M+1), 352 [(M+2)/2]. Anal. calcd for C43H38N6
.
Anal. calcd for C44H38N6O4 H2O: C, 72.1; H, 5.5; N,
11.4. Found: C, 72.2: H, 5.3: N, 11.2%.
N-[3-[4-(11-oxo-11H-indeno[1,2-b]quinoline-6-carbonyl-
amino)butyl]amino]propyl]-11-oxo-11H-indeno[1,2-b]-
quinoline-6-carboxamide (5l). Obtained in 69% yield, mp
(MeOH) 240±242 ꢀC (dec., after darkening >200 ꢀC);
1H NMR (CDCl3) d 1.5±2.1 (m, 6H, C-CH2), 2.80±3.0
(m, 4H, CH2NH), 3.6±3.8 (m, 4H, CH2NHCO), 7.35±7.46
(m, 2H), 7.55±7.70 (m, 6H), 7.80±8.0 (m, 4H), 8.30±8.35
(m, 2H), 8.75±8.85 (m, 2H), 10.8±11.0 (br s, 2H, NH).
ESMS: m/z 660.2 (M+1). Anal. (after vacuum drying
at 50 ꢀC/3 days) calcd for C41H33N5O4.3H2O: C, 69.0;
H, 5.5; N, 9.8. Found: C, 68.7; H, 5.1; N, 10.2%.
.
O4 0.5H2O: C, 72.6; H, 5.5; N, 11.8. Found: C, 72.5; H,
5.4; N, 11.9%.
In vitro growth delay assays
Murine P388 leukemia cells, Lewis lung carcinoma cells
(LLTC), and human Jurkat leukemia cells (JLC), toge-
ther with their amsacrine and doxorubicin-resistant
derivatives (JLA and JLD, respectively), were obtained
and cultured as described.21,22 Growth inhibition assays
were performed by culturing cells at 4.5Â103 (P388), 103
(LLTC), and 3.75Â103 (Jurkat lines) per well in micro-
culture plates (150 mL per well) for 3 (P388) or 4 days in
the presence of drug. Cell growth was determined by
[3H]TdR uptake (P388)23 or the sulforhodamine assay.24
Independent assays were performed in duplicate, and
coecients of variation were ca. 25%.
11,110-[(O,O0-Hexane-1,6-diyl)bisisonitroso]bis-[N-[2-(di-
methylamino)ethyl]-11H-indeno[1,2-b]quinoline-6-carbox-
amide (6). A solution of the known12 amide 4 (0.4 g) and
O,O0-1,6-hexanediylbishydroxylamine dihydrochloride
(0.12 g) in 5% HCl (10 mL) was heated under re¯ux for
3 h. The solution was cooled to room temperature and
basi®ed to pH 10 with 10% NaOH. The oily residue
which formed was extracted into CHCl3, washed with
water (2Â10 mL) and dried over MgSO4. Removal of
the solvent gave 6 as an orange oil (0.39 g, 84%). The
1H NMR was complex and poorly resolved. ESMS: m/z
803 (M+1), 402 ((M+2)/2). A hygroscopic perchlorate
salt was prepared in isopropanol and dried in vacuo,
and had mp 167±169 ꢀC. Anal. calcd for C48H50
In vivo colon 38 tumor assay
Colon 38 tumors were grown subcutaneously from
1 mm3 fragments implanted in one ¯ank of mice
(anaesthetised with pentobarbitone 90 mg/kg). When
tumors reached a diameter of approximately 4 mm (7±8
days), mice were divided into control and drug treat-
ment groups (5 mice/group), with similar average tumor
volumes in each group. Drugs were administered as
solutions of the hydrochloride salts in distilled water
and were injected in a volume of 0.01 mL/g body weight
in two equal injections administered 1 h apart. The mice
were monitored closely and tumor diameters were mea-
.
.
N8O4 2HClO4 4H2O: C, 53.6; H, 5.6; N, 10.4. Found:
C, 54.0: H, 5.3: N, 10.1%.
N-[3-[4-[3-(1,3-Dioxo-1H-benz[de]isoquinolin-2(3H)-yl)-
propyl]piperazin-1-yl]propyl]-11H-11-oxoindeno[1,2-b]-
quinoline-6-carboxamide (7). A solution of 1,8-naphtha-

L. W. Deady et al. / Bioorg. Med. Chem. 8 (2000) 977±984
983
Figure 1. Averaged growth delay data for control (*) and treated mice bearing advanced subcutaneous colon 38 tumors. A: 5e at doses of 20 mg/
Kg (~) and 13.3 mg/Kg (!). B: 5h at doses of 5.0 mg/Kg (~) and 3.3 mg/Kg (!). C: LU79553 at a dose of 5.9 mg/Kg (!). The indicated drug
dose was administered intraperitoneally three times at four-day intervals to groups of ®ve mice. Higher doses than those indicated were toxic to at
least one animal per group.
sured with callipers three times a week. Tumor volumes
were calculated as 0.52Âa2Âb, where a and b are the
minor and major tumor axes and data plotted on a
semilogarithmic plot as mean tumor volumes (‹SEM)
versus time after treatment. The growth delay was cal-
culated as the time taken for tumors to reach a mean
volume 4-fold higher than their pre-treatment volume
(Figure 1).
5. Brana, M. F.; Castellano, J. M.; Perron, D.; Maher, C.;
Conlon, D.; Bosquet, P. F.; George, J.; Qian, X.-D.; Robin-
son, S. P. J. Med. Chem. 1997, 40, 449.
6. Hernandez, L.; Cholody, W. M.; Hudson, E. A.; Resau, J.
H.; Pauly, G.; Michejda, C. J. Cancer Res. 1995, 55, 2338.
7. Cholody, W. M.; Hernandez, L.; Hassner, L.; Scudiero, D.
A.; Djurickovic, D. B.; Michejda, C. J. J. Med. Chem. 1995,
38, 3043.
8. (a) Chaires, J. B.; Leng, F. F.; Przewloka, T.; Fokt, I.; Ling,
Y. H.; Perez-Soler, R.; Priebe, W. J. Med. Chem. 1997, 40,
261. (b) Leng, F. F.; Priebe, W.; Chaires, J. B. Biochemistry,
1998, 37, 1743.
Acknowledgements
9. Gamage, S. A.; Spicer, J. A.; Atwell, G. J.; Finlay, G. J.;
Baguley, B. C.; Denny, W. A. J. Med. Chem. 1999, 42, 2383.
10. Spicer, J. A.; Gamage, S. A.; Atwell, G. J.; Finlay, G. J.;
Baguley, B. C.; Denny, W. A. Anti-Cancer Drug Design 1999,
14, 281.
We thank Ms. D. Greenhalgh for the P388 cell line
data, Dr. L. Zhuang for the colon 38 assay, and Biota
Cancer Research Pty Ltd for funding this work.
11. Spicer, J. A.; Gamage, S. A.; Atwell, G. J.; Finlay, G. J.;
Baguley, B. C.; Denny, W. A. J. Med. Chem. 1997, 40, 1919.
12. Deady, L. W.; Kaye, A. J.; Finlay, G. J.; Baguley, B. C.;
Denny, W. A. J. Med. Chem. 1997, 40, 2040.
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