Synthesis of substituted indeno[1,2-b]quinoline-6-carboxamides, [1]benzothieno[3,2-b]quinoline-4-carboxamides and 10H-quindoline-4-carboxamides: Evaluation of structure-activity relationships for cytotoxicity

Article abstract of DOI:10.1016/S0968-0896(00)00179-6

New substituted indeno[1,2-b]quinoline-6-carboxamides, [1]benzothieno[3,2-b]quinoline-4-carboxamides and 10H-quindoline-4-carboxamides were prepared from methyl 2-amino-3-formylbenzoate by a new Friedlander synthesis. Evaluation of these carboxamides for cytotoxicity in a panel of cell lines showed that small lipophilic substituents in the non-carboxamide ring, in a pseudo-peri position to the side chain, significantly increased cytotoxic potency while retaining a pattern of cytotoxicity consistent with a non-topo II mode of action. The methyl-substituted indeno[1,2-b]quinoline-6-carboxamide demonstrated substantial effectiveness (20-day growth delays) in a sub-cutaneous colon 38 in vivo tumor model. This is comparable to that reported for the dual topo I/II inhibitor DACA that is in clinical trial. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00179-6

Bioorganic & Medicinal Chemistry 8 (2000) 2461±2466
Synthesis of Substituted Indeno[1,2-b]quinoline-6-carboxamides,
[1]Benzothieno[3,2-b]quinoline-4-carboxamides and 10H-
Quindoline-4-carboxamides: Evaluation of Structure±Activity
Relationships for Cytotoxicity
Junjie Chen,a Leslie W. Deady,a,* Jose Desneves,a Anthony J. Kaye,a
Graeme J. Finlay,b Bruce C. Baguleyb and William A. Dennyb
aChemistry Department, La Trobe University, Bundoora, Victoria, Australia 3083
bAuckland Cancer Society Research Centre, Faculty of Medical and Health Sciences,
The University of Auckland, Private Bag 92019, Auckland 1000, New Zealand
Received 24 April 2000; accepted 12 June 2000
AbstractÐNew substituted indeno[1,2-b]quinoline-6-carboxamides, [1]benzothieno[3,2-b]quinoline-4-carboxamides and 10H-quin-
doline-4-carboxamides were prepared from methyl 2-amino-3-formylbenzoate by a new Friedlander synthesis. Evaluation of these
carboxamides for cytotoxicity in a panel of cell lines showed that small lipophilic substituents in the non-carboxamide ring, in a
pseudo-peri position to the side chain, signi®cantly increased cytotoxic potency while retaining a pattern of cytotoxicity consistent
with a non-topo II mode of action. The methyl-substituted indeno[1,2-b]quinoline-6-carboxamide demonstrated substantial e€ec-
tiveness (20-day growth delays) in a sub-cutaneous colon 38 in vivo tumor model. This is comparable to that reported for the dual
topo I/II inhibitor DACA that is in clinical trial. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
The neutral-chromophore DNA intercalators TAS 103
(1)1,2 and DACA (2),3,4 currently both in clinical trial as
anticancer drugs,5 7 are dual topoisomerase I/II (topo I/
II) inhibitors, although the cytotoxicity of TAS 103 has
recently been attributed entirely to its topo II e€ects.8
We have previously reported9 11 on the 11-oxo-11H-
indeno[1,2-b]quinoline-6-carboxamides (e.g., 3a). These
tetracyclic quinoline derivatives showed similar patterns
of activity to DACA in a panel of topo II-de®cient
human leukemia lines.9 They also had similar structure±
activity relationships to DACA for ring substitution,
with small lipophilic substituents (Me, Cl) on the carbon
peri to the chromophore nitrogen being the most e€ec-
tive.10 Substitution with small lipophilic groups at this
position is known3 to give analogues with tighter DNA
binding; recent crystallographic studies12,13 of drug/oli-
gonucleotide complexes suggest this is probably through
van der Waals interaction of the substituent with the
cytosine forming the intercalation site.
Prompted by the good activity of 3a and the above
understanding of the substituent structure±activity rela-
tionships in this and related series, we have now investi-
gated further 4-methyl and 4-methoxy analogues (3b±3g),
exploring the e€ects of additional ring A substitution, in
order to identify a candidate for possible further devel-
opment. The bene®cial e€ect of a 4-methyl substituent
*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)00179-6

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J. Chen et al. / Bioorg. Med. Chem. 8 (2000) 2461±2466
has also been evaluated in two closely-related chromo-
phores; benzothieno[3,2-b]quinolines (4a, 4b) and quin-
dolines (5a, 5b) with S and NR respectively in place of
the carbonyl group in the B ring of 3a.
carcinoma,15 and three human leukemia (Jurkat) lines
that have been described in detail previously.16,17 JLC is
the wild-type (sensitive) line, JLA is resistant to the topo
II agents (85-fold resistant to amsacrine) because of a
reduced level of topo II, and JLD is similarly resistant
(13-fold) to doxorubicin. IC50 values for the compounds
in the P388, LLTC and JLA lines are given in Table 1,
together with ratios of IC50 values against JLC and the
other two Jurkat lines (ratios JLA/JLC and JLD/JLC).
Values of these ratios of less than about 2-fold suggest a
non-topo II mediated mechanism of action. Thus the
dual topo I/II inhibitor DACA (2) has ratios of 1.9 and
Results and Discussion
Chemistry
In previous studies,9,10 the tetracyclic quinoline deriva-
tives were constructed by P®tzinger synthesis between
isatin-7-carboxylic acid and the appropriate bicyclic
ketone. Some shortcomings of this approach became evi-
dent: decarboxylation of the intermediate 10-carboxylic
acids were successful only on a small scale, certain
methoxy substituted compounds tended to demethylate
during the decarboxylation process and formation of [1]
benzothieno[3,2-b]quinoline analogues were low yield-
ing under the strongly basic P®tzinger conditions. In a
signi®cant improvement, our recent synthesis of methyl
2-amino-3-formylbenzoate14 6 allowed the tetracycles
reported here to be prepared more successfully, by
Friedlander condensation of 6 with indanone analogues
under mild, base catalyzed conditions (Scheme 1). 1-
Indanones 7±10, a sulfur analogue 11 and precursors 12
and 13 of indole carbonyl compounds were all employed.
In the preparation of the carbonyl containing com-
pounds 19±22 from the methylene bridged precursors
14±17, no single oxidant suced for all, and suitable
agents were found by trial and error (see Experimental).
The new amides of Table 1 were prepared by generating
an intermediate imidazolide in situ from the precursor
acids 18±24 and 1,10-carbonyldiimidazole, and reacting
this with N,N-dimethylethylenediamine (Scheme 2).
Table 1. Cell growth inhibition data for indeno[1,2-b]quinoline-6-
carboxamides
IC50 (nM)a
P388b LLc JLC JLA/JLC JLD/JLC
IC50 ratios
d
Compound
X
Y
3ae
3bf
3c
3d
3ef
3f
3g
4ae
4b
5ae
5b
2g
CO
CO
CO
CO
CO
H
4-Me
1,4-diMe
2,4-diMe
4-OMe
110
14
240
700
23
91 180
15 35
55 122
1.2
2.1
0.7
0.9
2.2
2.4
1.0
1.5
1.3
1.0
1.1
1.9
0.9
0.9
0.9
0.8
0.8
1.0
0.9
1.6
1.2
1.1
1.5
2.3
12
23
27
30
71
68
CO 1,4-diOMe
CO 2,3,4-triOMe 250
S
53
64 118
66 170
H
1,4-diMe
H
46
S
230 104 171
370 290 450
59
98 189 580
2.5 1.5 5.4 56
20
NH
NH
DACA
4-Me
33
77
3
TAS 103 (topo II)
75
74
13
Amsacrine (topo II)h
Doxorubicin (topo II)h
Camptothecin (topo I)
12
22
33
37
9.6
5.6
85
4.5
2.0
15
13
Structure±activity relationships for growth inhibition
1.4
The compounds were evaluated in culture using a panel
of cell lines; murine P388 leukemia, murine Lewis lung
aIC50; concentration of drug to reduce cell number to 50% of control
cultures (see text).
bMurine P388 leukemia.
cMurine Lewis lung carcinoma.
dHuman Jurkat leukemia.
eData from ref 9.
fData from ref 10.
gData from ref 4.
hData from ref 30.
Scheme 1. (i) t-BuOK/t-BuOH (14±16), piperidine/EtOH (18); (ii)
NaOH/aq. EtOH; (iii) Na2Cr2O7/3 M H2SO4; (iv) SeO2/dioxan;
(v) KMnO4/aq. Na2CO3.
Scheme 2. (i) CDI/dioxan; (ii) H2N(CH2)3NMe2.

J. Chen et al. / Bioorg. Med. Chem. 8 (2000) 2461±2466
2463
Figure 1. E€ect of 3b (A) and irinotecan (B) on the growth of colon 38 tumors. (A) Mice (with subcutaneously implanted tumors of approximately
4 mm diameter) were treated with either solvent as control (*), or with 3b at doses of 30 mg/kg (&) or 45 mg/kg (!) on an intermittent schedule (every
4 daysÂ3). (B) Mice were treated as above with either solvent as control (*), or with irinotecan at doses of 20 (&), 30 (!), 45 (~) or 65 (*) mg/kg.
In vivo studies
2.3 respectively, while the ``classical'' topo I inhibitor
camptothecin shows ratios of 2.0 and 1.4, and TAS 103
(recently8 shown to functionally be a topo II inhibitor)
has much larger ratios (56 and 75) (Table 1).
Because of the superior growth inhibitory properties of
3b, this compound was selected for in vivo studies, to
further gauge the value of the general class of indeno
[1,2-b]quinoline-6-carboxamide dual topo I/II inhibi-
tors. Figure 1(A) shows it has substantial e€ectiveness
against subcutaneously implanted colon 38 tumors, with
a 20-day growth delay when given at 45 mg/kg on an
intermittent dose schedule (3 doses, 4 days apart). This
is comparable to that reported for DACA (5.5 days at
65 mg/kg) using the same schedule.18 The indenoquino-
line 3a is also active in this model, with a growth delay
of 7 days at 90 mg/kg, but on a di€erent, single-dose
schedule.9 The pure topo I inhibitor irinotecan gave a
growth delay of ca 5 days using the 4 daysÂ3 schedule,
with little evidence of a dose-response over the dose
range 65±20 mg/kg (Fig. 1(B)). This may be because
irinotecan is metabolized in vivo to the active form
SN-38.19
All of the compounds in Table 1 had similar potencies
in both the wild-type and resistant Jurkat lines, sug-
gesting a mode of action not primarily dependent on
topo II. As reported previously,10 substitution of the
parent compound 3a with a 4-methyl group (to give 3b)
resulted in substantially increased potency (5±8-fold) in
all the cell lines. This parallels data reported for DACA
analogues, where the (structurally equivalent) 5-sub-
stituted derivatives were more cytotoxic than DACA
itself.4 Compounds 3c and 3d explore the e€ects of
substituents in addition to the 4-Me, and show that the
2,4-diMe (but not the 1,4-diMe) is broadly comparable.
Similarly,10 the 4-OMe (3e) was substantially more
e€ective than 3a, as was the 1,4-diOMe (3f) but not the
2,3,4-triOMe (3g) derivatives.
Compounds 4b and 5b were prepared to evaluate the
consistency of the e€ect of a 4-methyl group in the rela-
ted [1]benzothieno[3,2-b]quinoline-4-carboxamide and
10H-quindoline-4-carboxamide subclasses of hetero-
cycles. The parent [1]benzothieno[3,2-b]quinoline (4a) is
more potent than the indenoquinoline,9 but the 1,4-
diMe derivative 4b was less e€ective (the 4-Me analogue
could not be prepared). In the 10H-quindoline subseries,
the 4-Me analogue 5b was substantially more e€ective
than the parent 5a, but still not as e€ective as the 4-Me
indeno[1,2-b]quinoline analogue 3b.
Conclusions
Substitution of indeno[1,2-b]quinoline-6-carboxamide
(3a) and the related 11H-benzothieno[3,2-b]quinoline-6-
carboxamide (4a) and 10H-quindoline-4-carboxamide
(5a) at the pseudo-peri position to the carboxamide side
chain provided analogues of signi®cantly increased
cytotoxic potency. The indeno[1,2-b]quinoline-6-carb-
oxamide analogue (3b) also had substantial in vivo
activity in the refractory colon 38 model. This substitu-

2464
J. Chen et al. / Bioorg. Med. Chem. 8 (2000) 2461±2466
tion pattern seems generally bene®cial in the general
class of tetracyclic carboxamides.
6,9-Dimethyl[1]benzothieno[3,2-b]quinoline-4-carboxylic
acid (18). To a solution of 6 (0.21 g) and 11 (0.18 g) in
EtOH (10 mL) under a nitrogen atmosphere was added
piperidine (0.08 g), and this solution was heated under
re¯ux for a further 18 h. The solvent was removed
under reduced pressure, and the residue was triturated
with light petroleum (bp 40±70 ꢀC). The solid which
formed was ®ltered o€ (0.32 g), mixed with 10% aqu-
eous NaOH (20 mL), heated under re¯ux for 5 h, then
cooled and the solution was washed with CHCl3 (20 mL).
The aqueous layer was acidi®ed with concentrated HCl
to give 18 as a purpleꢀsolid (0.24 g, 77%), mp >295 ꢀC
Experimental
1H NMR spectra were obtained at 300 MHz, in DMSO-
d6 unless stated otherwise, and are referenced to Me4Si.
In the listings, proton counts for aromatic protons
(which have not been assigned) are given only for unre-
solved multiplets; the other aromatic signals are single
proton doublets and triplets with J=6±8 Hz, except the
pyrido ring proton, a singlet. Microanalyses were per-
formed at the Campbell Microanalytical Laboratory,
University of Otago, New Zealand.
1
(forms needles >240 C). H NMR (DMSO-d6/1 drop
CF3CO2H) d 2.53 (s, 3H, CH3), 3.02 (s, 3H, CH3), 7.39
(d), 7.51 (d), 7.86 (t), 8.42 (d), 8.61 (d), 9.36 (s).
2,3,4-Trimethoxy-11H-indeno[1,2-b]quinoline-6-carboxy-
lic acid14 17 and 4,7-dimethoxy-1-indanone20 9 were
prepared as reported. 4,7-Dimethylbenzothiophen-3(2H)-
one 11 was prepared by a literature procedure21 (for
benzothiophen-3(2H)-one) from 2,5-dimethylthiophenol
in 59% overall yield, mp 81±86 ꢀC (from light petroleum
(bp 60±90 ꢀC)). 3-Acetoxy-1-acetylindole 12 and 3-acet-
oxy-1-acetyl-4-methylindole 13, mp 89±91 ꢀC, were pre-
pared by a general method22 from the appropriate
precursor N-(carboxymethyl)anthranilic acids, also avail-
able by a general procedure.23
2,4-Dimethyl-11H-indeno[1,2-b]quinoline-6-carboxylic
acid (15). To a solution of 6 (0.5 g) and 8 (0.55 g) in t-
BuOH (not dried) (5 mL) was added 0.35 M t-BuOK
solution (20 mL). This was re¯uxed for 30 min, cooled
and poured onto light petroleum (bp 40±70 ꢀC). The
solid which separated was ®ltered o€, dissolved in water
(20 mL) and acidi®ed with concentrated HCl to give 15
as a pale orange solid (0.65 g, 81%), mp 199±201 ꢀC. 1H
NMR d 2.42 (s, 3H, CH3), 2.81 (s, 3H, CH3), 4.13 (s,
2H, CH2), 7.19 (s), 7.41 (s), 7.78 (t), 8.35 (d), 8.55 (d),
8.71 (s), 16.9 (br s, 1 H, CO2H).
5,7-Dimethyl-1-indanone (8). A mixture of dry dimethyl-
amine hydrochloride (7.2 g), paraformaldehyde (5 g),
2,4-dimethylacetophenone (10 g) and concentrated HCl
(2mL) in ethanol (30 mL) was heated under re¯ux with
vigorous stirring for 16h, and the solvent was then
removed under reduced pressure. The residue was treated
with 10% aqueous Na2CO3 (100mL) and extracted with
ether (3Â100 mL). The combined extracts were dried
(MgSO4) and the ether was removed under reduced
pressure to give 3-(dimethylamino)-1-(2,4-dimethylphe-
nyl)propan-1-one as a golden oil (12.75 g, 92%). This
was then reacted as reported24 for 7-methyl-1-indanone to
give 8, mp 77±78 ꢀC (lit.25 mp 76±77 ꢀC), in 54% overall
1,4-Dimethyl-11H-indeno[1,2-b]quinoline-6-carboxylic
acid (14). This was prepared (from 6 and 7) as for 15,
as a pale orange solid (84%), mp 277±279 ꢀC. 1H NMR d
2.32 (s, 3H, CH3), 2.68 (s, 3H, CH3), 3.87 (s, 2H, CH2),
7.17 (d), 7.24 (d), 7.70 (t), 8.25 (d), 8.46 (d), 8.61 (s).
1,4-Dimethoxy-11H-indeno[1,2-b]quinoline-6-carboxylic
acid (16). This was prepared (from 6 and 9) as for 15,
as a pale orange solid (56%), mp 263±265 ꢀC (dec.). H
1
NMR d 3.87 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 4.01 (s,
2H, CH2), 7.09 (d), 7.18 (d), 7.77 (t), 8.33 (d), 8.53 (d),
8.72 (s).
1
yield. H NMR (CDCl3) d 2.33 (s, 3H, CH3), 2.54±2.65
(m, 5H), 3.10 (t, 2H, J=6Hz, CH2), 6.89 (s), 7.05 (s).
10H-Quindoline-4-carboxylic acid (23). To a solution of
6 (0.18 g, 1.0 mmol) and 3-acetoxy-1-acetylindole 12
(0.22 g, 1.0 mmol) in ethanol (10 mL) was added 10%
sodium hydroxide solution (2 mL) and the mixture was
re¯uxed, under nitrogen, for 1 h. After being cooled, the
mixture was taken to pH 2 with concentrated HCl to
give 23 (0.23 g, 88%), mp >300 ꢀC. 1H NMR d 7.37 (t),
7.66±7.79 (m, 3H), 8.37 (d), 8.50 (d), 8.53 (d), 8.70 (s),
12.0 (s, CO2H).
4,7-Dimethyl-1-indanone (7). A solution of p-xylene
(15 g) and propionyl chloride (10.4 g) was added over
10 min to a stirred and cooled (4 ꢀC) mixture of AlCl3
(10 g) in CS2 (30 mL). A further portion of AlCl3 (5 g)
was then added over 5 min and the whole was immedi-
ately poured onto ice/water (100 mL), stirred for 1 h,
then extracted with CHCl3. The extract was washed
with water, dried (MgSO4), the solvent was removed,
and the residue was distilled to give 20,50-dimethylpro-
piophenone (71%), bp 132 ꢀC/20 mm Hg. A Willgerodt
reaction (as for propiophenone26 but with 16 h re¯ux)
gave a mixture of 3-(2,5-dimethylphenyl)propanoic acid
and 2,5-dimethylbenzoic acid. Recrystallization from
light petroleum (bp 40±70 ꢀC) removed the less soluble
benzoic acid, and evaporation of the ®ltrate gave the
propionic acid (36%), mp 45±46 ꢀC (lit.27 45±46 ꢀC).
This was then converted to the acid chloride and
cyclized with AlCl3 to give 7 (92%), mp 77±79 ꢀC (lit.27
78±79 ꢀC).
6-Methyl-10H-quindoline-4-carboxylic acid (24). This
was obtained in 80% yield (from 6 and 3-acetoxy-1-
acetyl-4-methylindole 13) as for 23 (re¯ux 30 min), mp
>300 ꢀC. H NMR d 2.94 (s, 3H, CH3), 7.09 (d), 7.45
1
(d), 7.57 (t), 7.69 (t), 8.43 (d), 8.49 (d), 8.59 (s), 11.95 (s,
NH), 17.24 (br s, CO2H).
2,4-Dimethyl-11-oxo-11H-indeno[1,2-b]quinoline-6-car-
boxylic acid (20). To a boiling mixture of 15 (0.28 g) in
3 M H2SO4 (10 mL) was added, in small portions, a
solution of Na2Cr2O7 (0.70 g) in 3 M H2SO4 (13 mL).
The whole was then heated and stirred for a further 3 h,

J. Chen et al. / Bioorg. Med. Chem. 8 (2000) 2461±2466
2465
cooled, water (75 mL) was added and the solid which
The following amides were made by this method:
formed was ®ltered o€ toꢀgive 20 as a pale yellow solid
CH3), 2.74 (s, 3H, CH3), 7.46 (s), 7.50 (s), 7.79 (t), 8.39
(d), 8.50 (d), 8.79 (s).
1
(0.16 g, 54%), mp >295 C. H NMR d 2.41 (s, 3H,
N-[2-(Dimethylamino)ethyl]-1,4-dimethyl-11-oxo-11H-
indeno[1,2-b]quinoline-6-carboxamide (3c). In 27% yield
after chrꢀomatography (alumina; CHCl3), Rf=0.24, mp
1
196±198 C (from MeCN). H NMR (CDCl3) d 2.32 (s,
1,4-Dimethyl-11-oxo-11H-indeno[1,2-b]quinoline-6-car-
boxylic acid (19). This was prepared (from 14) as for
20, as ꢀa pale yellow solid (64%) (with ca. 5% of 14), mp
>295 C (with darkening >260 ꢀC). 1H NMR d 2.52 (s,
3H, CH3), 2.64 (s, 3H, CH3), 7.28 (d), 7.41 (d), 7.74 (t),
8.34 (d), 8.46 (d), 8.69 (s).
6H, N(CH3)2), 2.61±2.68 (m, 5H,CH3, CH2N), 2.87 (s,
3H, CH3), 3.71 (q, 2H, J=7 Hz, CH2NH), 7.18 (d), 7.33
(d), 7.62 (t), 7.97 (d), 8.39 (s), 8.85 (d), 10.8 (br s, 1H,
NH). Anal. calcd for C23H23N3O2 0.75H2O: C, 71.4; H,
6.3; N, 10.9. Found: C, 71.1; H, 5.8; N, 10.7.
.
N-[2-(Dimethylamino)ethyl]-2,4-dimethyl-11-oxo-11H-
indeno[1,2-b]quinoline-6-carboxamide (3d). In 60%
1,4-Dimethoxy-11-oxo-11H-indeno[1,2-b]quinoline-6-car-
boxylic acid (21). A mixture of 16 (0.2 g) and selenium
(IV) oxide (0.2g) in dioxan (10 mL) was heated under
re¯ux, with vigorous stirring, for 1.5 h and then ®ltered
while hot. The solvent was removed from the ®ltrate under
reduced pressure. The residue was dissolved in CHCl3,
®ltered, and the ®ltrate was washed with water, dried
and evaporated to dryness. This left 21 as a yellow solid
yield, mp 215±217 ꢀC (from MeCN). H NMR (CDCl3)
1
d 2.30 (s, 6H, N(CH3) ), 2.41 (s, 3H, CH3), 2.61 (t, 2H,
J=7 Hz, CH2N), 2.84 (s, 3H, CH3), 3.71 (q, 2H, J=
7 Hz, CH2NH), 7.26 (s), 7.48 (s), 7.62 (t), 7.92 (d), 8.35
(s), 8.82 (d), 10.8 (br s, 1H, NH). Anal. calcd for
C23H23N3O2: C, 74.0; H, 6.2; N, 11.3. Found: C, 73.7;
H, 6.3; N, 11.3.
(0.12 g, 57%), mp 198±200 ꢀC. H NMR d 3.91 (s, 3H,
1
OCH3), 3.98 (s, 3H, OCH3), 7.30 (d), 7.48 (d), 7.80 (t),
8.38 (d), 8.56 (d), 8.75 (s).
N-[2-(Dimethylamino)ethyl]-1,4-dimethoxy-11-oxo-11H-
indeno[1,2-b]quinoline-6-carboxamide (3f). In 21% yield
after chromatography (alumina; CHCl3:MeOH, 98:2),
Rf=0.66, mp 185±187 ꢀC (softens 170 ꢀC) (from CHCl3/
2,3,4-Trimethoxy-11-oxo-11H-indeno[1,2-b]quinoline-6-
carboxylic acid (22). Indene 17 (0.2 g) was added to a
solution of Na2CO3 (0.2 g) in water (20 mL) and stirred
at 55 ꢀC until dissolution was complete. Potassium per-
manganate (0.16 g) was added and the mixture was
heated and stirred until a drop on ®lter paper showed
no permanganate color (ca. 2 min). The mixture was
®ltered through Celite, which was washed with 10%
Na2CO3, then water and the ®ltrate was acidi®ed to pH
2 with concentrated HCl. The solid which formed was
®ltered o€ to give 22 as a yellow solid (0.08 g, 38%), mp
1
hexane). H NMR (CDCl3) d 2.49 (s, 6H, N(CH3)2),
2.98 (m, 2H, CH2N), 3.9±4.0 (m, 5H, OCH3, CH2NH),
4.16 (s, 3H, OCH3), 7.05 (d), 7.24 (d), 7.62 (t), 7.97 (d),
8.40 (s), 8.83 (d), 11.8 (br s, 1H, NH). Anal. calcd for
.
C23H23N3O4 H2O: C, 65.2; H, 6.0; N, 9.9. Found: C,
65.1; H, 5.8; N, 9.7.
N-[2-(Dimethylamino)ethyl]-6,9-dimethyl[1]benzothieno
[3,2-b]quinoline-4-carboxamide (4b). In 27% yield after
chromatography (alumina; CHCl3), Rf=0.34, mp 192±
275±278 ꢀC (formed needles at ca. 240 ꢀC). H NMR d
194 ꢀC (from MeCN). H NMR (CDCl3) d 2.32 (s, 6H,
1
1
3.93 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 4.03 (s, 3H,
OCH3), 7.33 (s), 7.79 (t), 8.38 (d), 8.57 (d), 8.77 (s),
16.37 (br s, 1 H, CO2H).
N(CH3)2), 2.57 (s, 3H, CH3), 2.65 (t, 2H, J=7 Hz,
CH2N), 3.16 (s, 3H, CH3), 3.78 (q, 2H, J=7 Hz,
CH2NH), 7.28 (d), 7.35 (d), 7.69 (t), 8.03 (d), 8.71 (s),
8.90 (d), 11.2 (br s, 1H, NH). Anal. calcd for C22H23
N3OS 0.25H2O: C, 69.2; H, 6.2; N, 11.0. Found: C,
69.2; H, 6.0; N, 11.0.
.
Preparation of N-[2-(dimethylamino)ethyl]-2,3,4-trime-
thoxy-11-oxo-11H-indeno[1,2-b]quinoline-6-carboxamide
(3g): example of the amidation reaction. Oxo acid 22
(0.40 g, 1.09 mmol) and 1,10-carbonyldiimidazole (0.5 g)
in dry dioxan (20 mL) were heated under re¯ux until
dissolution was complete. The solvent was removed
under reduced pressure and the residue was dissolved in
CH2Cl2 (30 mL). The organic layer was washed twice
with warm water (20 mL), and dried over MgSO4. A
solution of N,N-dimethylethylenediamine (0.12 g, 1.36
mmol) in CH2Cl2 (3 mL) was added and the whole was
stirred at room temperature for 16 h then washed with
10% Na2CO3 solution (2Â20 mL), warm water (2Â
20 mL), dried (MgSO4) and the solvent was removed.
Column chromatography of the residue (alumina;
CHCl3) gave 3g (0.11 g, 24%), Rf=0.31, mp 189±191 ꢀC
(from MeCN). 1H NMR (CDCl3) d 2.61 (s, 6H,
N(CH3)2), 2.99 (m, 2H, CH2N), 3.97±4.04 (m, 8H,
OCH3, CH2NH), 4.19 (s, 3H, OCH3), 7.19 (s), 7.60 (t),
7.93 (d), 8.29 (s), 8.80 (d), 11.6 (br s, 1H, NH). Anal.
N-[2-(Dimethylamino)ethyl]-6-methyl-10H-quindoline-4-
carboxamide (5b). Imidazolide formation was given
26 h and subsequent amination 18 h to a€ord the amide
in 25% yield, mp 226±228 ꢀC (from MeCN). H NMR
1
(CDCl3) d 2.59 (s, 6H, N(CH3)2), 2.77 (s, 3H, CH3), 2.98
(t, J=5.8 Hz, 2H, CH2), 3.89 (q, J=5.8 Hz, 2H, CH2),
6.46 (m, 1H), 6.77±6.79 (m, 2H), 7.31 (t), 7.54 (s), 7.57
(d), 8.57 (d), 11.12 (s, NH), 11.65 (br t, NH). Anal.
.
calcd for C21H22N4O 0.25H2O: C, 71.9; H, 6.5; N, 16.1.
Found: C, 71.6; H, 6.3; N, 16.1.
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.16,17 Growth inhibition
assays were performed by culturing cells at 4.5Â103
.
calcd for C24H25N3O5 0.25H2O: C, 65.5; H, 5.9; N, 9.6.
Found: C, 65.4; H, 6.1; N, 9.5.

2466
J. Chen et al. / Bioorg. Med. Chem. 8 (2000) 2461±2466
(P388), 103 (LLTC), and 3.75Â103 (Jurkat lines) per
well in microculture 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)28 or the sulfo-
rhodamine assay.29 Independent assays were performed
in duplicate, and coecients of variation for all assays
were <20%.
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In vivo colon 38 tumor assay
Colon 38 tumors were grown subcutaneously from
1 mm3 fragments implanted in one ¯ank of C57/Bl mice
(anesthetized 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-
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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 (ÆSEM)
versus time after treatment. The growth delay was calcu-
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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.
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