Synthesis and in vitro antitumor activity of an isomer of the marine pyridoacridine alkaloid ascididemin and related compounds

Article abstract of DOI:10.1016/S0968-0896(03)00483-8

The isomer (9H-quino[4,3,2-de][1,7]phenanthroline-9-one) (2) of the marine alkaloid ascididemin (9H-quino[4,3,2-de][1,10]phenanthroline-9-one) (1) has been synthesized in six steps from 1,4-dimethoxyacridine (10) with an overall yield of 12%. Different related compounds were prepared and tested in vitro at six different concentrations on 12 different human cancer cell lines of various histopathological types (glioblastomas and breast, colon, lung, prostate and bladder cancers). Almost all the compounds present cytotoxic activity of micromolar order.

Full text of DOI:10.1016/S0968-0896(03)00483-8

Bioorganic & Medicinal Chemistry 11 (2003) 4351–4356
Synthesis and In Vitro Antitumor Activity of an Isomer of the
Marine Pyridoacridine Alkaloid Ascididemin and Related
Compounds
Evelyne Delfourne,a,* Robert Kiss,b Laurent Le Corre,a Joumaa Merza,a Jean Bastide,a
Armand Frydmanc and Francis Darroc,y
a
´
Centre de Phytopharmacie, FRE-CNRS 2605, Universite de Perpignan, 52 Avenue de Villeneuve, 66860 Perpignan Cedex, France
Laboratoire d’Histopathologie, Faculte de Medecine, Universite Libre de Bruxelles, 808 Route de Lennik, 1070 Bruxelles, Belgium
b
´
´
´
cCephalon France, Centre de Recherches, 19 avenue du Pr Cadiot, BP 22, 94701 Maisons-Alfort Cedex, France
Received 29 April 2003; accepted 18 July 2003
Abstract—The isomer (9H-quino[4,3,2-de][1,7]phenanthroline-9-one) (2) of the marine alkaloid ascididemin (9H-quino[4,3,2-
de][1,10]phenanthroline-9-one) (1) has been synthesized in six steps from 1,4-dimethoxyacridine (10) with an overall yield of 12%.
Different related compounds were prepared and tested in vitro at six different concentrations on 12different human cancer cell lines
of various histopathological types (glioblastomas and breast, colon, lung, prostate and bladder cancers). Almost all the compounds
present cytotoxic activity of micromolar order.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
analogues. We report herein our quite different
approach to prepare these compounds along with their
in vitro antitumor activity.
Pyridoacridine alkaloids constitute an important class
of natural products isolated from marine organisms,
many of which possess cytotoxic activity.1 Ascididemin
1, identified by Kobayashi et al. in 1988,2 was one of the
first example of these compounds. From this date, dif-
ferent related compounds have been synthesized in
order to discover new potential anticancer drugs or to
understand the mechanisms involved in their antitumor
activities.3 The synthesis of an isomer of ascididemin,
compound 2, was previously reported both by Cuerva et
al.4 and Alvarez et al.,5 this compound was described as
having a cytotoxic activity closely related to that of the
natural product. In a recent paper, Cuerva et al. cor-
rected their synthesis to yield ascididemin instead of its
isomer 2 (Fig. 1).6
Chemistry
The synthesis of 2 (Scheme 1) started with the known
anthranilic compound 8, readily prepared by condensa-
tion of 2-chlorobenzoic acid and 2,5-dimethoxyaniline.8
Acid 8 was converted into its corresponding methyl-
ketone 9 in high yield (92%) with methyllithium. The
cyclisation of 9 in polyphosphoric acid, resulted quan-
titatively into the acridine derivative 10, which was oxi-
dized by CAN to give in 93% yield the acridinedione 11.
Diels–Alder cycloaddition of propenal-N,N-dimethyl-
hydrazone to this last compound gave selectively in
31% yield, the cycloadduct of which structure 12 was
assigned by comparison with the other tetracyclic
regioisomer obtained by Bracher as an intermediate in
the synthesis of ascididemin.9 The formation of the last
cycle was achieved using Bracher’s methodology9 invol-
ving, in a first step, dimethylformamide-diethylacetal in
DMF under nitrogen to form the intermediate enamine
13, the second step corresponding to cyclization of this
As part of our work taking aim at designing new anti-
tumor compounds derived from marine pyr-
idoacridines,7 we are now interested in compound 2 and
*Corresponding author. Tel.: +33-468-662251; fax: +33-468-662223;
e-mail: delfourn@univ-perp.fr
yPresent address: Unibioscreen SA, 40 avenue J. Wybran, 1070 Brus-
sels, Belgium.
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00483-8

4352
E. Delfourne et al. / Bioorg. Med. Chem. 11 (2003) 4351–4356
enamine. Attempts to perform this enamine cyclization
with ammonium chloride did not work in the solvents
usually used (methanol or acetic acid) but proceeded
correctly in water, the yield of the two reactions being
47%. In summary, the synthesis of compound 2 was
realized in six steps with an overall yield of 12%.
transformed in 17% into the dimethylamino compound
7 by reductive formylation.
In vitro determination of the drug-induced inhibition of
human cancer cell line growth
For each of the different compounds under study, six
concentrations were tested on 12different human cancer
Our efforts in substitution of compound 2 focused to
obtain analogues substituted on ring D which have
shown the better cytotoxicities in the ascididemin series.
The different compounds 3–7 were obtained using com-
pound 2 as starting material (Scheme 2).
In this way, compound 3 was prepared in 24% yield by
nitration of 2 with fuming HNO3 whereas direct bro-
mination of 2 gave compound 5 (32%), substituted in
position 5. Catalytic hydrogenation of 3 led in 49%
yield to the amino-derivative 4. The treatment of 5 by
sodium azide in DMF directly yielded the correspond-
ing amino-derivative 6 (48%). This last compound was
Scheme 2. (a) Fuming HNO3, 100 ꢀC, 1.5 h, 24%; (b) H2, 10% Pd/C,
MeOH, 2h, 49%; (c) Br 2 (16 equiv), CH3COOH, 100 ꢀC, 4 h, 32%;
(d) NaN3, DMF, 105 ꢀC, 6 h, 48%; (e) formaldehyde, NaBH3CN,
CF3COOH, 0 ꢀC to rt, 17%.
Figure 1.
Scheme 1. (a) Cu2O, Cu, K2CO3, diglyme, reflux, overnight, 89%; (b) MeLi, THF, reflux, 4.5 h, 92%; (c) polyphosphoric acid, 100 ꢀC, 1 h, 100%;
(d) CAN, CH2Cl2/MeOH, 0 ꢀC, 20 min, 93%; (e) acrolein-N,N-dimethylhydrazone, Ac2O, toluene, 95 ꢀC, 30 min, 31%; (f) DMF–DEA, DMF,
120 ꢀC, 30 min; (g) NH4Cl, H2O, reflux, 25 min, overall yield (f+g) 47%.

E. Delfourne et al. / Bioorg. Med. Chem. 11 (2003) 4351–4356
4353
Table 1. Characterization of the in vitro cytotoxic-related antitumor effects (IC50 value in mM)
Compd
Cell lines
HCT-15
U-87MG
U-373MG
SW 1088
T24
J82
LoVo
MCF7
T-47D
A-549
0.20.06
A-427
0.008
0.08
7
0.3
0.7
0.5
NT
PC-3
0.09
1
2
3
4
5
6
7
0.07
0.8
>10
4
3
0.6
NT
0.5
0.8
9.0
6
0.8
0.4
NT
0.6
3
4
0.7
4
0.6
NT
0.8
0.1
26
0.09
0.8
0.5
NT
0.3
1
5
4
10
9
0.06
0.4
0.9
0.7
1
0.07
0.9
9
0.5
0.7
4
0.6
0.7
7
0.9
9
10
9
4
0.9
9
1
5
2
7
NT
0.2
0.5
0.7
NT
>10
>10
NT
>10
>10
NT
NT
NT
NT
The IC50 value constitutes the concentration of the compound which inhibits the growth of the human cancer cells by 50% as compared to the
control value. Six concentrations ranging from 10 mM to 0.1 nM were assayed on 12different human cancer cell lines for each compound under
study. The drug-induced effects at cell line growth level were determined by means of the MTT colorimetric assay.
cell lines including various histopathological types
(glioblastomas, and breast, colon, lung, prostate and
bladder cancers). We made use of the colorimetric MTT
assay, which indirectly assesses the effect of potentially
anticancer compounds on the overall growth of adher-
ent cell lines.10 The IC50 values, that is the concentration
which reduced the mean growth value of the 12cell lines
by 50%, was determined for each drug, in comparison
with the mean control growth value. Table 1 illustrates
the individual IC50 values of the different compounds
obtained for each of the 12cell lines under study.
depending on the cell line assayed, than their bromo and
amino analogues of the ascididemin series. Similar
observations were also made for the nitro (compound 3)
and the amino (compound 4) derivatives in position 7 of
ring D as compared to their counterpart in the ascidi-
demin series.
Conclusion
In conclusion, this work was aimed to propose a novel
synthetic route for the isomer (9H-quino[4,3,2-
de][1,7]phenanthroline-9-one (2) of the natural marine
alkaloid ascididemin (9H-quino[4,3,2-de][1,10]phenan-
throline-9-one (1) that showed cytotoxic properties.
Ring D-modified ascididemin isomer (2)-analogues were
evaluated for selective cytotoxicity on a panel of human
solid tumor cells in an attempt to determine their phar-
maceutical utility. In vitro, within the limits of the
study, compounds exhibited cytotoxic effects of the
same order than the parent compound suggesting that
ring D-positions 5 and 7 of the ascididemin isomer ser-
ies could be modulated without losing the cytotoxic
activity.
Discussion
The isomer of ascididemin was found as potent or
slightly less potent (with 1 log difference) than the nat-
ural product ascididemin on the panel of human cell
lines: human glioblastoma (U-87MG, U-373MG and
SW1088) and human breast (T-47D and CF-7), colon
(Lovo and HCT-15), non small-cell lung (A549 and A-
427), prostate (PC-3) and bladder (T24 and J82) carci-
noma cell lines, tested depending on the strain sensitiv-
ity. On the non small-cell lung A 549 cell line, the sole
cell line which data can be compared with other groups,
although similar results were reported for isomer of
ascididemin 2 with IC50 equal to 0.02 mM,5 0.3 mM11 and
0.2 mM (present study), quite divergent results from ours
(IC50=7 mM) were reported by Alvarez et al.5
(IC50=0.004 mM) for the isomer of ascididemin 2.
Experimental
Chemical synthesis
Assays
experimental
conditions
(solubilisation,
purity. . .) or strain sensitivity may possibly explain the
observed difference.
1H and 13C NMR spectra were obtained with a JEOL
400 MHz spectrometer, with the chemical shifts in the
remaining protons of the deuterated solvents serving as
internal standards. IR spectra were obtained with a
Perkin-Elmer (1600 series FTIR) spectrometer. Mass
spectra (MS) were recorded on an automass Unicam
spectrometer. Reagents were purchased from commer-
cial sources and used as received. Chromatography was
performed on silicagel (15–40 mm) by means of the sol-
vent systems indicated below. The purity of the different
compounds was evaluated on chromatographic systems
consisting either of a Kromasil Si, 5-mm column
(250 mm  4.6 mm), isooctane/EtOH/MeOH 80:10:10
at 2mL/min flow rate, 260 nm (system I), or Zorbax
NH2, 5 mm column (150 mm  4.6 mm), isooctane/
EtOH/MeOH 80:10:10 at 1 mL/min flow rate, 260 nm
(system II).
Ring D-modified analogues with substitutions in posi-
tion 5 or 7 of compound 2 skeleton exhibit similar
cytotoxic effects than the parent compound. Therefore,
both positions of the ascididemin isomer (compound 2)
series could be modulated without losing the cytotoxic
activity.
Within the limits of the two studies, the ascididemin
isomer (compound 2) analogues were found slightly less
potent when compared to their counterparts in the
ascididemin series, IC50 of which were already reported
by our group.6b As an example, compounds 5 and 6
substituted in position 5 by a bromo or an amino group
exhibited IC50 values higher, with one or 2log difference

4354
E. Delfourne et al. / Bioorg. Med. Chem. 11 (2003) 4351–4356
N-(2,5-Dimethoxyphenyl)anthranilic acid (8). A mixture
of 2-chlorobenzoic acid (9.2 g, 60 mmol), dimethoxyani-
line (10 g, 65 mmol), Cu (0.96 g), Cu2O (0.96 g) and
K2CO3 (10.4 g) in diglyme (120 mL) was refluxed over-
night. The reaction media was concentrated, made
alkaline by addition of 1 N NaOH and diethyl ether was
added. After filtration, the alkaline solution was washed
with ether (3 Â 300 mL) and acidified with concentrated
HCl, and extracted with AcOEt (3 Â 300 mL). The
combined extracts were washed with H2O, dried over
MgSO4 and evaporated to dryness in vacuo. The residue
was purified by flash-chromatography (CH2Cl2) to give
the expected product as a yellow solid (14.5 g, 89%), mp
(20 mL) and NaHCO3 saturated solution (20 mL) were
added and stirring was continued. The mixture was
extracted with CH2Cl2 (3 Â 100 mL), the combined
extracts were dried over MgSO4 and concentrated in
vacuo to give the expected quinone as a brown powder
ꢀ
1
(0.87 g, 93%), mp >260 C. H NMR (CDCl3) 3.22 (s,
3H); 7.09 (d, 1H, J=10.3 Hz); 7.18 (d, 1H, J=10.3 Hz);
7.78 (dd, 1H, J=8.5 and 8.5 Hz); 7.91 (dd, 1H, J=8.5
and 8.5 Hz); 8.32(d, 1H, J=8.5 Hz); 8.43 (d, 1H,
J=8.5 Hz). 13C NMR (CDCl3) 15.87; 124.40; 125.41;
126.30; 129.61; 132.32; 132.52; 137.88; 141.61; 147.05;
148.23; 151.23; 183.43; 186.69. IR (CHCl3) 1701;
1661 cmÀ1
.
138 ꢀC. H NMR (CDCl3) 3.77 (s, 3H); 3.85 (s, 3H);
1
6.57 (dd, 1H, J=8.8 and 2.9 Hz); 6.77 (ddd, 1H, J=1.9,
8.1 and 7.5 Hz); 6.87 (d, 1H, J=9.2Hz); 7.04 (d, 1H,
J=2.9 Hz); 7.3–7.4 (m, 2H); 8.05 (dd, 1H, J=7.7 and
1.1 Hz); 9.35 (br.s, 1H). 13C NMR (CDCl3) 55.76; 56.45;
107.30; 107.71; 112.00; 112.26; 114.70; 117.53; 130.78;
132.60; 134.09; 145.98; 147.71; 153.75; 172.95. IR
6-Methyl-1,11-diazanaphtacene-5,12-dione (12). A solu-
tion of quinone 11 (5.02g, 22.5 mmol), acrolein- N,N-
dimethylhydrazone (3.3 g, 33.8 mmol) acetic anhydride
(24.5 mL) in toluene (300 mL) was stirred at 95 ꢀC, in
the dark, and under nitrogen atmosphere for 30 min.
10% Pd/C (2.6 g) was added and the reaction mixture
was warmed at 95 ꢀC for 35 min. After concentration in
vacuo, the crude product was purified by flash-chroma-
tography (CH2Cl2/MeOH 98:2, and 99:1) to give the
tetracyclic compound which was washed with ether:
beige solid (1.9 g, 31%), mp >260 ꢀC. 1H NMR
(CDCl3) 3.32(s, 3H); 7.78–7.83 (m, 2H); 7.95 (ddd, 1H,
J=8.4, 7.7 and 1.5 Hz); 8.39 (dd, 1H, J=8.8 and
1.5 Hz); 8.51 (dd, 1H, J=7.7 and 1.5 Hz); 8.68 (dd, 1H,
J=8.1 and 1.9 Hz); 9.16 (dd, 1H, J=4.8 and 1.9 Hz).
13C NMR (CDCl3) 16.67; 124.59; 125.44; 128.39;
129.76; 129.89; 132.25; 132.54; 132.88; 135.93; 148.00;
148.59; 148.73; 152.48; 155.31; 180.81; 184.37. IR
(CHCl3) 3327; 1685 cmÀ1
.
2-(20,50-Dimethoxyphenylamino)acetophenone (9). MeLi
(1.4 M/Et2O, 140 mL, 196 mmol) was added to a mix-
ture of compound 8 (17.6 g, 64.5 mmol) in THF
(140 mL), at 0 ꢀC under nitrogen atmosphere. The reac-
tion mixture was refluxed for 4 h 30 min, H2O (400 mL)
was added and the mixture was extracted with ether (3
 400 mL). The combined extracts were dried over
MgSO4, and the solvant was removed in vacuo. The
crude product was purified by flash-chromatography
(CH2Cl2) to give the expected product as a yellow solid
(16.1 g, 92%), mp 79 ꢀC. 1H NMR (CDCl3) 2.64 (s, 3H);
3.76 (s, 3H); 3.84 (s, 3H); 6.55 (dd, 1H, J=8.8 and
2.9 Hz); 6.73 (dd, 1H, J=1.4 and 7.5 Hz); 6.85 (d, 1H,
J=8.8 Hz); 7.04 (d, 1H, J=2.9 Hz); 7.3–7.4 (m, 2H);
7.81 (dd, 1H, J=1.5 and 8.0 Hz); 10.5 (br.s, 1H). 13C
NMR (CDCl3) 28.14; 55.68; 56.32; 107.03; 107.66;
111.99; 114.75; 116.83; 120.06; 130.65; 132.39; 134.32;
145.92; 146.62; 153.59; 200.97. IR (CHCl3) 3350;
(CHCl3) 1703; 1663 cmÀ1
.
9-H-Quino[4,3,2-de][1,7]phenanthrolin-9-one (2). Dime-
thylformamide diethylacetal (0.18 mL, 1.05 mmol) was
added dropwise under nitrogen atmosphere to a sus-
pension of tetracyclic compound 12 (83 mg, 0.3 mmol)
in anhydrous DMF (2mL). The reaction mixture was
warmed at 120 ꢀC for 30 min. After concentration in
vacuo, H2O (90 mL) and NH4Cl (0.49 g) were added
and the mixture was refluxed for 25 min. The mixture
was extracted with CH2Cl2 (3 Â 50 mL). The combined
organic layers were dried over MgSO4 and con-
centrated. The crude product was purified by flash-
chromatography (CH2Cl2/MeOH 98:2) to give the pen-
tacyclic compound as a yellow solid (40 mg, 47%), mp
1642cm À1
.
1,4-Dimethoxy-9-methylacridine (10).
compound 9 (11.2g, 40 mmol) and polyphosphoric acid
A
mixture of
ꢀ
(102g) was warmed at 100 C for 1 h. H2O (400 mL) was
added and the mixture was neutralyzed with 5 M NaOH
(370 mL), and extracted with CHCl3 (3 Â 300 mL). The
combined extracts were dried on MgSO4 and con-
centrated in vacuo. The crude product was purified by
flash-chromatography (CH2Cl2) to give quantitatively
the tricyclic derivative as a orange-brown solid, mp
>260 ꢀC. H NMR (CDCl3) 7.78 (dd, 1H, J=8.1 and
1
4.8 Hz); 7.97 (ddd, 1H, J=8.0, 7.4 and 1.2Hz); 8.04
(ddd, 1H, J=8.0, 7.4 and 1.2Hz); 8.51 (d, 1H,
J=5.9 Hz); 8.69 (dd, 2H, J=8.0 and 1.5 Hz); 9.08 (dd,
1H, J=4.8 and 1.9 Hz); 9.13 (d, 1H, J=5.9 Hz); 9.27 (d,
1H, J=1.9 and 8.1 Hz). 13C NMR (CDCl3) 115.15;
121.76; 122.28; 127.13; 127.16; 129.65; 130.82; 132.21;
132.60; 132.99; 136.88; 139.91; 144.85; 148.00; 148.03;
151.75; 151.84; 179.94. IR (CHCl3) 1692cm À1. HRMS
calcd for C18H9N3O (M+H)+: 283.0745. Found
283.0749. tr: 7.44 min (100% purity) using system II.
ꢀ
1
136 C. H NMR (CDCl3) 3.36 (s, 3H); 3.96 (s, 3H);
4.09 (s, 3H); 6.68 (d, 1H, J=8.0 Hz); 6.89 (d, 1H,
J=8.4 Hz); 7.54 (m, 1H); 7.73 (m, 1H); 8.32(d, 1H,
J=8.4); 8.36 (d, 1H, J=8.8 Hz). 13C NMR (CDCl3)
17.78; 55.66; 56.13; 102.43; 105.18; 120.25; 124.28;
125.62; 126.59; 129.44; 130.81; 142.45; 144.23; 147.21;
149.46; 151.45. IR (CHCl3) 1685; 1661 cmÀ1
.
9-Methylacridine-1,4-dione (11). A solution of com-
pound 10 (1 g, 3.95 mmol) and cerium ammonium
nitrate (7 g, 17.6 mmol) in a mixture CH2Cl2/H2O
(25 mL/12 mL) was stirred at 0 ꢀC for 20 min. H2O
7-Nitro-9-H-quino[4,3,2-de][1,7]phenanthrolin-9-one (3).
A mixture of compound 2 (200 mg, 0.7 mmol) and fum-
ing HNO3 (13 mL) was warmed at 100 ꢀC for 1 h 30 min.
The reaction mixture was poured into ice (40 g), made

E. Delfourne et al. / Bioorg. Med. Chem. 11 (2003) 4351–4356
4355
alkaline with NH4OH (30 mL) and extracted by a mix-
ture CHCl3/MeOH 95:5 (3 Â 300 mL). The combined
organic layers were dried over MgSO4 and concentrated
in vacuo. The crude product was purified by flash-
chromatography (CHCl3/MeOH 95:5) to give the nitro
compound as a brown solid (55 mg, 24%), Pf >260 ꢀC.
1H NMR (DMSO-d6) 7.93 (dd, 1H, J=8.1 and 4.4 Hz);
8.18 (dd, 1H, J=8.4 and 7.7 Hz); 8.56 (dd, 1H, J=7.7
and 1.1 Hz); 8.96 (d, 1H, J=5.9 Hz); 9.0 (dd, 1H, J=4.4
and 1.5 Hz); 9.15 (dd, 1H, J=8.1 and 1.4 Hz); 9.25 (d,
1H, J=5.9 Hz); 9.29 (dd, 1H, J=8.4 and 1.1 Hz). IR
(CHCl3) 1700 cmÀ1. HRMS calcd for C18H8N4O3
(M+H)+: 328.0596. Found 328.0590; tr: 12.58 min
(96.4% purity) using system I with isooctane/EtOH
80:20 and flow rate 1 mL/min.
The precipitate was washed with CHCl3/MeOH 98:2(4
 10 mL) to give the amino-compound as a violine solid
(60 mg, 48%), mp >260 ꢀC. 1H NMR (CDCl3) 6.69
(2H); 7.40 (dd, 1H, J=9.0 and 2.2 Hz); 7.76 (d, 1H,
J=2.2 Hz); 7.87 (dd, 1H, J=8.1 and 4.4 Hz); 8.11 (d,
1H, J=9.0 Hz); 8.45 (d, 1H, J=5.9 Hz); 8.97 (dd, 1H,
J=4.4 and 1.8 Hz); 9.04 (d, 1H, J=5.9 Hz); 9.16 (dd,
1H, J=8.1 and 1.8 Hz); tr: 9.73 min (100% purity) using
system I.
5-Dimethylamino-9-H-quino[4,3,2-de][1,7]phenanthrolin-
9-one (7). To a solution of compound 6 (4.18 g,
28 mmol) in trifluoroacetic acid (1 mL) was added at
0 ꢀC, formaldehyde (37% aqueous, 0.113 mL,
1.52mmol) and NaBH 3CN (26 mg, 0.41 mmol). The
mixture was allowed to warm up to room temperature,
made alkaline with 5 N NaOH, and extracted with
CH2Cl2 (2 Â 20 mL). The combined extracts were dried
over MgSO4, concentrated in vacuo and the crude pro-
duct was purified by flash-chromatography (CH2Cl2/
MeOH 95:5) to give the dimethylamino-compound as a
violine solid (4 mg, 17%), mp >260 ꢀC. 1H NMR
(CDCl3) 3.28 (s, 6H); 7.45 (dd, 1H, J=2.8 and 9.2 Hz);
7.56 (d, 1H, J=2.8 Hz); 7.71 (dd, 1H, J=8.1 and
4.4 Hz); 8.35 (d, 1H, J=5.6 Hz); 8.45 (d, 1H, J=9.2Hz);
8.99 (d, 1H, J=5.6 Hz); 9.03 (dd, 1H, J=4.4 and
1.9 Hz); 9.23 (dd, 1H, J=8.1 and 1.9 Hz). IR (CHCl3)
7-Amino-9-H-quino[4,3,2-de][1,7]phenanthrolin-9-one (4).
A mixture of nitro-derivative 3 (160 mg, 0.54 mmol) and
10% Pd/C (107 mg) in MeOH (80 mL) was stirred under
a hydrogen atmosphere for 2h. After filtration through
Celite and evaporation in vacuo, the crude product was
purified by flash-chromatography (CHCl3/MeOH 95:5)
to give the amino compound as a violine solid (71 mg,
49%), mp >260 ꢀC. H NMR (DMSO-d6) 6.59 (br. s,
1
2H); 7.20 (d, 1H, J=7.7 Hz); 7.70 (dd, 1H, J=7.7 and
7.7 Hz); 7.91 (dd, 1H, J=8.1 and 4.4 Hz); 8.02(d, 1H,
J=7.7 Hz); 8.72(d, 1H, J=5.7 Hz); 9.00 (dd, 1H,
J=4.4 and 1.8 Hz); 9.07 (d, 1H, J=5.7 Hz); 9.16 (dd,
1H, J=8.1 and 1.5 Hz). 13C NMR (DMSO-d6) 110.01;
113.35; 117.61; 117.74; 124.66; 128.69; 133.01; 133.61;
133.63; 133.88; 137.93; 143.09; 148.39; 148.63; 149.01;
149.41; 152.63; 180.03. IR (CHCl3) 3420; 3315;
1672cm À1. HRMS calcd for C18H10N4O (M+H)+:
298.0855. Found 298.0862; tr: 4.52 min (100% purity)
using system II with isooctane/EtOH/MeOH 70:20:10.
1755, 1715 cmÀ1
.
HRMS calcd for C20H14N4O
(M+H)+: 326.1167. Found 326.1161; tr: 12.48 min
(100% purity) using system I.
In vitro characterization of the drug-induced effects on
human cancer cell line growth
Twelve human tumor cell lines were obtained from the
American Type Culture Collection (ATCC, Manassas,
VA, USA). These included three glioblastomas
(SW1088, U-373 MG and U-87 MG), two colon (HCT-
15 and LoVo), two non small-cell-lung (A549 and A-
427), two bladder (J82 and T24), one prostate (PC-3)
and two breast (T-47D and MCF7) cancer models. The
ATCC numbers of these cell lines are HTB 12
(SW1088), HTB 14 (U-87 MG), HTB 17 (U-373 MG),
CCL225 (HCT-15), CCL229 (LoVo), CCL 185 (A549),
HBT 53 (A-427), HTB1 (J82), HTB4 (T24), HTB133
(T-47D), HTB22 (MCF7) and CRL1435 (PC-3). The
cells were cultured at 37 ꢀC in sealed (airtight) Falcon
plastic dishes (Nunc, Gibco, Belgium) containing
Eagle’s minimal essential medium (MEM, Gibco) sup-
plemented with 5% fetal calf serum (FCS). All the
media were supplemented with a mixture of 0.6 mg/mL
glutamine (Gibco), 200 IU/mL penicillin (Gibco),
200 IU/mL streptomycin (Gibco) and 0.1 mg/mL genta-
mycin (Gibco). The FCS was heat-inactivated for 1 h at
56 ꢀC.
5-Bromo-9-H-quino[4,3,2-de][1,7]phenanthrolin-9-one
(5). A mixture of compound 2 (166 mg, 0.59 mmol) and
bromine (0.5 mL, 9.7 mmol) in acetic acid (8.3 mL) was
warmed at 100 ꢀC for 4 h. After concentration in vacuo,
the crude product was washed with CHCl3, made alka-
line with NaHCO3 saturated solution and extracted
with CHCl3 (3 Â 30 mL). The combined extracts were
dried over MgSO4 and concentrated in vacuo. After
recristallization in CHCl3, the bromo-compound was
obtained as a yellow solid (69 mg, 32%), mp >260 ꢀC.
1H NMR (CDCl3) 7.78 (dd, 1H, J=8.1 and 4.8 Hz);
8.11 (dd, 1H, J=8.8 and 2.0 Hz); 8.41 (d, 1H,
J=5.6 Hz); 8.53 (d, 1H, J=8.8 Hz); 8.81 (d, 1H,
J=2.0 Hz); 9.07 (dd, 1H, J=4.8 and 1.5 Hz); 9.14 (d,
1H, J=5.6 Hz); 9.25 (dd, 1H, J=8.1 and 1.5 Hz). 13C
NMR (CDCl3) 116.10; 116.94; 124.64; 125.79; 125.84;
128.37; 133.53; 134.16; 134.62; 135.41; 136.95; 144.61;
146.78; 147.64; 149.03; 149.47; 153.02; 180.71. IR
(CHCl3) 1694 cmÀ1. HRMS calcd for C18H8N3OBr
(M+H)+: 360.9851. Found 360.9852; tr: 10.02 min
(98.2% purity) using system I with flow rate 1 mL/min.
The 12cell lines were incubated for 24 h in 96-microwell
plates (at a concentration of 40,000 cells/mL culture
medium) to ensure adequate plating prior to the deter-
mination of the cell growth. This process was carried
out by means of the colorimetric MTT assay, as detailed
previously.12,13 This assessment of cell population
growth is based on the capability of living cells to
5-Amino-9-H-quino[4,3,2-de][1,7]phenanthrolin-9-one (6).
A mixture of compound 5 (150 mg, 0.42mmol) and
NaN3 (210 mg, 3.2 mmol) in DMF (4.5 mL) was
warmed at 105 ꢀC for 6 h. After concentration in vacuo,
H2O (6 mL) was added and the mixture was filtered.

4356
E. Delfourne et al. / Bioorg. Med. Chem. 11 (2003) 4351–4356
reduce the yellow product MTT (3-(4,5)-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St
Louis, MO, USA) to a blue product, formazan, by a
reduction reaction occurring in the mitochondria. The
number of living cells is directly proportional to the
intensity of the blue, which is quantitatively measured
by spectrophotometry on a DIAS microplate reader
(Dynatech Laboratories, Guyancourt, France) at a 570-
nm wavelength (with a reference of 630 nm). Each
experiment was carried out in sextuplicate. We validated
the MTT-related data using two alternative techniques,
namely direct cell counting and the genomic incor-
poration of tritiated thymidine (data not shown).
Barrows, L. R.; Copp, B. R. Tetrahedron Lett. 2000, 41, 1667.
(b) Lindsay, B. S.; Christiansen, H. C.; Copp, B. R. Tetra-
hedron 2000, 56, 497.
4. Cuerva, J. M.; Cardenas, D. J.; Echavarren, A. M. Chem.
Commun. 1999, 1721.
5. Alvarez, M.; Feliu, L.; Ajana, W.; Joule, J. A.; Fernandez-
Puentes, J. L. Eur. J. Chem. 2000, 849.
6. Cuerva, J. M.; Cardenas, D. J.; Echavarren, A. M. J.
Chem. Soc., Perkin Trans. 1 2002, 11, 1360.
7. (a) Delfourne, E.; Darro, F.; Bontemps-Subielos, N.; Dec-
aestecker, C.; Bastide, J.; Frydman, A.; Kiss, R. J. Med.
Chem. 2001, 44, 3275. (b) Delfourne, E.; Darro, F.; Portefaix,
P.; Galaup, C.; Bayssade, S.; Bouteille, A.; Le Corre, L.; Bas-
tide, J.; Collignon, F.; Lesur, B.; Frydman, A.; Kiss, R. J.
Med. Chem. 2002, 45, 3765.
8. Ionescu, M.; Mester, I. Rev. Roum. Chim. 1969, 14, 789.
9. Bracher, F. Heterocycles 1989, 29, 2093.
10. Weinstein, J. N.; Kohn, K. W.; Grever, M. R.; Viswa-
nadhan, V. N.; Rubinstein, L. V.; Monks, A. P.; Scudiero,
D. A.; Welch, L.; Koutsoukos, A. D.; Chiausa, A. J.; Paull,
K. D. Science 1992, 258, 447.
Six concentrations ranging from 10À5 to 10À9 M were
assayed for each of the compounds under study (see
Table 1).
11. Mean graphs depicting the dose–response data for com-
pounds submitted to the NCI can be viewed, by searching for
the NSC number of the compound at the NCI web site: http://
dtp.nci.nih.gov/docs/cancer/searches/cancer_open_compounds.
html.
12. Pauwels, O.; Kiss, R.; Pasteels, J. L.; Atassi, G. Pharm.
Res. 1995, 12, 1011.
13. Camby, I.; Salmon, I.; Danguy, A.; Pasteels, J. L.;
Brotchi, J.; Martinez, J.; Kiss, R. J. Natl. Cancer Inst. 1996,
88, 594.
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3. (a) Matsumoto, S. S.; Sidford, M. H.; Holden, J. A.;