Synthesis and in vitro and in vivo antitumor activity of 2-phenylpyrroloquinolin-4-ones

Article abstract of DOI:10.1021/jm049387x

In our search for potential new anticancer drugs, we designed and synthesized a series of tricyclic compounds containing the antimitotic 2-phenylazaflavone chromophore fused to a pyrrole ring in a pyrroloquinoline structure. Compounds 8, 18, 19, 22, 23, 25 and 26, when tested against a panel of fourteen human tumor cell lines, showed poor in vitro cytotoxic activity, whereas 20, 21 and 24 showed significant activity (IC₅₀ 0.7 to 50 μM). Steroid hormone-sensitive ovary, liver, breast and adrenal gland adenocarcinoma cell lines displayed the highest sensitivity (IC₅₀ 0.7 to 8 μM). Compound 24 blocked cells in the G₂/M phase of the cell cycle and induced a significant increase in apoptotis. Compounds 20, 21 and 24 proved to alter microtubule assembly and stability, displaying a cytoplasmic microtubule network similar to that caused by Vincristine. In vivo, administration of compound 24 to Balb/c mice inhibited the growth of a syngenic hepatocellular carcinoma.

Full text of DOI:10.1021/jm049387x

J. Med. Chem. 2005, 48, 3417-3427
3417
Synthesis and in Vitro and in Vivo Antitumor Activity of
2-Phenylpyrroloquinolin-4-ones
Maria Grazia Ferlin,*,† Gianfranco Chiarelotto,† Venusia Gasparotto,†,‡ Lisa Dalla Via,† Vincenzo Pezzi,§
Luisa Barzon,‡ Giorgio Palu`,‡ and Ignazio Castagliuolo‡
Department of Pharmaceutical Sciences, and Department of Histology, Microbiology and Medical Biotechnologies,
University of Padova, and Department of Pharmaco-Biology, University of Calabria, Italy
Received July 30, 2004
In our search for potential new anticancer drugs, we designed and synthesized a series of
tricyclic compounds containing the antimitotic 2-phenylazaflavone chromophore fused to a
pyrrole ring in a pyrroloquinoline structure. Compounds 8, 18, 19, 22, 23, 25 and 26, when
tested against a panel of fourteen human tumor cell lines, showed poor in vitro cytotoxic activity,
whereas 20, 21 and 24 showed significant activity (IC50 0.7 to 50 µM). Steroid hormone-sensitive
ovary, liver, breast and adrenal gland adenocarcinoma cell lines displayed the highest sensitivity
(IC50 0.7 to 8 µM). Compound 24 blocked cells in the G2/M phase of the cell cycle and induced
a significant increase in apoptotis. Compounds 20, 21 and 24 proved to alter microtubule
assembly and stability, displaying a cytoplasmic microtubule network similar to that caused
by Vincristine. In vivo, administration of compound 24 to Balb/c mice inhibited the growth of
a syngenic hepatocellular carcinoma.
Introduction
In the field of anticancer drugs, microtubules are still
a valid target, and it is probable that antimitotic drugs
will continue to be important chemotherapeutic agents,
especially as more selective approaches are developed.1
Growing understanding of their mechanisms of action,
and the synergy shown in combination therapies, to-
gether with the desire to obtain orally available ana-
logues and the need to overcome neurotoxicity and the
development of antimitotic resistance have all produced
ever increasing interest, aimed at discovering more
effective agents and therapeutic strategies.
Most of the antimitotic compounds used in clinical
practice are either naturally occurring molecules or their
synthetic analogues, such as vinca alkaloids, taxol,
colchicine, podophyllotoxin and combretastatin A-4.2
Interestingly, it has recently been reported that some
natural flavonoids (Figure 1, 2-phenyl-benzopyran-4-
ones I) also exert potent cytotoxic activity against
several cancer cell lines, both in vitro and in vivo by
interfering with tubulin polymerization.3,4 However,
since these natural compounds interact with various
Figure 1.
biological targets,5-8 their antitumor properties may be
the result of multiple activities antioxidant,5 anti-
ones, II) and a series of bioisosteric bicyclic diaza
analogues, quinazolinones2 (Figure 1, III) and naphthyri-
dinones2,20-22 (Figure 1, IV). All these classes of com-
pounds have strong antimitotic effects and appear to
interact specifically with the colchicine binding site on
â tubulin.19,23 SAR and QSAR studies have also indi-
cated very stringent structural and spatial requirements
for the antimitotic activity of 2-phenylazaflavones,
indicating that specific substitutions on the side phenyl
ring, together with others on the quinolinone ring, may
be important for their antiproliferative activity.19,24
As part of our search for synthetic pyrroloquinoline-
derivative compounds with antitumor activity,25,26 we
designed and synthesized a series of 2-phenylpyrrolo-
mutagenic,5,40 pro-apoptotic,9 antitopoisomerase10 and
antiestrogenic.11-16
To enhance specifically the antimitotic activity of
flavonoid compounds, some authors have synthesized
new azaflavones2,17-19 (Figure 1, 2-phenylquinolin-4-
* To whom correspondence should be addressed. Prof. Maria Grazia
Ferlin, Department of Pharmaceutical Sciences, Faculty of Pharmacy,
University of Padova, Via Marzolo 5, 35131 Padova, Italy. Tel:
00390498275718. Fax: 00390498275366. E-mail: mariagrazia.ferlin@
unipd.it.
† Department of Pharmaceutical Sciences, University of Padova.
‡ Department of Histology, Microbiology and Medical Biotechnolo-
gies, University of Padova.
§ University of Calabria.
10.1021/jm049387x CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/13/2005

3418 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Scheme 1. Retrosynthetic Routes
Ferlin et al.
Scheme 2. Synthesis of Aminoindoles
quinolinones (Figure 1, V): these may be viewed as the
antimitotic 2-phenylazaflavone pharmacophore modified
by fusion to a pyrrole ring. This structural modification,
suitable for further substitutions, in view of controlled
phamacodynamic and pharmacokinetic testing, will be
useful for more detailed SAR information about this
class of molecules. Although we expected that the new
compounds would exert antimitotic activity, it should
be recalled that enlargement of a bicyclic structure to a
planar tricyclic one may result in additional mecha-
nisms of action, such as antitopoisomerase I and II
activity. Furthermore, looking at the structural similar-
ity of the newly synthesized 2-phenyl-1H,7H-pyrrolo-
[2,3-h]quinolin-4-ones (Figure 1, VII) with flavones and
7,8-benzoflavones6 (Figure 1, VI), a certain interaction
with estrogen aromatase enzyme may also be hypoth-
esized. This paper reports the synthesis of nine new
tricyclic derivatives, characterized by the pyrrolo[2,3-
h]quinolin-4-one nucleus bearing a methyl group or a
variously substituted phenyl ring in position 2. All
compounds were evaluated for their in vitro antiprolif-
erative activity on human tumor cell lines, and in vivo
on a syngenic hepatocellular carcinoma in Balb/c mice
(24). In addition, to obtain some preliminary insights
on the mechanism(s) of action, their effects on cell cycle
and microtubule dynamics, programmed cell death
(apoptosis) and aromatase and topoisomerase I and II
enzymatic activity were also examined.
Scheme 3. Synthetic Route to 1H,7H-Pyrrolo
[2,3-h]quinoline-4-one (8)
be disconnected to o-nitrotoluidine by two retrosynthetic
steps of the Madelung indole synthesis variation re-
ported by Bergman and Sand.30
Scheme 2 shows the preparation of aminoindoles 1
and 4. For compound 1, 2,6-dinitrotoluene, treated with
dimethylformamide dimethylacetal (DMFDMA) in N,N-
dimethylformamide (DMF), produced the dimethyl-
aminostyrene derivative in an almost quantitative yield
already after only 4 h. This was subjected to reductive
cyclization with TiCl3 to give 4-aminoindole: we chose
this reagent, because it provided a higher yield with
respect to other methods (H2, Pd/C) over a shorter
reaction time (minutes instead of hours).28 For com-
pound 4, we first obtained 4-nitroindole from 2-methyl-
3-nitroaniline, which was reacted with triethyl ortho-
formate in the presence of p-toluensulfonic acid to give
the imidate ester. Treatment of the latter with diethyl-
oxalate and potassium ethoxide produced 4-nitroindole
2.30,31 As regards experimental procedures and reaction
products 1 and 2, no evident differences were noted
compared with those previously reported.
Results and Discussion
Chemistry. On the basis of our previous experience
with pyrroloquinoline nucleus synthesis25,26 and in view
of the retrosynthetic pathways briefly shown in Scheme
1, 4-aminoindole was chosen as the starting material,
leading to the tricyclic structure of 2-substituted-pyr-
rolo[2,3-h]quinolin-4-one by a retro-Conrad-Limpach27
disconnection at the N-1 and C-2 bonds in the C ring.
As 4-aminoindole or its precursor 4-nitroindole are
commercially available but extremely expensive, we
prepared them ourselves. 4-Aminoindole28 may be dis-
connected to a vinyl intermediate and the latter brought
to 2,6-dinitrotoluene via a retro-Leimgruber-Batcho
reaction. Leimgruber-Batcho synthesis29 is a two-step
method providing indoles which are only substituted on
the benzene ring. The 4-nitroindole precursor, useful for
the synthesis of N7-alkylated tricyclic derivatives, can
Next, 2 was alkylated to the N1-methyl derivative 3
by CH3I and KOH in acetone at room temperature,
which turned out to be a more advantageous method32
than the usual one (CH3I, NaH).33,34 Again, the following

2-Phenylpyrroloquinolin-4-ones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3419
Scheme 4a
a (a) Synthesis of â-ketoesters 9 and 10. (b) General route to 2-substituted-pyrroloquinolinones.
Table 1. In Vitro Cytotoxic Activities of 2-Substituted-pyrroloquinolin-4-ones 8, 18-26 Compared with Vincristine Sulfate Salt
cytotoxicity, IC50 (µM)
compd
Aro
C8305
A-172
Mia Paca 2
PT45
HT-29
8
>100
>100
>100
100
10
>100
>100
>100
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4
>100
>100
100
>100
>100
>100
60
18
19
20
21
22
23
24
25
26
>100
80
8
5
100
10
8
100
50
>100
80
>100
>100
10
80
>100
0.1
>100
>100
5
>100
>100
0.01
>100
>100
100
>100
>100
50
>100
>100
0.1
4
70
>100
>100
>100
<0.0001
vincristine
sulfate salt
0.1
Table 2. In Vitro Cytotoxic Activities of 2-Substituted-pyrroloquinolin-4-ones 8, 18-26 Compared with Vincristine Sulfate Salt
cytotoxicity, IC50 (µM)
compd
Hep 3B
Hep G2
PLC/PRF/5
SW13
NCI-H295
Ovcar-3
Igrov-1
MCF-7
8
>100
>100
50
>100
>100
10
>100
>100
80
>100
>100
>100
40
>100
>100
20
>100
>100
15
>100
>100
30
18
19
20
21
22
23
24
25
26
>100
50
4
4
8
3
0,9
50
60
0,7
10
8
2
5
4
1
1
7
2
2
3
4
30
100
>100
2
>100
>100
7
80
50
60
60
50
60
50
80
70
2
3
1
3
1
>100
>100
0.1
50
>100
0.1
50
>100
0.1
50
30
30
50
>100
>100
0.1
>100
0.01
>100
0.01
>100
0.01
vincristine
sulfate salt
0.001
reduction reaction was performed with TiCl3, leading
to almost pure N-methyl-4-aminoindole (4) in good
yields.
sation of 2-acetylthiophene and m-methoxyacetyl ben-
zene with anhydrous diethyl carbonate in the presence
of sodium hydride.
Synthesis of unsubstituted 1H,7H-pyrrolo[2,3-h]-
quinolin-4-one (8) was carried out with a four-step
pathway (Scheme 3). Starting from 4-aminoindole, we
constructed the tricyclic compound 8 by means of the
Gould-Jacob reaction,35 a method used to obtain the
quinolin-4-one nucleus which may subsequently lead to
2,3-unsubstituted derivatives. Condensation of 1 with
diethyl ethoxymethylenemalonate to 5, thermal cycliza-
tion to 6, ester saponification to 7 and a final decar-
boxylation reaction produced pyrroloquinolinone 8.
The syntheses of 2-substituted pyrroloquinolin-4-ones
are outlined in Scheme 4.
(b) Following Conrad-Limpach synthesis27 of the
quinoline nucleus, 4-aminoindoles 1, 4 were condensed
with â-keto-esters a-d and 9, 10 to give enamine
derivatives 11-17, which were subjected to thermal
cyclization to give final compounds 18-24.
m-Nitro derivative 23 was also reduced by TiCl3 to
the corresponding amino compound 25 and then to the
more stable dihydrochloride 26.
Biology. In Vitro Cytotoxic Activity of 2-Phenyl-
pyrroloquinolin-4-ones. Tables 1 and 2 show the in
vitro cytotoxic activity of compounds 8, 18-26, tested
against vincristine sulfate salt as reference on a panel
of 14 human tumor cell lines, derived largely from solid
tumors including liver, pancreas, colon, central nervous
system, thyroid, adrenal gland, ovary and breast.
(a) Ethyl thiophencarbonyl acetate20 9 and ethyl
m-methoxybenzoyl acetate36 10 were prepared, accord-
ing to a method reported in the literature,37 by conden-

3420 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Ferlin et al.
Figure 2. Effects of compound 24 on cell cycle dynamics as determined by Flow Cytometry. Figure 2A. DNA fluorescence flow
cytometric profiles of propidium iodide-stained ovary carcinoma Ovcar-3 cells before treatment. Figure 2B. As above, after 24 h
incubation with compound 24 (5 µM).
As expected, compounds 8 and 18, carrying respec-
tively a hydrogen and a methyl group in position 2, were
devoid of activity, confirming the importance of a
2-phenyl ring for quinolin-4-one moiety cytotoxicity.19
For 2-phenyl derivatives, compounds 19, 22, 23, 25 and
26 had negligible cytotoxic effects against all cell lines
tested, as their IC50 were generally above 50 µM. This
is consistent with literature17 data, which reports a loss
of activity for compounds carrying strong electron-
withdrawing groups such as the NO2 group (22, 23) or
electron-donating groups, especially if they are inserted
in ortho and para positions, whereas the presence of a
free phenyl ring or one substituted with 3′-OCH3
enhances cytotoxic activity, as observed for compounds
20, 21 and 24.
Since compounds 20, 21 and 24 were 50-fold more
active against hormone-responsive cell lines, like liver,
ovary, breast and adrenal gland adenocarcinomas (IC50
0.7 to 8 µM, Table 2) than against nonresponsive ones
(cf. IC50 values in Tables 1 and 2), this result revealed
a considerable selective effect, which 2-phenylaza-
flavones and their analogues do not show.18
Figure 3. Apoptosis cell death. Ovcar-3 cells were incubated
for 24 h with compound 24 (1-5 µM) and presence of histone-
associated DNA fragments in cytoplasm was quantified by
ELISA assay. Results are expressed as means ( SE (optical
density values at 420 nm) from two different experiments
carried out in triplicate. *P < 0.01 vs control (untreated cells).
treatment. The capacity of a drug to block cells in the
G2/M phase of the cell cycle is consistent with disruption
of the mitotic spindle by interaction with the protein
tubulin. Drugs which interfere with tubulin, and there-
fore with cell division, can be shown to cause this effect
by flow cytometry.38
Furthermore, in all hormone-sensitive cell lines tested,
a sub-G0 peak (10.73%, Figure 2B) was observed after
24 h, indicating the presence of apoptotic cells with
fragmented DNA.39 Similar profiles (data not shown)
were obtained for cells exposed to methotrexate, a well-
known pro-apoptotic agent. To confirm the potential pro-
apoptotic effect of 2-phenylpyrroloquinolin-4-ones on
hormone-sensitive cell lines, we quantified the presence
of histone-associated-DNA complexes in the cytoplasm,
a well-accepted marker of apoptosis, in cells exposed for
24-48 h to 1-5 µM of compound 24. As shown in Figure
3, incubation of Ovcar-3 cells with compound 24 for 24
h induced a significant increase in histone-DNA com-
plexes in the cytoplasm, indicating induction of cell
death via an apoptotic pathway.
We may therefore state that the fusion of a pyrrole
ring at the 7,8 position of the 2-phenylquinolin-4-one
pharmacophore leads to molecules which retain their
cytotoxic activity and may also interfere with estrogen-
supported tumor cell growth.
For insights into the mechanism(s) of action of these
new compounds, we assessed the effects of the most
cytotoxic 20, 21 and 24, on cell cycle and microtubule
dynamics, on induction apoptosis and on CYP19 (aro-
matase) and topoisomerase I and II enzymatic activity.
Antimitotic Activity. On the basis of the chemo-
sensitivity profiles of compounds 20, 21 and 24, hormone-
sensitive cell lines were selected as prototypes, to
explore their mode of action on induced cell death.
Changes in SW13, HepG2, MCF-7 and Ovcar-3 cellular
proliferation (DNA content and distribution) during
treatment with compound 24, used for the assays, were
monitored by flow cytometry over a period of 48 h, a
sufficient time interval for these cell lines to complete
their cycle (data not shown).
To further explore the antimitotic activity of com-
pounds 20, 21 and 24, their effects on microtubule
dynamics were evaluated in Ovcar-3 (Figure 4A) and
MCF-7 (Figure 4B) cell lines, stained with TRITC-
labeled anti-â-tubulin antibody. As shown in Figure 4,
the three compounds were able to destabilize micro-
tubules both in the interphase and during mitotic
division, causing a loss of the normal microtubule
As shown in Figure 2, compound 24 induced a
significant increase in cells blocked in the G2/M phase
of the cell cycle: the percentage of cells found in the
G2/M phase was 38.87% (Figure 2B), compared with
16.84% for untreated cells (Figure 2A) after 24 h of

2-Phenylpyrroloquinolin-4-ones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3421
Figure 4. Immunofluorescence images of Ovcar-3 and MCF-7 cells revealed by staining with TRITC-labeled anti-â-tubulin
antibody. Ovcar-3 (Figure 4A) and MCF-7 (Figure 4B) cells were incubated for 18 h with medium drug-free (a1, b1), compounds
(5 µM) 20 (a2, b2), 21 (a3), 24 (a4) and vincristine (5 µM) (a5, b3). Cells were then fixed, stained for â-tubulin to visualize mitotic
spindle and interphase microtubules and analyzed by fluorescence microscopy. Treatment with compound 20 on Ovcar-3 (a2)
and MCF-7 (b2) cell lines, and compounds 21, 24 on Ovcar-3 cells (a3 and a4), caused loss of the normal microtubule network,
like that of vincristine (a5, b3) but much less evident.
network similar to that induced by vincristine but much
less evident. In particular, after treatment with com-
pounds 20, 21 and 24 (5 µM) for 18 h, the microtubule
network appeared to be “rarefied” and fragmented.
The ability of the 2-phenylpyrroloquinolinone de-
rivatives to induce the mitotic arrest and apoptosis
is most likely attributable to their antimicrotubule
activity.
Inhibition of Aromatase. To verify aromatase
activity, initially hypothesized for the structural simi-
larity of pyrrolo[2,3-h]quinolinones with the potent
aromatase inhibitors flavones and 7,8-benzoflavones6,41
(Figure 1, VI) and then reinforced by observed selectiv-
ity, the effects of compounds 20, 21 and 24 on enzyme
activity were investigated by applying the tritiated
water release assay42 to cell line H295R. Figure 5 shows
that compounds 20, 21 and 24 inhibited enzyme activity
in a dose-dependent way, both in basal conditions
(Figure 5A) and after induction with forskolin (Figure
5B), a substance which up-regulates CYP19 activity and
gene transcripts by adenylate cyclase activation.43 Lim-
ited exposure (3 h) of these compounds exerted good
inhibitory activity although to a lesser extent than
letrozole, one of the most potent aromatase inhibitors
currently used in therapy.44
Inhibition of Topoisomerases. We also investi-
gated the ability of 20, 21 and 24 to inhibit the cat-
alyzed reactions of nuclear enzymes topoisomerase I
(relaxation) and topoisomerase II (decatenation). In
detail, none of the tested compounds appeared able to
interfere with the relaxation ability of topoisomerase I
(data not shown). The effects on the decatenation
reaction catalyzed by topoisomerase II are shown in
Figure 6. In particular, 20 was able to inhibit signifi-
cantly the formation of decatenated topoisomerase II
products. In the same experimental conditions, 24

3422 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Ferlin et al.
Figure 6. Antitopoisomerase II activity. Topoisomerase II
decatenation assay in the presence of tested compounds: lane
1, KDNA control (no enzyme); lane 2, KDNA and topo-
isomerase II; lanes 3-5, same as lane 2 but with 0.8 µM 20,
21 and 24, respectively; lane 6, decatenated KDNA standard;
lane 7, linear KDNA standard.
As we did not note any death, weight changes and
histological abnormalities in liver and kidneys of the
treated hosts, 24 appeared to be nontoxic under our
experimental conditions. According to these results, the
selectivity of the antimitotic effect, by means of which
the 2-phenylpyrroloquinolin-4-ones 20, 21 and 24 ex-
erted their cytotoxic activity on the tested cell lines, may
be attributed to their ability to inhibit the aromatase.
This statement is also supported by recent binding
models proposed for aromatase inhibitors, whether
steroidal or not, and flavonoid compounds, illustrating
some similarities on a molecular bases.45,46 Some of
these structural elements are also present in 2-phenyl-
pyrroloquinolinones: the 4-oxo group ensures the in-
teraction of the molecule with the heme prosthetic group
of aromatase, and the pyrrole NH group simulates the
7-hydroxy group on the A ring of flavonoids, consider-
ably enhancing their inhibitory potency. As flavones,
substitutions on the 2-phenyl ring generally decrease
the aromatase inhibitory potency of pyrroloquinolinone
24 (m-OCH3) in comparison with 20 (Figure 5). The
specific angular geometry of pyrrolo[2,3-h]quinolinone
is the same as that 7,8-benzoflavone, which is a powerful
inhibitor of aromatase.
Figure 5. Inhibition of aromatase activity in H295R cells by
compounds 20, 21 and 24. H295R cells were cultured for 24 h
in DMEM-F12 in absence (A) or in the presence (B) of FSK
(25 µM). One hour before addition of substrate [1â-3H(N)]-
androst-4-ene-3,17-dione (0.5 µM), compounds 20, 21 and 24
and letrozole (L) were added to cells at concentrations shown.
After a further 2-h exposure, aromatase activity was assessed
using modified tritiated water method. Results are expressed
as pmoles [3H]H2O released per hour and were normalized for
mg protein (pmol/h/mg protein). Values represent means (
SEM of three different experiments, each performed in trip-
licate. (b) P < 0.01 compared with control (C) cells.
induced weak inhibition of enzymatic activity; 21 ap-
peared unable to exert any detectable effect.
Last, our findings on 2-phenylpyrroloquinolinones are
consistent with a number of SARs and QSARs which
have appeared during the past decade which reveal
higher aromatase inhibitory activity for natural and
synthetic flavones and flavanones carrying a phenyl ring
in position 2, than for isoflavones and isoflavanones
(phenyl in position 3), which are known to bind to
estrogen receptors.6,16
In Vivo Tumor Growth Inhibition. We tested the
effects of compound 24 on tumor growth by using a
typical transplanted model applied to a syngenic hepato-
cellular carcinoma in Balb/c mice. Indeed, 24 showed
significant in vitro cytotoxic activity (IC50 3 µM) against
BNL 1ME A.7R.1, a stabilized murine hepatocarcinoma
cell line which is comparable to other hormone-respon-
sive cell lines. As shown in Figure 7A, in mice implanted
subcutaneously with 107 BNL 1ME A.7R.1 cells, a daily
intraperitoneal injection of 24 (50 mg/kg) caused sig-
nificant inhibition of tumor growth (65%) with respect
to the control groups. Any tumor growth differences
were not noted in those mice, which were treated both
with phosphate-buffered saline and PEG suspension
(vehicle). Daily administration of 24 (50 mg/kg), started
5 days after tumor implant, that is when the tumor
mass had already been developed, caused 40% growth
inhibition with respect to those mice which were given
only vehicle (Figure 7B).
Conclusions
When tested in vitro on a panel of 14 human tumor
cell lines, some 2-phenylpyrroloquinolin-4-ones showed
high and significant selective cytotoxic effect on hormone-
sensitive cell lines. In view of the in vitro results and
in vivo considerable growth inhibition of the hormone-
sensitive BNL 1ME A.7R.1 tumor cell line implanted
in Balb/c mice, these new compounds open new perspec-
tives in the field of selective anticancer agents. Today,
we may state that they belong to the antimitotic cancer
drugs, as confirmed by flow cytometry and immuno-

2-Phenylpyrroloquinolin-4-ones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3423
Figure 7. Antitumor activity of compound 24 observed in Balb/c mice carrying syngenic hepatocarcinoma. Male mice were injected
subcutaneously with 107 BNL 1ME A.7R.1 cells, a syngenic hepatocellular carcinoma cell line, at their dorsal region. Figure 7A:
starting on the second day, groups of animals (n ) 8-12 per group) were administered daily ip with saline (circles), vehicle
(squares) or 50 mg/kg body weight of 24 (triangles). Tumor size was measured daily over 10 days using calipers, and tumor
volume (V) was calculated using the rotational ellipsoid formula: V ) A × B2/2, where A is the longer diameter and B the shorter
diameter. Figure 7B: administration of vehicle (circles) or 50 mg/kg body weight of 24 (squares) was initiated 5 days after injection
with tumor cells, and this treatment was continued for 7 days. Tumor size was measured daily as described above. Results are
expressed as means ( SE. * indicates P < 0.01 vs vehicle or untreated animals.
Aldrich Chimica, Acros and Riedel-de Haen (TiCl3, 15% HCl
solution), and solvents from Carlo Erba, Fluka and Lab-Scan.
Anhydrous DMSO was obtained by distillation under vacuum
and stored on molecular sieves.
fluorescence microscopy analysis, but unlike their par-
ent 2-phenylquinolinones, they are selective for estrogen-
sensitive cell lines, due to their aromatase inhibitory
activity, as confirmed by the tritiated water release
assay. Among the most active compounds 20, 21 and
24, only the unsubstituted 2-phenylpyrroloquinolinone
20 showed significant inhibitory activity of topo-
isomerase II.
All these results prompt us to design and synthesize
more new pyrroloquinolinones in order to develop phar-
macophoric elements directed toward mutual antimi-
totic action and aromatase inhibition with the ultimate
aim of discovering new, more active, and more selective
anticancer compounds.
4-Aminoindole28 (1). In a 50 mL round-bottomed flask, a
solution of 2,6-dinitrotoluene (4.630 g, 25.4 mmol) and N,N-
dimethylformamide dimethylacetal (6.8 mL, d ) 0.89, 50.8
mmol) in anhydrous DMF (15 mL) was refluxed for 4 h, after
which the starting material had completely disappeared, as
shown by TLC analysis, leaving a spot at Rf ) 0.25 (dichloro-
methane). The mixture was then evaporated to dryness and
the resulting reddish-black oil treated with a 1:1 CH2Cl2/n-
hexane mixture. After standing overnight at 4 °C, a red
crystalline solid had formed, which was collected by filtration,
washed with aqueous methyl alcohol and dried under vacuum.
This turned out to be 2,6-dinitro-trans-â-dimethylamino-
styrene, obtained in quantitative yields, and was directly used
in the next reduction reaction (TiCl3, 15% HCl solution) to give
4-aminoindole 1.
Experimental Section
4-Nitroindole30,31 (2). In a one-necked flask equipped with
a Claisen condenser were placed 2-methyl-3-nitro-aniline (6.00
g, 39.4 mmol), p-toluensulfonic acid (0.032 g, 0.2 mmol) and
anhydrous triethyl orthoformate (9.1 mL, d ) 0.891, 54.7
mmol); the mixture was heated at 120 °C until all the ethanol
had distilled off. Then, following vacuum distillation at 150
°C and 0.6 mmHg, the imidate ester (6.5 g, 79.23%) was
collected.
Without further purification, the imidate ester was used in
the next reaction: in a 100 mL flask, 3.0 g (14.4 mmol) of
imidate ester in 11.0 mL of anhydrous DMSO was treated with
3.0 mL of diethyl oxalate (21.7 mmol, d )1.076) in 7 mL of
anhydrous DMF and 1.58 g (18.8 mmol) of potassium ethoxide
at 0 °C. After 24 h at 40 °C, the reaction mixture was poured
onto ice/water and the nitroindole separated as a brown
crystalline product, subsequently purified by flash chroma-
tography (CH2Cl2). Rf 0.55 (TLC, CH2Cl2).
A. Chemistry. Melting points were determined on a Gal-
lenkamp MFB 595 010M/B capillary melting point apparatus
and are not corrected. Infrared spectra were recorded on a
Perkin-Elmer 1760 FTIR spectrometer using potassium bro-
mide pressed disks; all values are expressed in cm-1. UV-vis
spectra were recorded on a Perkin-Elmer Lambda UV/vis
spectrometer. 1H NMR spectra were recorded on Varian
Gemini (200 MHz) and Bruker (300 MHz) spectrometers, using
the indicated solvents; chemical shifts are reported in δ (ppm)
downfield from tetramethylsilane as internal reference. Cou-
pling costants are given in hertz. In the case of multiplets,
the chemical shift was measured starting from the ap-
proximate center. Integrals were satisfactorily in line with
those expected on the basis of compound structure. Elemental
analyses were performed in the Microanalytical Laboratory,
Department of Pharmaceutical Sciences, University of Padova,
using a Perkin-Elmer elemental analyzer model 240B; results
fell in the range of calculated values ( 0.4%. The analytical
data are presented in detail for each final compound. Mass
spectra were obtained with a Mat 112 Varian Mat Bremen
(70Ev) mass spectrometer and Applied Biosystems Mariner
System 5220 LC/Ms (nozzle potential 250.00). Column flash
chromatography was performed on Merck silica gels (250-
400 mesh ASTM); chemical reactions were monitored by
analytical thin-layer chromatography (TLC) using Merck silica
gel 60 F-254 glass plates with a 9:1 dichloromethane/methanol
mixture as eluant, unless otherwise specified.
N1-Methyl-4-nitroindole32 (3). To a stirring solution of 1
g (6.2 mmol) of 4-nitroindole in 30 mL of acetone were added
1.728 g of powdered KOH at 0 °C, giving a dark-red color, and
then 0.77 mL (12.3 mmol) of CH3I. After 2 h stirring at room
temperature, the starting material had disappeared and a new
spot formed at Rf ) 0.51 (TLC, benzene/n-hexane/ethyl acetate
1:1:0.3). Following the addition of 180 mL of benzene to the
reaction mixture, a precipitate formed, which was then
eliminated by filtration. The filtrate was washed first with a
NaCl saturated solution and then with water, dried over
Na2SO4, and evaporated to dryness. The yellow residue was
purified by flash chromatography, yielding 0.930 g (85.14%).
Solutions were concentrated in a rotary evaporator under
reduced pressure. Starting materials were purchased from

3424 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Ferlin et al.
N1-Methyl-4-aminoindole28 (4). In a 150 mL flask, 0.396
g (2.2 mmol) of 3 was dissolved in 40 mL of acetic acid/water
(2:1 mixture) by heating; to this orange solution were then
added 11.56 mL of TiCl3 15% in HCl 5-10% (13.5 mmol)
(Riedel-de Hae¨n) at room temperature. The resulting brown
mixture was stirred at the same temperature for about 2 h,
the reaction being monitored by TLC (CH2Cl2/ethyl acetate 8:2,
Rf 0.65). Last, water was added to the light yellow mixture,
and a first extraction was made (CH2Cl2/methanol 9:1) to
remove the starting product; the aqueous phase was made
alkaline by concentrated NaOH and, following the addition of
30% NH3, a second extraction was made with the same solvent
mixture, to retrieve the amino derivative. The organic phase
was washed with a saturated NaCl solution and H2O and then
dried with anhydrous Na2SO4. After solvent evaporation, 0.279
g of a brownish-red oil appeared which was purified by flash
chromatography (CH2Cl2/ethyl acetate 8:2), yielding 0.195 g
of a pure light yellow oil (60.6%).
2-[(1H-Indol-4-ylamino)methylene]malonic Acid Di-
ethyl Ester (5). In a 50 mL round-bottomed flask were placed
0.265 g (2.00 mmol) of 4-aminoindole 1 and 0.405 mL (2.00
mmol, d ) 1.07) of diethyl ethoxymethylenemalonate, and the
mixtrue was heated at 130 °C for about 3 h. Any unreacted
diethyl ethoxymethylenemalonate was removed under vacuum,
leaving 0.500 g of a brown solid product (yield 83%); mp 122
°C (ethanol); Rf ) 0.83 (CH2Cl2/ethyl acetate).
was needed in order to remove completely the unreacted
diethyl carbonate and retrieve the desired product (130 °C,
0.6 mmHg) as a dense light orange oil (10.442 g, yield 89%);
Rf ) 0.38 (n-hexane/ethyl acetate 8:2).
3-(3-Methoxy-phenyl)-3-oxopropionic Acid Ethyl Es-
ter36 (10). This compound was prepared following the same
method used for compound 9, as reported in the literature.37
General Procedure for Synthesis of 3-(1H-Indol-4-
ylamino)-3-substitutedacrylic Acid Ethyl Esters (11-17).
In a 50 mL round-bottomed flask, 3-4 mmol of aminoindole 1
or 4 in 10-20 mL of absolute ethanol were condensed with an
equimolar quantity of â-keto-ester (commercial a-d or pre-
pared 11, 12) in the presence of 1 mL of glacial acetic acid
and 100 mg of drierite. The mixture was refluxed for about
24 h, the reaction being monitored by TLC analysis (dichloro-
methane/ethyl acetate 9:1). As the reaction did not come to
completion, after 24 h the mixture was cooled and filtered to
remove the drierite; the resulting solution was evaporated to
dryness under vacuum and the residue purified by flash
chromatography and recrystallization from a suitable solvent.
3-Methyl-3-(1H-indol-4-ylamino)acrylic Acid Ethyl Es-
ter (11). Brown oil, yield 51% (flash chromatography); Rf )
0.86 (dichloromethane/ethyl acetate 9:1).
3-Thienyl-3-(1H-indol-4-ylamino)acrylic Acid Ethyl Es-
ter (12). Yellow solid product purified by flash chromatogra-
phy, yield 45.35%; mp 130-133 °C; Rf ) 0.92 (dichloromethane/
ethyl acetate 9:1).
3-Phenyl-3-(1H-indol-4-ylamino)acrylic Acid Ethyl Es-
ter (13). Brown crystals, yield 50.31%; mp 135-136 °C; Rf )
0.9 (dichloromethane/ethyl acetate 9:1).
3-Phenyl-3-(1-methyl-indol-4-ylamino)acrylic Acid Eth-
yl Ester (14). Dark-red vitreous solid by flash chromatogra-
phy, yield 53%; Rf ) 0.96 (dichloromethane/ethyl acetate 8:2);
mp 55-60 °C.
4-Oxo-4,7-dihydro-1H-pyrrolo[2,3-h]quinolin-3-carbox-
ylic Acid Ethyl Ester (6). A 0.500 g (1.7 mmol) amount of 5
was added to 50 mL of boiling diphenyl ether portionwise; after
20 min refluxing, the precipitate formed by cooling was
collected by filtration, washed many times with diethyl ether
and recrystallized from ethanol, yielding 0.360 g of pure
tricyclic product (yield 83%); mp 280 °C (dec); Rf ) 0.45 (ethyl
acetate/methanol, 8:2).
3-(p-Nitrophenyl)-3-(1H-indol-4-ylamino)acrylic Acid
Ethyl Ester (15). Brownish-red amorphous solid by flash
chromatography, yield 59%; mp 143-145 °C; Rf ) 0.88
(dichloromethane/ethyl acetate 9:1).
3-(m-Nitrophenyl)-3-(1H-indol-4-ylamino)acrylic Acid
Ethyl Ester (16). Orange-red crystals obtained by flash
chromatography and recrystallization from methanol. Yield
47%; mp 133-134 °C; Rf ) 0.90 (dichloromethane/ethyl acetate
9:1).
3-(m-Methoxyphenyl)-3-(1H-indol-4-ylamino)acrylic
Acid Ethyl Ester (17). Vitreous dark-red solid by flash
chromatography. Yield 43.10%; mp 50-60 °C; Rf ) 0.87
(dichloromethane/ethyl acetate 9:1).
General Procedure for Synthesis of 2-Substituted-
1H,7H-pyrrolo[2,3-h]quinolin-4-one Derivatives (18-24).
In a two-necked round-bottomed flask, 50 mL of diphenyl ether
were heated to boiling temperature; 1-2 mmol of acrylates
11-17 were then added portionwise, and the mixture was
refluxed for 30 min. After cooling to 60 °C, the resulting
precipitate was collected by filtration and washed many times
with diethyl ether. In all cases, the collected products were
purified by flash chromatography (ethyl acetate/methanol 9:1).
2-Methyl-1H,7H-pyrrolo[2,3-h]quinolin-4-one (18). Light
brown solid from methanol, yield 70%; mp 302 °C (dec); Rf )
0.13 (ethyl acetate/methanol 8:2).
4-Oxo-4,7-dihydro-1H-pyrrolo[2,3-h]quinolin-3-carbox-
ylic Acid (7). In a 100 mL round-bottomed flask were placed
0.260 g (1.01 mmol) of 6 and 30 mL of 2 N NaOH; the
suspension was heated to complete solution and was then
refluxed for 6 h, the reaction being monitored by TLC analysis
(ethyl acetate/methanol, 8:2). After cooling, the reaction
mixture was acidified to pH 5 by acetic acid/water 1:2. A brown
precipitate formed, which was collected, washed with water
and dried at 60 °C, yielding 0.185 g of a crystalline solid (yield
80%); mp 320 °C (dec); Rf ) 0.71 (ethyl acetate/methanol, 8:2).
1H,7H-Pyrrolo[2,3-h]quinolin-4-one (8). In a two-necked
100 mL round-bottomed flask, 50 mL of diphenyl ether were
heated to boiling temperature, and, following portionwise
addition of acid 7 (0.185 g-0.81 mmol), the solution was
refluxed for 20 min. After cooling, the formed precipitate was
collected, washed many times with diethyl ether and dried,
providing 0.120 g of a solid product (yield 80%); mp 351 °C
(dec); Rf ) 0.38 (ethyl acetate/methanol, 8:2).
3-Oxo-3-thiophen-2-yl-propionic Acid Ethyl Ester20 (9).
A 250 mL two-necked, round-bottomed flask equipped with a
magnetic stirrer was fitted with a 100 mL pressure-equalizing
constant-rate dropping funnel and a condenser, the top of
which was connected to a mineral oil trap, to prevent air from
entering the vessel during reaction and to monitor gas
development. In the flask were subsequently placed 7.131 g
(178.3 mmol) of sodium hydride (60% in mineral oil), which
was then washed four times with 20 mL portions of benzene.
Following the addition of 100 mL of fresh benzene and then
14.04 g (14.4 mL, d ) 0.975, 118.9 mmol) of anhydrous diethyl
carbonate, the mixture was heated under stirring. When it
reached the boiling point, a solution of 2-acetylthiophene (6.42
mL, d ) 1.168, 59.4 mmol) in benzene was added dropwise,
slowly (1 h). When this addition was complete, the reaction
mixture was refluxed until all hydrogen gas formation stopped.
On cooling, a pasty, pink solid product separated; this was
dissolved by adding 15 mL of glacial acetic acid dropwise,
followed by 45 mL of ice-cold water. Once the organic layer
was separated, the aqueous phase was extracted with benzene,
and the organic extract washed with cold water, dried with
anhydrous Na2SO4 and evaporated. Distillation under vacuum
2-Thienyl-1H,7H-pyrrolo[2,3-h]quinolin-4-one (19). Light
brown solid from methanol, yield 35%; mp 293-294 °C (dec);
Rf ) 0.51 (ethyl acetate/methanol 8:2).
2-Phenyl-1H,7H-pyrrolo[2,3-h]quinolin-4-one (20). Crys-
talline product from ethanol, yield 69%; mp 319 °C (dec); Rf
0.37 (ethyl acetate/methanol 8:2).
2-Phenyl-N7-methyl-1H-pyrrolo[2,3-h]quinolin-4-one
(21). Light brown crystals, yield 37%; mp 319-320 °C; Rf )
0.26 (ethyl acetate/methanol 8:2).
2-(p-Nitrophenyl)-1H,7H-pyrrolo[2,3-h]quinolin-4-
one (22). Light brown crystals, yield 56%; mp >300 °C; Rf )
0.70 (dichloromethane/methanol 8:2).
2-(m-Nitrophenyl)-1H,7H-pyrrolo[2,3-h]quinolin-4-
one (23). Yellow solid product, yield 57%; mp >300 °C (dec);
Rf ) 0.36 (dichloromethane/methanol 8:2).

2-Phenylpyrroloquinolin-4-ones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3425
2-(m-Methoxyphenyl)-1H,7H-pyrrolo[2,3-h]quinolin-4-
one (24). Light crystals, yield 78%; mp 298-299 °C (dec); Rf
) 0.37 (dichloromethane/methanol 8:2).
24:1), followed by PBS, and finally incubated in an RNAse
solution (100 µg/mL in PBS). After 30 min at 37 °C, the cells
were incubated in a propidium iodide solution (PI, Sigma, 100
µg/mL in PBS) at room temperature for a further 30 min. To
determine the effects of compound 24 on cell cycle dynamics,
DNA fluorescence was measured by flow cytometry, analyzing
at least 15 000 events with Lysis II software (Becton, Dick-
inson) at 488/525 nm (excitation/emission wavelength).
All experiments were repeated 3-4 times, and DNA content
analysis was carried out using both logarithmic and linear
scales. Results were comparable, irrespective of the scale used,
and are shown on a logarithmic scale.
Apoptosis Assay. The capacity of compound 24 to induce
apoptosis in tumor cell lines was assessed by quantifying
histone-associated DNA fragments in the cytoplasm. OVCAR-3
cells were plated in quadruplicates at a concentration of 104
cells/well on 96-well plates. Following 24 h culture, compound
24 was added (1-5 µM) and, after 48 h incubation, cytoplasmic
histone-associated DNA fragments were quantified by a com-
mercially available cell death detection ELISA assay (Roche).
Indirect Immunofluorescence Detection of Micro-
tubule Perturbation. MCF-7 and Ovcar-3 cells (3 × 104)
were seeded on sterile microscope coverslips. After 24 h,
compounds 20, 21, 24 (5 µM) and vincristine (1 µM) were added
to the culture medium, and cells were incubated for 18 h. As
described previously,48,49 cells were fixed with 4% formaldehyde
in PBS for 10 min at room temperature, washed three times
with PBS, permeabilized in 0.2% Triton X-100 in PBS for 10
min at room temperature and placed in methanol at -20 °C
for 30 min. They were then washed with PBS, incubated for 1
h at 37 °C with 2% bovine serum albumin (BSA) and
subsequently with a mouse monoclonal anti-â-tubulin antibody
(diluted 1:400) for 1 h at 37 °C. Slides were washed three times
with PBS and incubated with a tetramethyl rhodamine
isothiocyanate (TRITC)-conjugated rat anti-mouse IgG (diluted
1:200 in a 2% BSA solution in PBS) for a further 1 h at 37 °C.
Slides were then washed repeatedly with PBS, mounted with
mounting medium and analyzed by cofocal microscopy (SP-2,
Leyca) under green light.
2-(m-Aminophenyl)-1H,7H-pyrrolo[2,3-h]quinolin-4-
one (25). In a 250 mL flask containing a solution of 0.400 g
(1.3 mmol) of compound 23 in 120 mL of acetic acid/water 2:1
solution was added 6.74 mL of TiCl3 15% in HCl 5-10% (7.9
mmol) (Riedel-de Hae¨n); the resulting mixture was stirred at
room temperature for 3 h (TLC analysis, ethyl-acetate/
methanol 8:2). Water was then added, and the mixture made
alkaline with concentrated NaOH, followed by 30% NH3. After
extraction with ethyl acetate, treatment with a saturated NaCl
solution of the organic layer and drying with anhydrous
Na2SO4, yielded of a material, subsequently purified by flash
chromatography (ethyl acetate/methanol 8:2) to give 0.240 g
of a light yellow crystalline product (yield 67%); mp 183-186
°C; Rf ) 0.49 (ethyl acetate/methanol 8:2).
2-(m-Aminophenyl)-1H,7H-pyrrolo[2,3-h]quinolin-4-
one Dihydrochloride (26). A solution obtained by dissolving
amino derivative 25 in a minimum quantity of heated absolute
ethanol was saturated with dry HCl gas. After cooling to 0
°C, the dihydrochloride compound separated as a crystalline
solid. Yield 93%. anal. (C17H13N3Cl2): C, H, N, Cl.
B. Biology. Cell Lines and Culture Conditions. Human
cell lines, thyroid carcinoma (anaplastic C8305 and ARO),
colon carcinoma (HT-29), pancreas adenocarcinoma (Mia Paca
2 and PT45), hepatocellular carcinoma (Hep 3B, Hep G2, PLC/
PRF/5), glioblastoma (A-172), ovary carcinoma (OVCAR-3,
IGROV-1), breast cancer (MCF-7), adrenocortical carcinoma
(SW13 and NCI-H295) and a mouse hepatocellular carcinoma
cell line (BNL 1ME A.7R.1) were grown at 37 °C in a
humidified incubator in the presence of 5% CO2, except for
SW13, which needed no CO2 to grow.
C8305, A-172, Mia Paca 2, PT45, IGROV-1 and OVCAR-3
cell lines were cultured in RMPI medium supplemented with
10% heat-inactivated fetal bovine serum (FBS, Invitrogen), 100
U/mL penicillin G and 10 µg/mL streptomycin; NCI-H295 cells
were grown in RPMI supplemented with 2% FBS, 100 U/mL
penicillin G and 10 µg/mL streptomycin, 1% insulin, transfer-
rin, selenium-A (Gibco); ARO, Hep 3B, Hep G2, PLC/PRF/5,
MCF-7, HT-29 and BNL 1ME A.7R.1 cells were grown in
Dulbecco’s modified Eagle’s medium supplemented with 10%
heat-inactivated FBS, 100 U/mL penicillin G and 10 µg/mL
streptomycin, and SW13 was grown in Leibovitz medium
containing 10% FBS, 100 U/mL penicillin G and 10 µg/mL
streptomycin. All cell lines were purchased from American
Type Culture Collection.
In Vitro Cytotoxicity Assay. The cytotoxic activity of all
new compounds was determined using a standard 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)-
based colorimetric assay.7 Briefly, cell lines were seeded at a
density of 104 cells/well in 96-well microtiter plates (Costar).
After 24 h, exponentially growing cells were exposed to the
indicated compound at a final concentration ranging from 0.01
to 100 µM. After 72 h, cell survival was determined by the
addition of an MTT solution (10 µL of 5 mg/mL MTT in
phosphate saline buffer).
After 4 h, 100 µL of 10% SDS in 0.01 N HCl was added,
and the plates were incubated at 37 °C for a further 18 h;
optical absorbance was measured at 550 nm by a LX300 Epson
Diagnostic microplate reader. Survival ratios are expressed
in percentual values with respect to untreated cells. The IC50
value, i.e., the concentration of each compound required to
reduce absorbency by 50% versus control cells, was determined
from replicates of 6-8 wells from at least two independent
experiments.
Fluorescence-Activated Cell Sorting Analysis. Hep G2,
MCF-7, SW13 and Ovcar-3 cells were cultured for 24-48 h in
a drug-free medium or supplemented with compound 24 (1-5
µM), vincristine sulfate salt (1 µM) or methotrexate (1 µM).
As previously described,47 cells were harvested with a cell
scraper, washed twice with PBS and fixed in 70% cold ethanol
(30 min at -20 °C). Cells (106) were then washed once in
citrate phosphate buffer (0.2 N Na2HPO4 and 0.1 M citric acid,
Aromatase Activity Assay. Aromatase activity in sub-
confluent H295R cells was measured by the tritiated water
release assay, with 0.5 µM [1â-3H(N)]-androst-4-ene-3,17-dione
(25.3 Ci/mmol; DuPont NEN, Boston, MA) as substrate.42
Briefly, H295R cells were seeded at 106 cells/well on six-well
plates and, after 48 h, were cultured for 24 h in DMEM-F12
in the absence or presence of FSK (25 µM). One hour before
addition of the substrate, various concentrations of compounds
20, 21 and 24 and letrozole were added. After a further 2 h
exposure at 37 °C, 750 µL of incubation media was mixed with
5 volumes of chloroform and vortexed for 60 s to extract
unconverted substrate. The aqueous phase was increased to
1.5 mL with distilled water and centrifuged at 800g for 5 min.
3
An aliquot of H2O (1 mL) was placed in tubes containing 1
mL of 5% charcoal and 0.5% dextran T-70, vortexed for 60 s,
and centrifuged at 9000g for 30 min. After centrifugation, 1
3
mL of the H2O phase was quantified by counting in 5 mL of
Picofluor 15 premixed cocktail in a liquid scintillation counter.
The results were expressed as pmoles [3H]H2O released per
hour and were normalized for mg protein (pmol/h/mg protein).
Protein yield was determined by the Bradford method.50
Activity Assays on Topoisomerase I and II. Relaxation
Assay. Topoisomerase I activity was assayed by monitoring
the relaxation of supercoiled plasmid pBR322 DNA. Reactions
were performed in 22.5 µL of a reaction mixture containing
10 mM Tris-HCl pH 7.9, 1 mM EDTA, 150 mM NaCl, 0.1%
BSA, 0.1 mM spermidine, 5% glycerol, 0.25 µg pBR322 DNA
(Sigma Chemical Co.), a test compound as indicated and 2 U
of human topoisomerase I (TopoGEN, Inc.). Reactions were
incubated at 37 °C for 30 min and then stopped by the addition
of 2.5 µL of a solution containing 5% SDS and 5 mg/mL
proteinase K (Sigma Chemical Co.) prewarmed at 37 °C for
30 min. After this time, 5 µL of stop buffer (50% sucrose, 50
mM EDTA, 0.1% bromophenol blue, 0.01% SDS) was added.
Samples were analyzed by electrophoresis on a 1% agarose

3426 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Ferlin et al.
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gel in TAE buffer (40 mM Tris-acetate, 1 mM EDTA). Gels
were then stained with 1 µg/mL ethidium bromide and
subsequently destained with distilled water.
Decatenation Assay. Topoisomerase II activity was mea-
sured by the ATP-dependent decatenation of kinetoplast DNA
(KDNA). The assay was performed using the topoisomerase
II kit (TopoGEN, Inc.) and human topoisomerase II (TopoGEN,
Inc.). Briefly, 20 µL reaction volumes containing 0.15 µg of
KDNA, a test compound as indicated and 1 U of topoisomerase
II were incubated for 1 h at 37 °C. After this time, 4 µL of
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by electrophoresis on a 1% agarose gel containing ethidium
bromide 0.5 µg/mL in TAE buffer. Gels were then destained
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In Vivo Effects of Compound 24 on Tumor Growth.
The test was conducted with a syngenic hepatocellular carci-
noma model (BNL 1ME A.7R.1) in Balb/c mice. Male mice, 8
weeks aged, were purchased from Charles River (Calco, Lecco,
Italy), and tumors were induced by a subcutaneous injection
at both sides of 200 µL sterile PBS containing 107 BNL 1ME
A.7R.1 cells. Animals were randomly divided in three groups,
and starting on the second day, they were daily dosed
intraperitoneally (ip) with 500 µL of saline, vehicle and 24 (50
mg/kg body weight) suspended in 0.9% NaCl containing 5%
poly(ethylene glycol) and 0.5% Tween 80. Ten days later,
animals were sacrificed and the tumor size was measured.51
In another set of experiments, drug administration started
5 days after the injection of tumor cells and lasted 7 days. All
animals were then sacrificed and their tumors measured and
harvested for pathological examination. In these experiments,
the tumor size was measured every other day in anesthetized
animals.
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To measure the perpendicular diameter of each mass,
calipers were used. In particular, the tumor volume (V) was
calculated by the rotational ellipsoid formula: V ) A × B2/2,
where A is the longer diameter (axial) and B is the shorter
diameter (rotational). All experimental procedures were ac-
complished following guidelines recommended by the Institu-
tional Animal Care and Use Committee of Padua University.
Pathology. Mouse tissue specimens from the in vivo
experiments were fixed in buffered 4% formalin for 24 h, and
paraffin was embedded following dehydration. Cut sections (5
µm thick) were stained with hematoxylin/eosin and subjected
to routine histological examination. To evaluate inflammatory
infiltrates, tissue necrosis and presence of apoptotic cells, a
microscopical examination was also carried out.
Statistical Analysis. Results are reported as means (
standard error (M ( SE). Statistical analysis was performed
by using one-way variance analysis or Student’s t-test, as
appropriate. A P value of less than 0.05 was considered
statistically significant.
Supporting Information Available: Spectroscopic data
(IR, 1H NMR, 13C NMR, HRMS) and elemental analyses of all
target compounds 8, 18-25. This material is available free of
charge via the Internet at http://pubs.acs.org.
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