Synthesis and biological activity of 7-phenyl-6,9-dihydro-3H-pyrrolo[3,2-f] quinolin-9-ones: A new class of antimitotic agents devoid of aromatase activity

Article abstract of DOI:10.1021/jm0510676

The newly synthesized 7-phenyl-3H-pyrrolo[3,2-f]quinolinones 16-26 and previously 27 and 28 were assayed for their in vitro antiproliferative activity on tumor cell lines, and the lead compound 16 in vivo on a singenic hepatocellular carcinoma in Balb/c m

Full text of DOI:10.1021/jm0510676

1910
J. Med. Chem. 2006, 49, 1910-1915
Synthesis and Biological Activity of 7-Phenyl-6,9-dihydro-3H-pyrrolo[3,2-f]quinolin-9-ones: A
New Class of Antimitotic Agents Devoid of Aromatase Activity
Venusia Gasparotto,†,§ Ignazio Castagliuolo,§ Gianfranco Chiarelotto,† Vincenzo Pezzi,‡ Daniela Montanaro,‡ Paola Brun,§
Giorgio Palu`,§ Giampietro Viola,† and Maria Grazia Ferlin†,*
Department of Pharmaceutical Sciences, and Department of Histology, Microbiology and Medical Biotechnologies, UniVersity of PadoVa,
35131 PadoVa, Italy, and Department of Pharmaco-Biology, UniVersity of Calabria, Calabria, Italy
ReceiVed October 21, 2005
The newly synthesized 7-phenyl-3H-pyrrolo[3,2-f]quinolinones 16-26 and previously 27 and 28 were assayed
for their in vitro antiproliferative activity on tumor cell lines, and the lead compound 16 in vivo on a singenic
hepatocellular carcinoma in Balb/c mice. Results from FACS, immunofluorescence microscopy analysis,
tubulin polymerization assay, and tritiated water release assay for the CYP19 activity confirmed the new
compounds as potential anticancer agents acting by tubulin depolymerization, but devoid of aromatase activity
unlike their geometric [2,3-h] isomers.
Introduction
In chemotherapy, the search for multiacting drugs has always
been a very attractive strategy, to increase effectiveness and
selectivity and to overcome the drawbacks of toxicity and
resistance.1 In principle, molecules exhibiting several pharma-
cological properties simultaneously should display structural
features suitable for interacting with various biological targets
and consequently producing specific pharmacological effects.
In this case, effects should aim at inhibiting tumor growth. With
this aim in mind, we recently designed and characterized some
2-phenylpyrrolo[2,3-h]quinolin-4-ones2 IV (Figure 1), as tricy-
clic aza-analogues of flavones. Aza-flavones 2-phenyl-quinolin-
4-ones I (PQ),3-7 2-phenyl-quinazolinones II (PQZ),8-12 and
phenylnaphthyridinones III (PN)13-15 (Figure 1) have been
reported to possess potent in vitro cytotoxic activity but devoid
of any selectivity.16 Some of our phenylpyrroloquinolin-4-ones2
IV were confirmed as being quite cytotoxic, with significant
selectivity against estrogen-dependent growth cell lines. Studies
of the mechanism of action confirmed the ability of the new
tricyclic compounds to interfere with microtubule dynamics but
also to inhibit aromatase, a key enzyme involved in estrogen
biosynthesis.2 Aromatase inhibitors are well-established drugs
for treatment of estrogen-dependent breast cancer.17,18 The latter
unexpected activity was speculated to be due to the presence
of structural elements such as those present in flavones (side
phenyl ring, carbonyl group, and 7-NH group)19-21 and to their
tricyclic structure, with angular geometry similar to that of 7,8-
benzoflavones V, powerful aromatase inhibitors (Figure 1).22,23
Now, to confirm the specific angular geometry of 2-phenyl-
[2,3-h]pyrroloquinolin-4-ones IV as an element determining the
selectivity and power of their cytotoxic effects, we undertook
the synthesis and study of a series of geometric isomers in which
the pyrrole ring is fused in positions 5 and 6 instead of 7 and
8 of 2-phenyl-4-quinolinone (Figure 1).
This paper reports the synthesis of eleven compounds,
characterized by the pyrrolo[3,2-f]quinolin-9-one nucleus VI
bearing various substituents on phenyl ring in position 7 and/
Figure 1.
or on the tricycle. Final compounds were also assayed for their
in vitro antiproliferative activity on human and murine tumor
cell lines and in vivo on a singenic hepatocellular carcinoma in
Balb/c mice. To elucidate the mechanism(s) of action, the effect
on cytoskeleton antimitotic activity was studied by fluorescence
activated cell sorting analysis (FACS), immunofluorescence
microscopy analysis, and tubulin polymerization assay, and their
effect on CYP19 (aromatase) activity was examined by the
tritiated water release assay.
Results and Discussion
Chemistry. In sections A and B of Scheme 1, the synthesis
of ethyl substituted-benzoylacetates 1a-f and 7-phenyl-pyr-
roloquinolinones 16-26 are reported following a method
previously described24 and recently adopted by us.2 5-Amino-
indole derivatives 326 and 4,27 obtained by reduction of the
corresponding nitro compounds and the N1-diethylaminoethyl-
substituted 5 (from alkylation of 2), were condensed with ethyl
benzoylacetates 1a-f and commercial ethyl 3-nitrobenzoyl- and
benzoylacetates to give ethyl 3-indole-amino-3-phenylacrylates
6-15 in good yields. They were then cyclized to 7-phenyl-
pyrroloquinolin-9-ones 16-26 in boiling diphenyl ether (250
°C). Amino derivative 23 was obtained by reduction with TiCl3
reagent.
Biology. In Vitro Cytotoxic Activity. The in vitro cytotoxic
activity of newly synthesized 7-phenyl-6,9-dihydro-pyrrolo[3,2-
f]quinolin-9-ones 16-26, 7-methyl-6,9-dihydro-pyrrolo[3,2-f]-
quinolin-9-one 27, and 6,9-dihydro-pyrrolo[3,2-f]quinolin-9-one
28 was studied on a panel of nine human and two murine tumor
* To whom correspondence should be addressed. Tel: 00390498275718;
Fax: 00390498275366: e-mail: mariagrazia.ferlin@unipd.it.
† Department of Pharmaceutical Sciences, University of Padova.
§ Department of Histology, University of Padova.
‡ University of Calabria.
10.1021/jm0510676 CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/28/2006

Pyrroloquinolinone Antimitotic Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 1911
Scheme 1. Synthesis
Figure 2. Effects of phenylpyrroloquinolinones 16, 17, and 19 on
aromatase activity. H295R cells were cultured for 24 h in DMEM-F12
in absence (A) or 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 16, 17, 19, and letrozole (Let) were added to cells at the
specified concentrations. After a further 2-h exposure, aromatase activity
was assessed using a modified tritiated water method. Results are
expressed as pmol [3H]H2O released per hour and normalized for mg
protein (pmol/h/mg protein). Values represent means ( SEM of three
different experiments, each performed in triplicate.
cell lines by applying the MTT colorimetric assay.28 Compounds
were tested on a range of concentrations from 50 to 0.1 µM,
and the calculated IC50 values are listed in Table 1. As shown,
of all compounds tested, 16 and 17, carrying a free phenyl ring
or a 3′-methoxyphenyl group in position 7, were the most active,
with IC50 values ranging from 0.4 to 8 µM. This is consistent
with our previous data obtained with 2-phenyl-1,4-dihydro-
pyrrolo[2,3-h]quinolin-4-ones2 and with others previously re-
ported for antitubulin PQ, PN, and PQZ.3-16 Compounds 18,
19, 24, and 25 showed cytotoxic activity with IC50 values
ranging from 4 to 30 µM. 7-(Substituted-phenyl) 20-23,
7-methyl 27, and 7-unsubstituted 28, carrying a phenyl ring with
polar groups or lacking the phenyl ring, were only slightly or
not at all active, with IC50 values generally higher than 30 µM.
The chemically reactive cyano group was introduced at the
7-phenyl ring of pyrroloquinolinone (20, 21), as it is also present
in the most recent third-generation azole-type aromatase inhibi-
tors,29,30 but in this case it does decrease cytotoxic activity.
Table 1. In Vitro Cytotoxic Activity of 7-Substituted-6,9-dihydropyrrolo[3,2-f]quinolin-9-ones 16-28
cytotoxicity IC50 (µM)
compd
Hela
Hep G2
H295R
Ovcar-3
MCF-7
Aro
A549
HT-29
PT-45
4T1
BNL
16
17
18
19
0.7
0.7
6
8
6
10
0.4
0.7
8
6
7
20
0.8
2
7
0.7
1
7
1
2
6
6
2
6
8
0.5
0.7
7
2
4
25
20
3
4
6
5
6
5
10
5
5
9
6
4
20
21
22
23
8
40
10
30
30
>50
>50
>50
3
40
39
30
7
>50
>50
>50
>50
>50
30
40
20
45
8
>50
40
15
15
25
7
30
50
50
15
32
25
>50
50
40
50
5
>50
50
25
>50
15
>50
30
15
>50
24
6
10
7
8
20
8
25
5
7
6
18
8
20
12
6
5
9
5
26
>50
>50
>50
>50
0.3
0.2
>50
>50
>50
0.08
0.09
>50
>50
>50
0.01
0.02
>50
>50
>50
0.007
0.02
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.05
0.01
>50
>50
>50
>50
>50
>50
>50
27a
28a
Taxol
vincristine
>50
>50
>50
>50
0.008
0.005
0.0002
0.0001
0.006
0.03
0.005
0.004
0.2
0.02
0.2
0.08
a Reference 26.

1912 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Ferlin et al.
Figure 3. Flow-activating cell sorting analysis of phenylpyrroloquinolinones 16-26. Effect of compounds 16-26 on cell cycle progression of
Hep-G2 and Ovcar-3 cell lines was examined by staining cells with propidium iodide. Cells were treated with the different compounds at 10 µM
(Hep G2) and 5 µM (Ovcar-3) and, after 24 h incubation, the percentage of cells in each phase was determined by FACS analysis and calculated
using Lysis II software (Becton-Dickinson).
Compounds 22 (3′-NO2-phenyl) and 23 (3′-NH2-phenyl) were
not cytotoxic. Thus, for this series of pyrroloquinolones, too,
the presence of a phenyl ring is essential and, if unsubstituted,
is preferable, due to its antiproliferative action.
the polar nature of substitutions at the pyrrole ring of pyrrolo-
quinolinones. Polar groups do not in fact promote cytotoxicity,
perhaps by hindering or preventing useful interactions with
lipophilic regions surrounding active tubulin sites.31
It is interesting to note that compounds 25 (5 < IC50 <25
µM), carrying the short diethylaminoethyl polar chain linked
to the pyrrole N, and 26 (IC50 > 50 µM), with two oxygenated
groups in positions 2 and 4, had lower activity in comparison
with compounds 16 and 17 and to other analogues without polar
chains at the pyrrole ring (data to be published). We therefore
suggest an important structure-activity relationship regarding
Comparing the new 7-phenylpyrrolo[3,2-f]quinolin-9-ones
with the 2-phenylpyrrolo[2,3-h]quinolin-4-ones recently de-
scribed by us (IC50 ranging from 8 to 0.7 µM),2 we note that
some of the former are more potent. Compounds 16 and 17 do
show IC50 values under 1 µM for Hela, H295R, and PT-45
tumor cell lines (Table 1), but, looking at their cytotoxic profiles,
they do not display any selectivity for estrogen-supported tumor

Pyrroloquinolinone Antimitotic Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 1913
Figure 4. Immunofluorescence images of Ovcar-3 cells treated with 16. Ovcar-3 cells were incubated for 18 h with medium drug-free (a), compounds
16 5 µM (b), and vincristine 5 µM (c). Cells were then fixed and stained with anti-â-tubulin antibody, and microtubule distribution was visualized
by fluorescence microscopy. Compound 16 completely destabilized microtubules in both interphase and mitotic cells, causing an abnormal microtubule
network.
cell lines such as Ovcar-3, MCF-7, Hela, HepG2, and H295R,
unlike their analogues. This suggests that the new compounds
do not inhibit CYP-19 (aromatase) activity.
Aromatase Activity. The more cytotoxic compounds 16, 17,
and 19 were subjected to the tritiated water release assay,32 with
letrozole as reference compound. At concentrations of 5-10-
15 µM they did not show any inhibitory activity on aromatase.
Taken together, the cytotoxicity properties (Table 1) and
aromatase activity (Figure 2) of these compounds confirm that
their specific geometry is the discriminating factor for possible
selective effects in the pyrroloquinolinone family: derivatives
with [2,3-h] geometry are antimitotics and aromatase inhibitors
in estrogen-dependent tumor cell lines,2 whereas derivatives with
[3,2-f] geometry are only antimitotics (see below).
Flow-Activated Cell Sorting Analysis (FACS). To examine
the effect of the new derivatives on cell cycle progression, flow
Figure 5. Tubulin polymerization assay. “In vitro” microtubule
assembly of protein tubulin was followed fluorimetrically using a
microtubule polymerization assay kit. Microtubule proteins were
incubated with compound 16 at concentrations of 5, 2.5, 1.25, and 0.625
µM, and fluorescence (λex 355 nm; λem 450 nm) was measured every
activated cell sorting analysis (FACS) was performed. This
method highlights the effects of drugs on the distribution of
cells in specific phases: antimitotic drugs block cells in the
G2/M phase, causing an increment of the relative peak in the
DNA histogram.33,34 Eleven compounds, 16-26, were tested
on two cell lines (HepG2, Ovcar-3) at 5-10 µM. Figure 3
reports the results obtained after 24 h treatment for the active
compounds 16, 17, and 19. They present significant arrest of
the cell cycle in G2/M phase, raising the G2/M peak from 21.2%
(control) to 47.7% (16), 49.3% (17), and 40.3% (19) in Hep
G2. In Ovcar-3, the same compounds increased the G2/M peak
from 15.7% (control) to 62.0% (16), 65.3% (17), and 42.9%
(19). It is worthwhile to note that there is a good correlation
between the cytotoxicity and the ability to block the cell cycle
in G2/M of these compounds, as 16, 17, and 19 turned out to
be the most potent compounds in the MTT proliferation assay
too (Table 1).
Immunofluorescence Microscopy. To prove that these new
derivatives interfere with microtubule network, compound 16,
chosen as leader compound, was checked by immunofluores-
cence microscopy. As shown in Figure 4, compound 16
disrupted the tubulin network. Cells showed an evident char-
acteristic “rounded up” morphology, caused by breaking up of
microtubules in both interphase and mitotic cells after 18 h
treatment at 5 µM, as for vincristine.
minute for 60 min. Taxol and vincristine 3 µM were used as reference
compounds.
Figure 6. “In vivo” cytotoxic activity of compound 16. Male mice
were injected subcutaneously at their dorsal region with 107 BNL 1ME
A.7R.1 cells, a syngenic hepatocellular carcinoma cell line. Starting
on the second day, groups of animals (n ) 8-12 per group) were
administered daily ip with saline (circles), vehicle (squares), or 40 mg/
kg body weight of 16 (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 (axial) and B the shorter one (rotational). Results are expressed
as means ( SE. / indicates P < 0.01 vs vehicle or untreated animals.
at 5 and 2.5 µM, in a dose-dependent manner. We may therefore
state that 16 exerts its antimitotic activity by tubulin depolym-
erization.
Effect on Tubulin Polymerization. We explored the effect
of compound 16 on tubulin polymerization using a microtubule
polymerization assay kit (Cytoskeleton), with taxol and vinc-
ristine as reference compounds. It is well-known that, in this
assay, taxol causes a rise and vincristine a drop in the tubulin
polymerization curve, as they respectively promote or prevent
tubulin polymerization.35,36 In Figure 5, compound 16 shows
behavior similar to that of vincristine, displaying a good activity
In Vivo Tumor Growth Inhibition. We tested the effects
of compound 16 on tumor growth by using a typical transplanted
model applied to a syngenic hepatocellular carcinoma in Balb/c
mice because 16 showed significant in vitro cytotoxic activity
(IC50 3 µM) against BNL 1ME A.7R.1 cells. As shown in Figure
6, in mice implanted subcutaneously with 107 BNL 1ME A.7R.1
cells, a daily intraperitoneal injection of 16 (40 mg/Kg) caused
a significant inhibition of tumor growth (83.2%) as compared

1914 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Ferlin et al.
7-Thienyl-6,9-dihydro-3H-pyrrolo[3,2-f]quinolin-9-one (24).
Pale brown solid, yield 59%; mp>300 °C; Rf 0.63 (eluant ethyl
acetate/methanol 9:1).
7-Phenyl-3-(diethylaminoethyl)-6,9-dihydro-pyrrolo[3,2-f]-
quinolin-9-one (25). Pale yellow solid, yield 59%; mp 85-90 °C;
Rf 0.58 (eluant ethyl acetate/methanol 1:1).
2-Carboxymethyl-4-methoxy-7-phenyl-6,9-dihydro-3H-pyr-
rolo[3,2-f]quinolin-9-one (26). White crystalline solid, yield 35%;
mp 321-323 °C; Rf 0.68 (eluant ethyl acetate/methanol 9:1).
to mice receiving just vehicle. Further, any tumor growth
difference was not noted in mice treated with PBS and PEG
suspension. As we did not note any death, weight changes, and
histological abnormalities in liver and kidneys of the treated
hosts, 16 appeared to be nontoxic under our experimental
conditions.
Conclusions
A series of new 3H-pyrrolo[3,2-f]quinolin-9-ones (16-28)
was synthesized and studied as antiproliferative agents. 7-Phenyl
derivatives 16, 17, and 19 showed considerable cytotoxic activity
when subjected to the MTT assay on a panel of eleven human
and murine tumor cell lines (IC50 ranging from 0.4 to 8 µM).
To study their mechanism(s) of action, FACS analyses of
compounds 16-26 were performed and showed that they
provoke mitotic arrest. Immunofluorescence microscopy and
tubulin polymerization assay of 16, chosen as the lead com-
pound, revealed relevant antimicrotubule effects by tubulin
depolymerization. Compound 16 was tested “in vivo” in a
syngenic model of murine hepatocarcinoma in Balb/c mice,
showing remarkable antiproliferative activity, as it reduced the
tumor volume by 83.2% within 10 days treatment with respect
to the control group. In addition, compounds 16, 17, and 19
did not inhibit aromatase activity when subjected to the tritiated
water release assay in H295R cells, thus suggesting that [3,2-f]
pyrroloquinolinone geometry is not suitable for interfering with
CYP19 aromatase, unlike the [2,3-h] geometry of recently
described analogues. In conclusion, the mechanism of cytotox-
icity of phenylpyrroloquinolinones described here refers only
to antimitotic activity, as they share many features with
compounds reported as microtubule destabilizing agents, such
as the vinca alkaloids, vincristine and vinblastine.
Acknowledgment. V.G. is recipient of a fellowship from
I.O.V. (Istituto Oncologico Veneto, Padova, Italy) supported
in part from grants from I.O.V. to G. P.
Supporting Information Available: Procedures for the syn-
thesis of compounds 1a-f, 2, 5, 6-15, yields, chemical-physical
properties (Rf, mp), and spectroscopic data of all synthesized
compounds (IR, 1H NMR, 13C NMR), HRMS, the table of elemental
analyses of all target compounds 16-26, and the biological
experimental section. This material is available free of charge via
the Internet at http://pubs.acs.org.
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Pyrroloquinolinone Antimitotic Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 1915
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