3-Substituted 7-phenyl-pyrroloquinolinones show potent cytotoxic activity in human cancer cell lines

Article abstract of DOI:10.1021/jm070534b

A novel series of 3-alkyl-substituted 7-phenyl-3H-pyrrolo[3,2-f]quinolin-9- ones (7-PPyQs) was synthesized with the aim to optimize the cytotoxic activity of recently identified PPyQs, promising inhibitors of tubulin polymerization. All compounds inhibited the growth of 11 human tumor cell lines at submicromolar concentrations as well as two human resistant cancer sublines, A549-T12 and A549-T24. FACS analysis indicated that all compounds caused significant arrest of the A549 cell cycle in G₂/M phase at 0.1 and 1 μM and a good correlation between the cytotoxicity IC₅₀ and their ability to block the cell cycle was observed.

Full text of DOI:10.1021/jm070534b

J. Med. Chem. 2007, 50, 5509-5513
5509
Brief Articles
3-Substituted 7-Phenyl-Pyrroloquinolinones Show Potent Cytotoxic Activity in Human Cancer
Cell Lines
Venusia Gasparotto,†,§ Ignazio Castagliuolo,§ and Maria Grazia Ferlin*,†
Department of Histology, Microbiology and Medical Biotechnologies, and Department of Pharmaceutical Sciences, UniVersity of PadoVa, Italy
ReceiVed May 7, 2007
A novel series of 3-alkyl-substituted 7-phenyl-3H-pyrrolo[3,2-f]quinolin-9-ones (7-PPyQs) was synthesized
with the aim to optimize the cytotoxic activity of recently identified PPyQs, promising inhibitors of tubulin
polymerization. All compounds inhibited the growth of 11 human tumor cell lines at submicromolar
concentrations as well as two human resistant cancer sublines, A549-T12 and A549-T24. FACS analysis
indicated that all compounds caused significant arrest of the A549 cell cycle in G2/M phase at 0.1 and 1 µM
and a good correlation between the cytotoxicity IC50 and their ability to block the cell cycle was observed.
Introduction
Antimitotics are a class of compounds, mainly of natural
origin, which have long been used to treat a variety of
malignancies. Although they are sometimes considered “old
chemotherapeutics” with respect to modern anticancer ap-
proaches,1 at present they are valuable anticancer molecules in
which scientific interest is still very high.2-6 Their impressive
Figure 1. Structural similarities of colchicine, flavones, and 2-phenyl-
success in clinical use is due to their potent antiproliferative
effect (the strongest of commercial anticancer drugs) and to their
particular mechanism of action, because they effect microtubule
dynamics by targeting either the tubulin polymerization or the
microtubule depolymerization processes. The mechanism of
action of these antimitotic agents has an important advantage
over other antitumor compounds, as they avoid interfering with
or damaging the DNA macromolecule and, therefore, do not
cause any cell genome mutations.4 However, taxanes and Vinca
alkaloids, the most clinically effective antimitotics, suffer from
two major drawbacks: selection of clones within tumor cells7-9
and neurological toxicity.5 Therefore, during the past few
decades, efforts to identify novel microtubule inhibitors able to
overcome the multidrug resistance (MDRa) phenotype as well
as other modes of resistance9,10 and, to present improved
pharmacology profiles, have produced dozens of microtubule
targeting compounds, some of which are in clinical and
preclinical trials.11,12
quinolinones.
Figure 2. Structures of the two series of 2-phenyl-pyrroloquinolinone
derivatives previously studied.17,18
mechanism of action, consisting of inhibition of tubulin
polimerization by interaction with the colchicine binding site
on â-tubulin.13-16 Their ability to bind to this site has been
explained by the presence of a three-ring A-B-C system in
the molecular structures of both flavone/azaflavone and colchi-
cine (Figure 1), of which the A and C rings appear to be
responsible for useful interactions with the molecular target.
2-Phenylquinolin-4-one derivatives (2-PQs; Figure 1) are
known for their high antimitotic activity. They are either natural
or synthetic small molecules, structurally derived from the
flavone nucleus by isosteric substitution of the pyran oxygen
with a nitrogen atom. In comparison with flavone derivatives,
2-PQs show higher antiproliferative activity and a more specific
Recently, as a further development of 2-PQs, we synthesized
and studied two series of phenylpyrroloquinolinone (PPyQ)
derivatives showing different angular geometry, 2-phenyl- and
7-phenyl-PPyQs, in which the pyrrole ring is fused to the h-
and f-side of the quinoline nucleus, respectively17,18 (Figure 2).
The fused pyrrole ring may have rendered the new compounds
with mechanism specificity and improved cytotoxicity. In
addition, the azole moiety is suitable for furnishing several
substituted derivatives to modulate pharmacodynamics and
pharmacokinetics.
* To whom correspondence should be addressed. Prof. Maria Grazia
Ferlin, Department of Pharmaceutical Sciences, School of Pharmacy,
UniversityofPadova,ViaMarzolo5,35131Padova,Italy.Tel.: 00390498271603.
Fax: 00390498275366. E-mail: mariagrazia.ferlin@unipd.it.
† Department of Pharmaceutical Sciences.
§ Department of Histology, Microbiology and Medical Biotechnologies.
a Abbreviations: PPyQ, phenyl-pyrroloquinolinone; FACS, fluorescence-
activated cell-sorting analysis; MDR, multidrug resistance; PQ, phe-
nylquinolinone; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide.
In vitro and in vivo, all PPyQs showed significant antipro-
liferative activity by antimitotic effects, with 7-PPyQs being
broadly and slightly more active than 2-PPyQs. Interestingly,
2-PPyQs were selective for estrogen-sensitive tumor cell lines.17
10.1021/jm070534b CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/04/2007

5510 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Brief Articles
Scheme 1a
Recently, using a structure-based approach, Nguyen et al.19
described a common pharmacophore for a diverse set of small
molecules representative of some classes of colchicine site
inhibitors. In that study, the authors proposed that the various
classes were linked by seven pharmacophore points (three
hydrogen bond acceptors, one hydrogen bond donor, two
hydrophobic centers, and one planar group). None of the studied
inhibitors showed all the points, although three of them turned
out to be essential for inhibitory activity, a hydrogen bond
acceptor, a hydrophobic center and a planar group. In the light
of this report, we analyzed and compared PQ and our PPyQ
structures: they share a hydrogen acceptor (carbonyl group), a
hydrogen donor (quinolinone NH), two hydrophobic centers
(aromatic carbons), and two planar groups (phenyl and benzyl
rings). In addition, PPyQs show the hydrogen donor pyrrole
NH. However, this extra point does not significantly contribute
to increased cytotoxic activity in comparison with PQs.17,18
In this paper, we report how we optimized the cytotoxic
activity of previously studied 7-PPyQ series,18 which were the
most cytotoxic derivatives of the two series (general IC50 0.4-8
µM for 7-PPyQs, versus selective IC50 0.7-10 µM for 2-P-
PyQs). To improve cytotoxic activity, we took account of the
pharmacophore model of Nguyen et al.19 and the important role
of hydrophobic interactions surrounding the colchicine binding
site on â-tubulin at the R,â-tubulin interface.20 So, considering
that further hydrophobic interactions at the colchicine site may
have led to higher inhibitory activity and, therefore, to a greater
antimitotic effect, as a first approach we modified the 7-PPyQ
nucleus by linking a lipophilic group, consisting of an alkyl
side-chain, to the pyrrole N. To gain some SARs, we synthesized
and investigated the biological activity of eight 3-alkylated
7-PPyQ derivatives with side-chains of increasing length and
hindrance and carrying varying polar features (from methyl to
n-penthyl, cyclopropylmethyl, methoxymethyl, and benzyl).
Last, all compounds were assayed for their in vitro antiprolif-
erative activity on a panel of 11 human tumor cell lines. We
also examined whether there was any cross-resistance in two
selected cancer cell sublines (A549 T12 and A549 T24) to
taxol.21 To elucidate the mechanism(s) of action, the effect on
the cell cycle was studied by fluorescence-activated cell-sorting
analysis (FACS).
a Reagents and conditions (i) (a) CH3I, C2H5Br, n-C3H7Br, NaH,
anhydrous DMF; (b) n-C4H9Cl, n-C5H11Cl, cyclopropylmethylbromide,
BrCH2OCH3, benzylchloride, KOH, acetone; (ii) H2, Pd/C 10%, absolute
ethanol, 30-40 °C; (iii) ethyl benzoylacetate, CH3COOH, absolute ethanol,
refluxing; (iv) diphenyl ether, 250 °C.
32 was studied on a panel of 11 human tumor cell lines by
applying the MTT colorimetric assay.22 Compounds were tested
over a range of concentrations from 5 to 0.005 µM, and the
calculated IC50 values, that is, the concentration (µM) of
compound able to cause 50% of cell death with respect to the
control culture, are reported in Table 1. The results show that
all PPyQs inhibited the growth of all human cancer cell lines
from various tissues and organs, with IC50 values in the
submicromolar range. By comparing the cytotoxic activity of
the new 3-alkylated 7-PPyQs 25-32 with that of the unsubsti-
tuted 7-PPyQ (Table 1) and analogs recently described by us,18
we note that the chemical modification, consisting of inserting
a lipophilic group at the N-pyrrole ring, generated very potent
compounds, with IC50 values up to 100-fold lower than the
previous compounds and comparable to those of the reference
compounds, taxol and vincristine.
Compound 25, bearing a methyl group, was less active (IC50
0.09-1 µM) than 26-30 with longer side-chains (C2-C5, IC50
0.005-0.2 µM), with the bulky 30 (cyclopropylmethyl) being
highly cytotoxic as well. This indicates that the increase in
cytotoxic activity may depend on longer side chains that induce
the establishment of specific hydrophobic interactions at the
colchicine binding site into â- or R-tubulin. Indeed, it is known
that colchicine binds to the â-subunit at its interface with R-,
and interacting with both of them, it forms a stable reversible
complex. Tubulin conformation changes follow, which prevent
or slow polymerization.20,23
Compounds 31 and 32, with less apolar methoxy-, methyl-,
and more bulky benzyl- groups, respectively, were less active
(IC50 about 10-fold higher) than 26-30, again confirming that
the more hydrophobic 3-5 C linear and cyclopropylmethyl
chains fit and interact better with the hydrophobic cavities. This
is further confirmed by much higher IC50 values (5-20 µM)
found for 33 (Figure 3), a previously described 7-PPyQ,18 which
bears a diethylaminoethyl side-chain at N-pyrrole. This construct
was generated to increase the water solubility of this class of
compounds. As this chain possesses two hindering ethyl groups
and a ternary N, which may be protonated at internal cellular
pH, becoming positively charged, it is less suitable to interact
with hydrophobic cavities. The above qualitative structure-
cytotoxic activity relationship, referring to the nature and bulk
of 7-PPyQ N-substituents, despite the low number of compounds
Results and Discussion
Chemistry. Scheme 1 describes the straightforward synthesis
of new 3-alkylated PPyQs 25-32, accomplished following a
pathway previously described.18 Preliminarily, 1-subsituted
5-nitro-indole derivatives 1-8 were obtained by two different
alkylating methods to have higher yields and cleaner products:
(a) NaH in DMF was better for compounds 1-3 and (b) KOH
in acetone for compounds 4-8. Then 5-amino-indole derivatives
9-16, quantitatively obtained by catalytic reduction (H2, Pd/C
10%) at atmospheric pressure of the corresponding nitro-
compounds 1-8, were condensed with ethyl benzoyl-acetate,
in ethanol and with acetic acid as catalyst, to give ethyl 3-indole-
amino-3-phenyl-acrylates 17-24 in good yields. These were
thermally cyclized to final 3-substituted 7-PPyQs 25-32 in
boiling diphenyl ether (250 °C) with high yields. In all cases,
the final compounds showed [3,2-f] angular geometry (1H NMR
ortho-couplings of 4-H and 5-H protons) and a quinolinone
1
structure, as confirmed by H NMR (N-H at about δ 11.5),
13C NMR (CdO ranging from 184 to 194 δ) and IR (CdO
near 1610 cm-1) spectroscopic data.
Biology. In Vitro Cytotoxic Activity. The in vitro cytotoxic
activity of the newly synthesized 3-substituted 7-PPyQs 25-

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5511
Table 1. Cytotoxic Activity of 3-Substituted 7-PPyQs 25-32, 33,18 and Unsubstituted 7-PPyQ18
cytotoxic activity IC50 (µM)
cmpd
Hela
Hep G2
H295R
Ovcar-3
MCF-7
Aro
A549
HT-29
PT-45
OE19
OE33
7-PPyQa
0.7 (
0.2
8 (
1
0.4 (
0.2
6 (
2
0.8 (
0.3
0.7 (
0.1
1(
0.2
2 (
0.5
0.5 (
0.1
1 (
0.3
2 (
0.5
25
0.1 (
0.05
1 (
0.2
0.09 (
0.01
0.7 (
0.1
0.4 (
0.2
0.3 (
0.1
0.3 (
0.1
0.3 (
0.2
0.1 (
0.05
0.1 (
0.02
0.8 (
0.3
26
0.01 (
0.008
0.03 (
0.006
0.02 (
0.01
0.02 (
0.005
0.02 (
0.01
0.05 (
0.01
0.04 (
0.01
0.02 (
0.005
0.03 (
0.01
0.02 (
0.005
0.7 (
0.2
0.03 (
0.009
0.02 (
0.005
0.01 (
0.005
0.02 (
0.01
0.01 (
0.005
0.4 (
0.2
0.04 (
0.01
0.02 (
0.01
0.01 (
0.002
0.02 (
0.01
0.01 (
0.008
0.5 (
0.1
0.04 (
0.01
0.07 (
0.01
0.06 (
0.01
0.08 (
0.02
0.04 (
0.01
0.3 (
0.1
0.03 (
0.01
0.05 (
0.02
0.03 (
0.01
0.07 (
0.01
0.03 (
0.005
0.3 (
0.1
0.03 (
0.01
0.05 (
0.01
0.03 (
0.005
0.04 (
0.02
0.02 (
0.002
0.1 (
0.05
0.03 (
0.01
0.2 (
0.05
0.03 (
0.01
0.2 (
0.05
0.06 (
0.001
0.4 (
0.2
0.03 (
0.02
0.05 (
0.01
0.04 (
0.01
0.04 (
0.01
0.02 (
0.001
0.08 (
0.04
0.08 (
0.01
0.05 (
0.01
0.007 (
0.002
0.005 (
0.002
0.1 (
0.05
0.2 (
0.05
0.1 (
0.05
0.06 (
0.01
0.02 (
0.01
0.02 (
0.01
0.1 (
0.05
27
28
29
30
31
0.1 (
0.07
0.5 (
0.2
32
0.3 (
0.1
5 (
0.5
0.008 (
0.001
0.005 (
0.002
0.8 (
0.2 (
0.4 (
0.3 (
0.1
8 (
1
0.007 (
0.001
0.02 (
0.01
0.3 (
0.4 (
0.5 (
0.3 (
1.0 (
0.5
10 (
2.0
0.4 (
0.1
0.05 (
0.002
0.5 (
0.1
16 (
1.5
0.2 (
0.05
0.03 (
0.01
0.1
0.05
0.2
0.1
0.2
0.1
0.02
33a
taxol
vincr.
7 (
1
0.3 (
0.05
0.2 (
0.05
6 (
0.5
0.08 (
0.01
0.09 (
0.01
18 (
2
0.01 (
0.002
0.02 (
0.005
20 (
2.5
0.0002 (
0.0001
0.0003 (
0.0001
12 (
1.5
0.006 (
0.003
0.03 (
0.01
6 (
0.5
0.05 (
0.02
0.01 (
0.005
5 (
1
0.005 (
0.001
0.004 (
0.001
a Ref 18, Figure 3.
Table 3. Flow Cytometry: Percentage of A549 Cells in Each Phase of
the Cell Cycle at Concentrations of 1 and 0.1 µM
cmpd
% cells
S
G1
G2/M
concentration
(µM)
1
0.1
1
0.1
1
0.1
Figure 3. Structure of 3-(2-(diethylamino)-7-phenyl-3H-pyrrolo[3,2-
f]quinolin-9-one, 33.18
25
26
27
28
29
30
31
32
15.6
14.1
13.5
12.9
15.5
16.3
14.6
13.6
74.0
46.8
66.9
11.7
13.6
11.8
12.4
16.5
78.9
81.7
9.2
8.1
9.6
9.2
8.8
8.6
9.4
8.4
11.8
7.9
8.8
8.1
8.7
7.7
7.2
7.0
9.7
7.9
74.9
77.3
76.9
77.6
75.5
74.6
75.8
77.9
13.3
45.2
18.2
79.8
76.2
80.4
79.6
75.7
10.4
10.2
Table 2. Cytotoxic Activity of 7-PPyQs 25-32 Against Human Lung
Carcinoma Taxol-Resistant Cell Sublines
cytotoxic activity IC50 (µM)
cmpd
A549
A549-T12 (RI)a
A549-T24 (RI)a
25
26
27
28
29
30
31
32
0.3 ( 0.1
0.3 ( 0.2 (1)
0.2 ( 0.05 (0.6)
0.03 ( 0.02 (1)
0.04 ( 0.01 (1)
0.03 ( 0.01 (1)
0.04 ( 0.02 (1)
0.02 ( 0.005 (1)
0.2 ( 0.05 (2)
control
vincristine
0.03 ( 0.01
0.05 ( 0.02
0.03 ( 0.01
0.04 ( 0.02
0.02 ( 0.01
0.1 ( 0.05
0.4 ( 0.1
0.03 ( 0.01 (1)
0.04 ( 0.007 (1.2)
0.02 ( 0.005 (0.6)
0.05 ( 0.01 (0.8)
0.02 ( 0.01 (1)
0.1 ( 0.05 (1)
nd
nd
nd
between IC50 values calculated for cells and those arising from
chemosensitive ones. Comparable sensitivity between parental
and resistant cell lines was observed (RI ) 0.6-1.2 and 0.6-1
for 25-32), as opposed to the RI values of 10.2 and 17 for
taxol, on A549-T12 and A549-T24, respectively (Table 2).
Results indicate that 7-PPyQs escape the mechanism of taxol
resistance and further support the existence of differing mech-
anisms of action.
Fluorescence-Activated Cell-Sorting Analysis. To examine
the effect of the new derivatives 25-32 on cell cycle progres-
sion, FACS was performed. This method highlights the effects
of drugs on the distribution of cells in specific phases of the
cell cycle. Indeed, antimitotic drugs block cells in the G2/M
phase, causing an increment of the relative peak in the DNA
histogram.24,3 All compounds were tested on the A549 cell line
at concentrations of 0.1 and 1 µM. Table 3 shows the results
following after 24 h of treatment. All compounds tested at 1
µM caused a significant arrest of the cell cycle in G2/M phase,
raising the G2/M peak from 13.3% (control) to 77.9% (32). At
0.1 µM, compounds 26-30 again increased the G2/M peak from
13.3% (control) to 80.4% (28), whereas compounds 25, 31, and
32 did not. It is worth noting that, at 0.1 µM, there is a good
correlation between the cytotoxicity of 3-substituted 7-PPyQs
0.4 ( 0.02 (1)
0.06 ( 0.03 (10.2)
0.5 ( 0.2 (1.25)
0.102 ( 0.6 (17)
taxol
0.006 ( 0.003
a RI ) resistance index.
examined, allowed us to hypothesize the presence of a deep
hydrophobic pocket near the colchicine site, able to accept
hindering atom groups like linear aliphatic chains up to five
carbons, cyclopropylmethyl and a benzyl group without a loss
of potent inhibitory capacity.
In Vitro Cytotoxic Activity against Taxol-Resistant Hu-
man Tumor A549 Sublines. The very encouraging results
obtained against the in-house panel of cell lines prompted us
to test the in vitro cytotoxic activity of compounds 25-32 on
two additional human cell sublines characterized by acquired
resistance to taxol: human lung carcinoma A549-T12 and A549-
T24 cell lines.21 Cytotoxicity was assessed after a 72 h exposure
by the MTT test. The results showed that the new 7-PPyQ
derivatives are equally potent toward the parental A549 cells
and the two taxol-resistant sublines, A549-T12 and A549-T24
(IC50 0.02-0.5 µM; Table 2). Cross-resistance profiles were
evaluated by the resistance index (RI), and defined as the ratio

5512 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Brief Articles
(TLC) with Merck silica gel 60 F-254 glass plates with a 9:1
dichloromethane/methanol mixture as eluant, unless otherwise
specified. Solutions were concentrated in a rotary evaporator under
reduced pressure. Starting materials were purchased from Aldrich
Chimica and Fluka Riedel-de Haen (TiCl3, 15% HCl solution), and
solvents were purchased from Carlo Erba, Fluka, and Lab-Scan.
DMSO was made anhydrous by distillation under vacuum and
stored on molecular sieves. Colchicine, vincristine, and taxol were
purchased from Sigma.
and the ability to arrest the A549 cell cycle in G2/M, as
compounds 25, 31, and 32 turned out to be less potent in the
MTT proliferation assay, too (Table 1).
Conclusions
In the area of antimitotic agents interfering with the colchicine
binding site on â-tubulin, a series of 3-substituted 7-PPyQs were
synthesized and tested for cytotoxicity on 11 human tumor cell
lines and for their effects on cell cycle progression. These agents
show an high cytotoxic activity with IC50 values in the
nanomolar range, up to 100-fold lower than the 3-unsubstituted
analogs previously studied18 and, in some cases, lower than those
of reference compounds, taxol and vincristine (Table 1). When
assayed against taxol-resistant human lung carcinoma A549-
T12 and A549-T24 cell lines, the new 7-PPyQ derivatives were
equally potent (IC50 0.02-0.5 µM) toward the parental A549
cells (IC50 0,03-2 µM; Table 2). Furthermore, preliminary
experiments showed that the most active compound (30) exerted
significant cytotoxic activity (>30% inhibition) on multidrug-
resistant breast cancer MCF-7 MDR cells at concentrations up
to 1 µM, whereas vincristine was ineffective at this concentra-
tions. All these compounds caused G2/M phase arrest in the
cell cycle after a 24 h incubation (Table 2). Results obtained in
this study and the previous one on PPyQs contribute to
knowledge about structure-activity relationship, referring to the
pyrroloquinolinone nucleus. It is clear that two structural
features, that is, the phenyl in position 7 and a hydrophobic
group at N-pyrrole, are essential for potent cytotoxic activity
and efficient mitotic arrest. As the most active compounds turned
out to be those carrying more hindering side chains at N-pyrrole,
we also suggest the probable presence of a large, deep
hydrophobic pocket near the colchicine binding site, in which
useful hydrophobic interactions occur. Besides, preliminary data
from tubulin polymerization assays revealed relevant antimi-
crotubule effects as all new compounds caused significant
tubulin depolymerisation (data not shown). The most active
compound 30 displayed a behavior similar to colchicine. Indeed,
colchicin completely inhibited tubulin polymerization at 3 µM
and 30 exhibited an IC50 of 0.312 µM. In conclusion, these
overall results shown by 3-substituted 7-PPyQs further increase
our interest in PPyQs and prompts us to carry on mechanicistic
studies to better characterize biological targets at the molecular
level.
General Procedure for the Synthesis of 1-Substituted 5-Nitro-
indoles 1-3. In a three-necked, 100 mL, round-bottomed flask,
an amount corresponding to 37-55.5 mmol of NaH (60% disper-
sion in mineral oil) was placed and washed with toluene (3 × 10
mL). A solution of commercial 5-nitro-indole (12.3-18.5 mmol)
in 20-30 mL of dry DMF was dropped into the flask, and the
starting yellow solution changed to red, with the formation of H2
gas. After 30 min at room temperature, a solution of halo-derivative
in dry DMF was added, and the reaction mixture was left under
stirring for 20-48 h, monitoring ongoing reactions by TLC analysis
(eluent: toluene/n-hexane/ethyl acetate 1:1:0.3). At the end, the
solvent was evaporated and the residue was extracted with ethyl
acetate; the organic phase, washed with water and dried over
anhydrous Na2SO4, was concentrated until dryness under vacuum.
The raw products were purified by flash chromatography.
General Procedure for the Synthesis of 1-Substituted 5-Nitro-
indoles 4-8. To a stirring solution of 1 g (6.2 mmol) of commercial
5-nitro-indole in 30 mL of acetone, 1.728 g powdered KOH at
0 °C was added, giving a dark-red color, and then an excess of
halo-derivative (12.3 mmol) was added. The stirred mixture was
heated at 40 °C for 3 h, until the starting material disappeared at
TLC analysis (toluene/n-hexane/ethyl acetate 1:1:0.3). After cooling
to room temperature, 200 mL of toluene was added and the formed
suspension was left under stirring for 30 min. The insoluble solid
was then filtered off and the filtrate was washed several times with
NaCl-saturated water, dried over anhydrous Na2SO4, and evapo-
rated to dryness, yielding oily or solid reddish-orange products.
These were used in the following step without any purification.
General Procedure for the Synthesis of 1-Substituted 5-Amino-
indoles 9-16. A solution of a nitro-indole 1-8 (5.87 mmol) in
400 mL of ethanol was dropped into a suspension of 10% Pd/C
(125 mg) saturated with H2 in ethanol (200 mL). The mixture was
stirred at room temperature with hydrogen at atmospheric pressure
for 3 h, according to TLC analysis (ethyl acetate/n-hexane 1:3).
The catalyst was then filtered off, and the filtrate was evaporated
under reduced pressure to dryness to give the corresponding amino-
indole. The almost pure residue (90% yield) was used in the next
reaction without purification.
General Procedure for the Synthesis of Ethyl 3-(1-Substituted-
indole-amino)-3-phenyl-acrylates 17-24. In a 50 mL round-
bottomed flask, 3-4 mmol of 3-substituted-aminoindole 9-16 in
10-20 mL of absolute ethanol was condensed with an equimolar
quantity of commercial ethyl benzoylacetate and with 1 mL of
glacial acetic acid and 100 mg drierite. The mixture was refluxed
for about 24 h, with the reaction being monitored by TLC analysis
(dichloromethane/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 was purified by flash chromatog-
raphy and then recrystallized from a suitable solvent.
General Procedure for the Synthesis of 3-Substituted 7-
Phenyl-3H-pyrrolo[3,2-f]quinolin-9-(6H)ones 25-32. In a two-
necked, round-bottomed flask, 50 mL of diphenyl ether was heated
to boiling temperature; 1-2 mmol of acrylates 17-24 were then
added portionwise, and the mixture was refluxed for 30 min. After
cooling to 60 °C, the separated 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).
Materials and Methods
Chemistry. Melting points were determined on a Gallenkamp
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 bromide 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 a Bruker (300 MHz) spectrometer, using the indicated
solvents; chemical shifts are reported in δ (ppm) downfield from
tetramethylsilane as internal reference. Coupling constants are given
in Hertz. In the case of multiplets, chemical shift was measured
starting from the approximate center. Integrals were satisfactorily
in line with those expected on the basis of compound structure.
Elemental analyses were performed in the Microanalytical Labora-
tory, Department of Pharmaceutical Sciences, University of Padova,
on a model 240B Perkin-Elmer elemental analyzer model; results
fell in the range of calculated values (0.4%. Analytical data are
presented in detail for each final compound. Mass spectra were
obtained on a 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
In Vitro Cytotoxicity Assay. The cytotoxic activity of 3-alky-
lated 7-PPyQs 25-32 was determined using a standard (MTT)-
based colorimetric assay (Sigma), using vincristine and taxol as

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5513
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reference drugs. Briefly, cell lines were seeded at a density of 7 ×
103 cells/well in 96-well microtiter plates (Costar). After 24 h,
exponentially growing cells were exposed to the indicated com-
pounds at final concentrations ranging from 0.005 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 PBS). 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 on an LX300 Epson Diagnostic microplate reader. Survival
ratios are expressed in percentages with respect to untreated cells.
IC50 values were determined from replicates of 6-8 wells from at
least two independent experiments.
In Vitro Cytotoxic Activity against Taxol Human Tumor
A549 Sublines. Exponentially growing cells were resuspended in
either drug-free medium (A549) or in presence of 12 nM (A549-
T12) or 24 nM (A549-T24) taxol. The resistant cells require taxol
to maintain normal cell proliferation. Cells were seeded at a density
of 104 cells/mL (A549) or 3 × 104 cells/mL (A549-T12 and A549-
T24) in triplicate six-well plates and allowed to attach for 24 h
before the addition of the indicate drug concentration. After a 72
h incubation, cells were trypsinized and counted.
Fluorescence-Activated Cell-Sorting Analysis. A549 cells were
cultured for 24 h in a drug-free medium or supplemented with
compounds 25-32 (0.1-1 µM) and vincristine (0.1 µM). As
previously described,37,38 cells were trypsinized 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, 24:1), followed by
PBS, and finally incubated in a 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
compounds on cell cycle dynamics, DNA fluorescence was
measured by flow cytometry, examining at least 15 000 events with
Lysis II software (Becton Dickinson, Franklin Lakes, NJ) at 488/
525 nm (excitation/emission wavelengths). All experiments were
repeated 3-4 times and DNA content analysis was carried out on
both logarithmic and linear scales. Results were comparable,
irrespective of the scale used, and are shown on a logarithmic scale.
Statistical Analysis. Results are reported as means ( standard
error (M ( S.E.). Statistical analysis was performed by one-way
analysis of variance or Student’s t-test, as appropriate. A P value
of less than 0.05 was considered statistically significant.
Supporting Information Available: Yields, mps, rfs, spectral
data, cell lines, culture conditions, and FACS analysis histograms
for all compounds 1-32. This material is available free of charge
via the Internet at http:// pubs.acs.org.
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Horwith, S.; Wilson, L.; Jordan, M. A. Resistance to taxol in lung
cancer cells associated with increased microtubule dynamics. Proc.
Natl. Acad. Sci. U.S.A. 2001, 98, 11737-11741.
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S.; Sobel, A.; Knossow, M. Insight into tubulin regulation from a
complex with colchicine and a stathmin-like domain Nature 2004,
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