Discovery of a new anilino-3H-pyrrolo[3,2-f]quinoline derivative as potential anti-cancer agent

Article abstract of DOI:10.1016/j.ejmech.2007.04.008

The newly synthesized 1-[4-(3H-pyrrolo[3,2-f]quinolin-9-ylamino)-phenyl]-ethanone hydrochloride showed high antiproliferative activity by mixed mechanisms of action. The compound acts by forming an intercalative complex with DNA and inhibiting DNA topoiso

Full text of DOI:10.1016/j.ejmech.2007.04.008

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European Journal of Medicinal Chemistry 43 (2008) 429e434
http://www.elsevier.com/locate/ejmech
Short communication
Discovery of a new anilino-3H-pyrrolo[3,2-f]quinoline derivative as
potential anti-cancer agent
L. Dalla Via a, O. Gia a, V. Gasparotto a,b, M.G. Ferlin a,
*
a Department of Pharmaceutical Sciences, University of Padova, Via Marzolo 5, 35131 Padova, Italy
b Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, via Gabelli, 63, 35121 Padova, Italy
Received 13 December 2006; received in revised form 5 March 2007; accepted 10 April 2007
Available online 6 May 2007
Abstract
The newly synthesized 1-[4-(3H-pyrrolo[3,2-f]quinolin-9-ylamino)-phenyl]-ethanone hydrochloride showed high antiproliferative activity by
mixed mechanisms of action. The compound acts by forming an intercalative complex with DNA and inhibiting DNA topoisomerase II (topo II)
and by blocking the cell cycle in G2/M phase. Probable cell death by apoptosis is also suggested by flow cytometry analysis.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Pyrroloquinoline derivative; Antiproliferative activity; DNA interaction; Antimitotic effect
1. Introduction
characteristic of the known anti-cancer drug m-amsacrine
[9,10] or with the aniline bifunctional mustard from known
therapeutic alkylating chlorambucil and mephalan (Fig. 1)
[11]. Interestingly, these compounds are remarkably active
against cell lines from solid tumors like CNS-, melanoma-
and prostate-derived cells [9]. Continuing this research line,
we designed and synthesized other PQ-based molecules modi-
fied at the side aniline ring with various substituents, in order to
identify new lead compounds. In preliminary in vitro cytotoxic
screening assays, one of these turned out, interestingly, to exert
high antiproliferative activity against some human tumor cell
lines. This paper reports the synthesis of a 9-anilino-3H-
pyrrolo[3,2-f]quinoline derivative, its antiproliferative activity
on human tumor cell lines, and its ability to interact with
some intracellular targets.
In the field of chemotherapeutic drugs, the search for new,
more active, more selective and less toxic compounds is still
very intense, and new promising anti-cancer approaches are
being tested [1,2]. Currently, combined anti-cancer therapies
or multi-acting drugs are clinically preferred to traditional cy-
totoxic treatment, with the aim of overcoming resistance and
toxicity drawbacks. These events often prevent successful
treatment and are responsible for reduced survival times [3,4].
In discovering small anti-cancer molecules, a notable role is
played by polyheterocyclic structures, and among these, a grow-
ing attention focuses on the synthesis and study of the biological
properties of compounds containing various combinations of in-
dole or quinoline moieties. Recently, much interest has arisen
for the pyrroloquinoline (PQ) nucleus [5,6].
Over the past few years, we have synthesized novel PQ deriv-
atives with remarkable cytotoxic activity [7,8] and reported an ad-
vantageous ring-forming method for the synthesis of substituted
angular PQs. In particular, we synthesized and studied new deriv-
atives, characterized by a 3H-pyrrolo[3,2-f]quinoline planar nu-
cleus connected with the methansulfon-anisidine residue
2. Results and discussion
2.1. Chemistry
As shown in Scheme 1, 9-chloro-pyrrolo[3,2-f]quinoline
was obtained by the multi-step pathway previously reported
[9], starting from 5-aminoindole by means of the conventional
reaction with diethyl ethoxymethylene malonate [12], which
* Corresponding author. Tel.: þ39 049 8271603; fax: þ39 049 8275366.
E-mail address: mariagrazia.ferlin@unipd.it (M.G. Ferlin).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.04.008

430
L. Dalla Via et al. / European Journal of Medicinal Chemistry 43 (2008) 429e434
R'''
of a cervical adenocarcinoma [14], JR8 from lymph node me-
tastasis of poll scalp melanoma [15] and the OVCAR-3 line
was established from the malignant ascites of a patient with
progressive adenocarcinoma of the ovary [16]. Results, ex-
pressed as IC50, i.e., the concentration that is able to cause
the death of 50% of cells with respect to the control culture,
indicate significant antiproliferative capacity toward all cell
lines examined although with different sensitivities (Table 1).
In detail, a micromolar IC50 value was obtained for HeLa cells;
submicromolar and comparable results were found for the
others. As the pyrroloquinoline molecule has shown inability
to exert antiproliferative action [11], the effect of 2 is attributed
to the insertion of the substituted aniline moiety.
R'
HN
N
NH
R= H, CH3
R' = H, OCH3
R'' = NHSO2CH3, N(CH2-CH2-Cl)2
R
Fig. 1. Some cytotoxic 9-anilino-pyrrolo[3,2-f]quinolines previously reported
[9,11].
2.3. Linear flow dichroism studies
The interesting antiproliferative activity shown by 2 promp-
ted us to study its mechanism of action. A previous study had
evidenced the capacity of some derivatives, composed of the
PQ chromophore connected to an aniline bifunctional mustard,
for remarkable antiproliferative activity, enhanced with respect
to reference compounds. In particular, their ability to interact
with DNA through an intercalative mode of binding was dem-
onstrated [11]. On the basis of this knowledge, the ability of 2
to give rise to a molecular complex with DNA was assayed by
means of linear flow dichroism experiments. The spectra of
a solution of salmon testes DNA, alone and in the presence
of 2 at a [drug]/[DNA] ¼ 0.08, are reported in Fig. 2. Both
the dichroic spectra show a strong negative signal at
260 nm, typical of the macromolecule. Nevertheless, in the
presence of PQ derivative 2 (dashed line), a further signal ap-
pears at higher wavelengths (300e450 nm), indicating the oc-
currence of a molecular complex between 2 and DNA. The
negative sign also indicates the preferentially parallel orienta-
tion of the chromophore to the plane of DNA bases, in agree-
ment with an intercalative mode of binding.
furnished the corresponding enamine derivative. This was
thermally cyclized to 8-carbethoxy-pyrrolo[3,2-f]quinolin-9-
one, which was submitted to alkaline hydrolysis in boiling
NaOH 20%, then to decarboxylation in boiling diphenyl ether,
and then chlorinated with POCl3 at 70 ꢀC. Nucleophilic substi-
tution of 1 with p-amino-acetophenone in the presence of HCl
as catalyst afforded 4-acetyl-aniline compound 2, as mono-
hydrochloride.
2.2. Antiproliferative activity
Evaluation of the biological activity of derivative 2 was car-
ried out by an in vitro antiproliferative assay on four human
tumor cell lines: HL-60, HeLa, JR8 and OVCAR-3. HL-60
was derived from the peripheral blood leukocytes of a patient
with acute promyelocytic leukemia [13], HeLa from a biopsy
C2H5OOC
COOC2H5
O
H2N
NH
HN
a
b
2.4. Inhibition of topo II activity
C2H5OOC
N
H
N
H
N
H
99%
81%
The ability of derivative 2 to give rise to an intercalative
molecular complex with DNA suggested that the synthesized
chromophore may interfere with DNA topo II, the enzyme
which catalyzes the conversion of pBR322 plasmid DNA
from the supercoiled to the fully relaxed conformation [17].
Fig. 3 shows the effect of the new derivative on topo II relax-
ation ability. In detail, concentration-dependent inhibition was
observed: indeed, 2 can partially affect plasmid relaxation at
10 mM, and inhibition is practically complete at 50 mM (lanes
c and d, respectively). The high concentration required for
89%
c
O
O
NH
NH
d
HOOC
N
H
84%
N
H
e
82%
CH3
O
C
.HCl
NH
O
CH3
C
Cl
HN
N
f
NH
30%
N
NH2
2
Table 1
Cell growth inhibition in presence of new PQ derivative 2
1
Scheme 1. Synthetic route for synthesizing anilino-pyrroloquinoline derivative
2. Reagents and conditions: (a) diethyl ethoxymethylenemalonate, 130 ꢀC, 3 h;
(b) diphenyl ether, 250 ꢀC, 15 min; (c) NaOH, 2 N, 3 h, refluxing; (d) diphenyl
ether, 250 ꢀC, 25 min; (e) POCl3, 130 ꢀC, 1 h; (f) methanol, HCl cat., 70 ꢀC,
12 h.
Compd.
Cell lines IC50 (mM)
HL-60
HeLa
3.4 ꢁ 0.5
JR8
OVCAR-3
2
0.50 ꢁ 0.06
0.59 ꢁ 0.07
0.53 ꢁ 0.03

L. Dalla Via et al. / European Journal of Medicinal Chemistry 43 (2008) 429e434
431
0.000
-0.002
-0.004
polymerization block cells in the G2/M phase, causing an in-
crement of the relative peak in the DNA histogram [18]. Com-
pound 2 was tested on HeLa, JR8, and OVCAR-3 cell lines, at
1e10 mM. It showed a significant dose-dependent antimitotic
activity in all the three. For example, in OVCAR-3 (Fig. 4),
it caused an increase of the G2/M peak at 1 mM from 15.3%
(Fig. 4a, control) to 31.4% (Fig. 4b), at 5 mM to 57.8%
(Fig. 4c) and at 10 mM to 63.7% (Fig. 4d).
Note the good correlation between the cytotoxicity
and antimitotic effects of the compound, which was very
active in all proliferation assays of all three tested cell lines
(Table 1).
Also note the increases in apoptotic peaks at 1 and 5 mM of
compound 2 (Fig. 4b and c, 7.6% and 4.3% for cells in sub G0
state, respectively), in comparison with the control (Fig. 4a,
0.2%). At 10 mM, compound 2 did not show any increase in
apoptotic peak (Fig. 4d, 0.5%). Increases at lower doses indi-
cated that apoptosis had occurred, and this specific behaviour
e that is, percentage decreases concomitant with dose in-
creases e is in line with the most recent reports of drugs
able to target microtubules. At low doses, these drugs act by
suppressing spindle-microtubule dynamics and inducing apo-
ptotic cell death; at high concentrations they cause reduction
of the microtubuleepolymer mass and mitotic arrest in the
G2/M phase [19]. The small observed increases in apoptotic
peaks were probably due to the short duration of drugecell
contact (24e48 h).
Flow cytometry results suggest that a possible target for 2
may be microtubules. On the basis of structural consider-
ations, the PQ derivative may interact with the colchicine
site on b-tubulin. Indeed, in the molecular structure of 2
pharmacophore elements recurring in tubulin ligands for
the colchicine site have been identified, as recently reported
by Nguyen et al.[20]: three hydrogen bond acceptors
(carbonyl, quinoline nitrogen, amine nitrogen), one hydrogen
bond donor (indole nitrogen), hydrophobic centers (aromatic
carbons) and one planar group (phenyl). Nevertheless, the
exact target site accounting for the antimitotic effect and
pro-apoptotic activity of 2 must be confirmed by suitable
assays.
x 5
250
300
350
Wavelength (nm)
400
450
500
Fig. 2. Linear flow dichroism spectra for salmon testes DNA (straight line) and
compound 2 (dashed line) at [drug]/[DNA] ¼ 0.08.
inhibition suggests that this effect is probably not, alone, suffi-
cient to account for the notable antiproliferative activity exerted
on human cell lines (see Table 1). This result is in agreement
with previous data of some PQ derivatives, whose properties
of cell growth inhibition did not appear to be related mainly
to inhibition of topo II [9,10]. Further experiments were there-
fore performed, to identify other possible cellular targets of 2
which could contribute to the overall cytotoxic effect.
2.5. Cell cycle analysis
Flow cytometry analysis was used to examine the effect of
the new anilino-PQ 2 on cell cycle progression. The method
highlights the effects of drugs on the distribution of cells in
specific phases: drugs which interfere with tubulin
a
b
c
d
e
f
3. Conclusions
We have discovered a small PQ molecule which shows
cytotoxic activity at submicromolar concentrations in pre-
liminary assays. In the course of a mechanistic study, it
turned out to exert antiproliferative activity by multiple
mechanisms of action, i.e., intercalation into the DNA dou-
ble strand, inhibition of topo II, and mitotic arrest in the G2/
M phase. As multi-acting drugs are thought to be suitable
for chemotherapy because a single compound can attack
tumors through multiple cellular pathways, we believe that
9-(4-acetylanilino)-3H-pyrrolo[3,2-f]quinoline is interesting
enough to prompt further studies of its potential as a lead
compound.
Relaxed DNA
Supercoiled DNA
Fig. 3. Effect of derivative 2 on relaxation of supercoiled pBR322 DNA by hu-
man recombinant topo II: lanes a, DNA control (no enzyme); lane b, DNA and
topo II; lanes c and d, same as lane b with 10 and 50 mM 2, respectively; lane e
and f: same as lane b, with 8 mM m-amsacrine and solvent alone, respectively.

432
L. Dalla Via et al. / European Journal of Medicinal Chemistry 43 (2008) 429e434
4a
4b
Control
Compound 2, 1 µM
Sub Go: 7.6 %
GoG1: 51.9 %
S: 9.4 %
Sub Go: 0.2 %
GoG1: 71.3 %
S: 13.6 %
G2/M: 15.3 %
G2/M: 31.4 %
4c
4d
Compound 2,5 µM
Sub Go: 4.3 %
GoG1: 24.7 %
S: 13.6 %
Compound 2,10 µM
Sub Go: 0.5 %
GoG1: 24.5 %
S: 11.6 %
G2/M: 57.8 %
G2/M: 63.7 %
4e
4f
Vincristin, 1 µM
Sub Go: 0.5 %
GoG1: 46.8 %
S: 7.9 %
m-Amsacrine, 16 µM
Sub Go: 1.8 %
GoG1: 44.7 %
S: 12.7 %
G2/M: 45.2 %
G2/M: 41.3 %
Fig. 4. Cell cycle analysis e effect on cell cycle progression of anilino-PQ derivative 2 at 1, 5 and 10 mM on OVCAR-3 cell line, and vincristine and m-amsacrine
as references.
4. Materials and methods
values are expressed in cmꢂ1. UVevis spectra were recorded
1
on a Perkin-Elmer Lambda UVevis spectrometer. H NMR
4.1. General
and 13C NMR spectra were recorded on a Bruker AMX spec-
1
trometer at 300.13 MHz for H and 75.04 MHz for 13C, with
Melting points were determined on a Gallenkamp MFB 595
010 M/B capillary melting point apparatus, and are uncor-
rected. Infrared spectra were recorded on a Perkin-Elmer
1760 FTIR spectrometer as potassium bromide pressed discs;
the indicated solvents. Elemental analyses were performed
in the Microanalytical Laboratory, Department of Pharmaceu-
tical Sciences, University of Padova, on a Perkin-Elmer Ele-
mental Analyzer Model 240B. High-resolution mass spectra

L. Dalla Via et al. / European Journal of Medicinal Chemistry 43 (2008) 429e434
433
were obtained with an Applied Biosystems Mariner System
4.4. Linear flow dichroism
5220 LC/Ms (nozzle potential 250.00). Starting materials
used in the syntheses shown in the scheme were purchased
from Aldrich and Acros Organics.
Linear dichroism (LD) measurements were performed on
a Jasco J500A circular dichroism spectropolarimeter, con-
verted for LD and equipped with an IBM PC and a Jasco J
interface.
4.1.1. 1-[4-(3H-Pyrrolo[3,2-f]quinolin-9-ylamino)-phenyl]-
ethanone hydrochloride (2)
Linear dichroism was defined as:
To a solution of 9-chloro-3H-pyrrolo[3,2-f]quinoline [5] (1)
(0.245 g, 1.21 mmol) and HCl 37% (0.1 ml) in 30 ml of methanol,
a solution of 4-amino-acetophenone (0.164 g, 1.21 mmol) in
10 ml of methanol was added dropwise under stirring. After about
40 h at 70 ꢀC, the solvents had evaporated and the residue was
treated with water. The aqueous solution after extraction with ethyl
acetate, which removed unreacted reagents, was concentrated and
cooled overnight. The desired product, precipitated as hydrochlo-
ride, was collected and recrystallized from hot water. Yield 43%;
mp 194 ꢀC; Rf 0.38 (ethyl acetate/methanol 8:2); 1H NMR
LD ¼ A==ðlÞ ꢂ AtðlÞ
ðlÞ
where A// and At correspond to the absorbances of the sample
when polarized light is oriented parallel or perpendicular to
the flow direction, respectively. The orientation was produced
by a device designed by Wada and Kozawa [21] at a shear gra-
dient of 500e700 rpm, and each spectrum was accumulated
four times and recorded at 25 ꢀC.
A solution of salmon testes DNA (1.6 ꢃ 10ꢂ3 M) in ETN
buffer (containing 10 mM Tris, 10 mM NaCl, and 1 mM
EDTA, pH ¼ 7) was used.
(DMSO-d6) d 2.6 (s, 3H, CH3), 7.29 (d, 1H, J8,7 ¼ 6.5 Hz, 8-H),
0
0
0
0
0
7.50 (s, 1H, 1-H), 7.68 (d, 2H, J2 ,3 and J5 ,6 ¼ 8.4 Hz, 2 -H,
60-H), 7.80 (m,0 2H, 0 4-H and 2-H), 8.12 (d, 2H, J2 ,3 and
0
0
0
0
J5 ,6 ¼ 8.5 Hz, 3 -H, 5 -H), 8.24 (d, 1H, J5,4 ¼ 8.9 Hz, 5-H), 8.57
(d, 1H, J7,8 ¼ 6.9 Hz, 7-H), 9.8 (s, 1H, NH), 12.45 (bs, 1H, pyrrole
NH); 13C NMR (DMSO-d6) d 26 (CH3), 103 (9b-C), 104 (9a-C),
113 (1-C), 118 (8-C), 120 (3a-C), 123 (4-C and 2-C), 127 (10-C),
130 (20-C and 60-C), 132 (40-C), 133 (5-C), 135 (9-C), 138 (5a-
C), 143 (300-C and 500-C), 154 (7-C), 196 (O-C); HR MS calcd.
337.803; found m/z [M þ H]þ 302.126; anal. calcd. for
C19H16N3OCl: C, 67.56; H, 4.77; Cl, 10.50; N, 12.44; found: C,
67.32; H, 4.79; Cl, 10.45; N, 12.57.
4.5. Topo II assay
Topo II activity was measured by the relaxation of super-
coiled plasmid DNA. The relaxation assay was performed by
incubating in 20 ml reaction volumes of supercoiled pBR322
DNA (Fermentas Life Sciences, 0.25 mg) for 60 min at
37 ꢀC with topo II (USB Corporation, 1 U) in the absence or
presence of the test compound. After this, 4 ml of stop buffer
and 50 mg/ml of proteinase K (Sigma Chemical Co.) were
added and incubated for a further 30 min at 37 ꢀC.
4.2. Cell cultures
DNA samples were separated by electrophoresis on a 1%
agarose gel. The gel was stained with ethidium bromide
1 mg/ml in TAE buffer (40 mM Tris, 20 mM glacial acetic
acid, 1 mM EDTA, pH ¼ 8), transilluminated by UV light,
and fluorescence emission was visualised by a CCD camera
coupled to a Bio-Rad Gel Doc XR apparatus.
HL-60, JR8 and OVCAR-3 cells were grown in RPMI 1640
(Sigma Chemical Co.) supplemented with 15%, 10% and 10%
heat-inactivated fetal calf serum (FCS, Seromed), respectively.
HeLa cells were grown in Nutrient Mixture F-12 [HAM]
(Sigma Chemical Co.) supplemented with 10% heat-inacti-
vated FCS.
4.6. Flow cytometry
Penicillin 100 U/ml, streptomycin 100 mg/ml and ampho-
tericin B 0.25 mg/ml (Sigma Chemical Co.) were added to
the media. The cells were cultured at 37 ꢀC in a moist atmo-
sphere of 5% carbon dioxide in air.
HeLa, JR8 and OVCAR-3 cells were cultured for 24e48 h
in a drug-free medium or supplemented with test compound
(1e10 mM), vincristine sulfate salt (1 mM) or m-amsacrine
(16 mM). As previously described [22], cells were harvested
with a cell scraper, washed twice with PBS, and fixed in
70% cold ethanol (30 min at ꢂ20 ꢀ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 in-
cubated in an RNAse solution (100 mg/ml in PBS). After
30 min at 37 ꢀC, the cells were incubated in a propidium io-
dide solution (PI, Sigma, 100 mg/ml in PBS) at room temper-
ature for a further 30 minutes. To determine the effects of the
test compound on cell cycle dynamics, DNA fluorescence was
measured by flow cytometry, analyzing at least 15 000 events
with Lysis II software. Cells were analyzed using a FACSVant-
age flow cytometer (Becton Dickinson, Franklin Lakes, NJ) at
488/525 nm (excitation/emission wavelength).
4.3. Inhibition growth assay
HL-60 cells (4 ꢃ 104) were seeded into each well of a 24-
well cell culture plate. After incubation for 24 h, various con-
centrations of the test agent were added and incubated for
a further 72 h. HeLa, JR8 and OVCAR-3 (4 ꢃ 104) cells
were seeded into each well of a 24-well cell culture plate. Af-
ter incubation for 24 h, the medium was replaced with an
equal volume of fresh medium and various concentrations of
the test agent were added. The cells were then incubated in
standard conditions for a further 72 h.
A trypan blue assay was performed to determine cell viabil-
ity. Cytotoxicity data are expressed as IC50 values.

434
L. Dalla Via et al. / European Journal of Medicinal Chemistry 43 (2008) 429e434
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All experiments were repeated three to four times and DNA
content analysis was carried out by means of both logarithmic
and linear scales. Results were comparable, irrespective of the
scale used, and are shown on a logarithmic scale.
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