Synthesis and in vitro evaluation of 3H-pyrrolo[3,2-f]-quinolin-9-one derivatives that show potent and selective anti-leukemic activity

Article abstract of DOI:10.1002/cmdc.201000180

A series of new substituted 7-phenyl-3H-pyrrolo[3,2-f]quinolin-9-ones were synthesized and evaluated for their antiproliferative activity. The most active derivatives showed high selectivity against human leukemia cell lines and potently inhibited their g

Full text of DOI:10.1002/cmdc.201000180

DOI: 10.1002/cmdc.201000180
Synthesis and in vitro Evaluation of 3H-Pyrrolo[3,2-f]-
quinolin-9-one Derivatives That Show Potent and Selective
Anti-leukemic Activity
Maria Grazia Ferlin,*[a] Roberta Bortolozzi,[b] Paola Brun,[c] Ignazio Castagliuolo,[c]
Ernest Hamel,[d] Giuseppe Basso,[b] and Giampietro Viola[b]
A series of new substituted 7-phenyl-3H-pyrrolo[3,2-f]quinolin-
9-ones were synthesized and evaluated for their antiprolifera-
tive activity. The most active derivatives showed high selectivi-
ty against human leukemia cell lines and potently inhibited
their growth, with GI50 values in the nanomolar range. The
active compounds strongly blocked tubulin assembly and col-
chicine binding to tubulin. Their activities were equal to or
greater than that of the reference compound combretastatin
A-4. Flow cytometry studies showed that the two most active
compounds arrested Jurkat cells in the G2/M cell-cycle phase in
a concentration-dependent manner. This effect was associated
with apoptosis, mitochondrial depolarization, generation of re-
active oxygen species, activation of caspase-3, and cleavage of
the enzyme poly(ADP-ribose) polymerase.
Introduction
For several years we have studied the pyrroloquinoline nucleus
as a promising scaffold for antiproliferative agents,[1–3] leading
to the discovery of the 3H-pyrrolo[3,2-f]quinoline (PyQ) scaffold
(Figure 1) as the most promising framework for the design of
potent anticancer compounds. In
cine site.[3] Substituents on the phenyl ring at C7, either elec-
tron-withdrawing or -donating groups, had only minor effects
on activity.
In an effort to produce additional highly active analogues,
we synthesized and evaluated the effects of substitutions at
other positions of the 3H-pyrrolo[3,2-f]quinolin-9-one nucleus.
Herein we describe 3H-PyQ derivatives with small substituents
at the C1 position and with the side phenyl ring shifted from
C7 to C8. In addition to synthesizing the simple 8-phenyl-PyQ
33, substituents were also introduced onto template 33 to ex-
amine the effects on the bioactivity of the resulting com-
pounds. We also focused on increasing water solubility relative
to previous analogues by inserting suitable substituents at the
1-, 3-, and 7-positions.[3] Such compounds should be valuable
for in vivo studies on pharmacokinetics, metabolism, and toxic-
ity.
particular, we found several active
agents with N-pyrrole alkyl sub-
stituents that are derivatives of 7-
phenyl-3H-pyrrolo[3,2-f]quinolin-9-
ones (7-PPyQs).[3] We showed that
7-PPyQs exhibit strong cytotoxic
activity against a wide panel of
human tumor cell lines, including
multi-drug resistance (MDR)-posi-
tive cancer cells. Moreover, in vivo,
the 7-PPyQs exhibit significant in-
hibition of tumor growth (83%) in
Figure 1. 3H-Pyrrolo[3,2-f]qui-
nolin-9(6H)-ones; SARs are de-
scribed in the text.
The newly prepared agents were assayed for their in vitro
cytotoxicity against a panel of human solid tumors and leuke-
a syngenic hepatocellular carcinoma model in Balb/c mice.[2]
Structure–activity relationship (SAR) studies of various pyrro-
loquinoline derivatives allowed us to establish some structural
features that are crucial for antitumor activity. First, the [3,2-f]
angular geometry is essential for significant activity, as is the
carbonyl group at the 9-position and the side phenyl ring.
Moreover, optimal activity requires the presence of an N-alkyl
group on the pyrrole ring. Although active compounds were
shown to have substituents ranging from ethyl to n-pentyl
groups as well as a cyclopropylmethyl group, ionizable groups
such as dimethylaminoethyl at position N3 or oxygen-contain-
ing substituents at positions C2 and C4 caused severe loss of
activity. Because previously synthesized compounds[3] had
caused cells to arrest in the G2/M phase of the cell cycle, we
suspected that these compounds interact with tubulin, possi-
bly at the colchicine site. Thus, the SAR observations suggest-
ed the presence of a deep hydrophobic pocket near the colchi-
[a] Dr. M. G. Ferlin
Department of Pharmaceutical Sciences, Faculty of Pharmacy
University of Padova, Via Marzolo 5, 35131 Padova (Italy)
Fax: (+39)0498275366
E-mail: mariagrazia.ferlin@unipd.it
[b] Dr. R. Bortolozzi, Prof. G. Basso, Dr. G. Viola
Department of Pediatrics, Laboratory of Oncohematology
University of Padova (Italy)
[c] Dr. P. Brun, Prof. I. Castagliuolo
Department of Histology, Microbiology and Medical Biotechnologies
University of Padova (Italy)
[d] Dr. E. Hamel
Screening Technologies Branch, Developmental Therapeutics Program, Divi-
sion of Cancer Treatment and Diagnosis, National Cancer Institute at Fred-
erick, National Institutes of Health, Frederick, Maryland 21702 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000180.
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M. G. Ferlin et al.
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mic cell lines. The most active compounds were confirmed as
inhibitors of tubulin assembly through colchicine site binding,
and were found to have major effects on cell-cycle distribution
and induction of apoptosis.
rylate derivatives 8–10 were subjected to thermal cyclization
(diphenyl ether, 2508C) to yield compounds 11–13.
Scheme 2 illustrates the synthesis of 1,3-disubstitued 7-
PPyQs 17, 21, and 22. Starting from the known 5-nitroindole
4,[3] carbaldehyde 14 was prepared by holding at reflux in DMF
Results and Discussion
Chemistry
The new PPyQs were synthesized as shown in Schemes 1–3,
generally beginning with an appropriate 5-nitroindole deriva-
tive.[1–3] Scheme 1 shows the synthesis of 3,7-disubstituted
Scheme 2. Synthesis of 7-phenylpyrroloquinolinone derivatives 17, 21, and
22. Reagents and conditions: a) POCl3, DMF, reflux, 3–5 h; b) and f) H2, Pd/C
(10%), EtOAc, 408C, 10–24 h; c) and g) ethyl benzoylacetate, EtOH, Drierite,
reflux, 24 h; d) and h) Ph2O, reflux, 30 min; e) NH2OH·HCl, pyridine, 808C, 2h,
SeO2, Na2SO4, DMF, 808C, 2.5 h; i) CH3COOH/H2SO4, H2O, reflux, 16 h.
Scheme 1. Synthesis of pyrroloquinolinone derivatives 11–13. Reagents and
conditions: a) tBuOK, benzene, 808C, 6 h; b) NaH, ethyl acrilate or trimeth-
oxybenzyl chloride, DMF, 708C, 4 h and 24–48 h, respectively; c) H2, Pd/C
(10%), EtOH, room temperature, 3 h; d) ethyl benzoylacetate or 1a, EtOH,
Drierite, reflux, 24 h; e) Ph2O, reflux, 30 min.
and phosphoryl chloride. Compound 14 was catalytically re-
duced (Pd/C 10%) to 1-methyl-5-aminoindole 15, which then
underwent condensation to yield 16. The final cyclization reac-
tion, as in Scheme 1, yielded 1-methyl-3-ethyl-7-PPyQ 17. Carb-
aldehyde 14 was also transformed into the corresponding 1-
carbonitrile derivative 18 in a one-pot reaction with hydroxyl-
amine hydrochloride, pyridine, selenium dioxide, and magnesi-
um sulfate,[5] followed by catalytic reduction to yield amine 19.
Compound 19 was condensed with ethyl benzoylacetate to
yield compound 20, which was thermally cyclized to 1-cyano-
3-ethyl-7-PPyQ (21). The latter was transformed into 1-carboxy-
amide-7-PPyQ 22 by treatment with a 1:1:1 mixture of acetic
acid/sulfuric acid/water.
PPyQs 11–13 by starting from commercial 5-nitroindole, which
was first N-alkylated on the pyrrole ring to furnish derivatives
2–4.[3] The 5-nitroindole was alkylated by ethyl acrylate, trime-
thoxybenzyl bromide or bromoethane in the presence of
sodium hydride in N,N-dimethylformamide (DMF). Intermedi-
ates 2–4 were catalytically reduced (Pd/C 10%, H2, ethanol) to
amines 5–7.[3] These, in turn, were condensed with ethyl ben-
zoylacetate or ethyl 3-oxo-3-(pyridin-3-yl)propanoate (1). We
prepared the latter by modifying a published procedure[4] that
furnished the b-ketoester 1 in good yield. This compound is
present in solution as an equilibrium mixture of the diketo 1a
and enolic 1b forms, as shown by the 1H NMR spectrum
([D6]DMSO). This showed two singlets at d 6.08 (CH) and 12.52
(OH), consistent with an enolic form of the compound. The ac-
The synthetic route to 8-PPyQs 33–37 is similar to that previ-
ously reported for the synthesis of 3-phenylquinolinones
(Scheme 3).[6] Commercial ethyl phenylacetates (R2 =H, m-me-
thoxy, or p-bromo) were formylated with ethyl formate in the
presence of sodium hydride[7] to afford intermediates 23[6]–25.
These were condensed with 5-aminoindoles 7,[2] 26,[3] and 27[3]
in ethanol at room temperature to yield enamine compounds
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Anti-leukemic 3H-Pyrrolo[3,2-f]quinolin-9-ones
Scheme 3. Synthesis of 8-phenylpyrroloquinolinone derivatives 33–37. Reagents and conditions: a) NaH, ethyl formate, room temperature, 24 h; b) EtOH, room
temperature, 10–15 h; c) Ph2O, reflux, 15 min.
28–32. The crude products consisted of a mixture of
the Z and E isomers, as observed by thin-layer chro-
1
matography and H NMR analysis. These compounds
were not separated but underwent thermal cycliza-
tion (diphenyl ether, 2508C) to 8-PPyQs 33–37.
Figure 2. Structures of compounds 38,[3] CA-4, and colchicine taken as references in the
assays of antiproliferative activity, inhibition of tubulin polymerization and colchicine
binding, respectively.
Biological evaluation
In vitro antiproliferative activities
35–37. Clearly, either removal of the side phenyl group from
position 7 or shifting it to the 8-position of the tricyclic nucleus
is deleterious for activity. Three of the new 7-PPyQ com-
pounds, 11–13, are active in all cell lines, while an additional
three (17, 21, and 22) have moderate activity only in the leuke-
mic lines. In fact, compound 38 showed GI50 values 10–100-
fold lower for leukemia cells (GI50: 0.5–2 nm) than for the solid
tumor cells (GI50: 0.01–0.06 mm). This difference is even more
dramatic for the two most active new compounds, 11 and 12,
for which the GI50 values toward the leukemia cells are 0.5–
10 nm and toward the solid tumor cells, 0.4–1.4 mm. Both of
These new compounds were evaluated for their in vitro anti-
proliferative activity against a panel of human tumor cell lines,
including both solid and leukemic lines. The previously de-
scribed[3] 3-ethyl-7-PPyQ (38) was used as a reference com-
pound (Figure 2). After continuous drug exposure for 72 h, the
compound concentration required for 50% growth inhibition
(GI50) was determined by the MTT colorimetric assay (Table 1).
Of the compounds listed, the reference compound 38 showed
nanomolar and sub-nanomolar GI50 values in all cell lines
tested, whereas its regioisomer 3-ethyl-8-PPyQ 34 was inactive
(GI50 >50 mm), as were the other 8-PPyQ derivatives 33 and
Table 1. In vitro inhibitory effects on cell proliferation by compounds 11–13, 17, 20, 21, 33–37, and 38.
Compd
GI50 [mm][a]
HL-60
HT-29
ARO
Hep G2
IGROV
MCF-7
HeLa
ML2
K562
RS 4;11
Jurkat
11
12
13
0.48Æ
0.08
0.45Æ
0.10
0.85Æ
0.14
>50
>50
>50
>50
>50
>50
>50
>50
0.03Æ
0.01
0.55Æ
0.15
0.58Æ
0.15
0.45Æ
0.07
>50
>50
>50
>50
>50
>50
>50
>50
0.03Æ
0.01
0.52Æ
0.10
0.95Æ
0.15
0.50Æ
0.05
5.8Æ1.1
>50
>50
>50
>50
>50
0.62Æ
0.15
0.85Æ
0.20
0.65Æ
0.15
>50
>50
>50
ND
ND
ND
ND
ND
0.06Æ
0.01
0.58Æ 0.45Æ0.10 0.002Æ0.0001 0.005Æ0.002 0.19Æ0.06
0.002Æ
0.0004
0.0005Æ
0.00001
0.003Æ0.0001
0.18
1.40Æ 0.40Æ0.05 0.002Æ0.0001 0.01Æ0.002 0.03Æ0.007
0.001Æ
0.50
0.0001
0.85Æ 0.40Æ0.05
0.03Æ0.002
0.3Æ0.04
0.24Æ0.03
0.04Æ0.007
0.05Æ0.004
0.15
17
21
22
33
34
35
36
37
38[3]
>50
>50
>50
ND
4.5Æ0.8
>50
>50
17.5Æ2.3
0.21Æ0.01
0.32Æ0.03
>50
14.4Æ2.3
0.55Æ0.03
0.09Æ0.02
>50
27.3Æ0.34
0.46Æ0.008
1.7Æ0.5
>50
6.5Æ1.0
0.13Æ0.02
0.03Æ0.002
ND
ND
ND
ND
ND
0.002Æ
0.0003
8.0Æ1.2
0.3Æ0.01
0.05Æ0.006
ND
ND
ND
ND
ND
0.0005Æ
0.00002
>50
ND
>50
>50
>50
>50
ND
>50
>50
>50
>50
>50
>50
0.05Æ
0.01
ND
>50
>50
>50
>50
ND
>50
>50
>50
>50
0.04Æ
0.01
0.01Æ
0.008
0.0005Æ
0.00002
0.001Æ
0.0003
0.001Æ
0.0001
[a] GI50: compound concentration required to inhibit tumor cell proliferation by 50%; data are expressed as the mean ÆSE from the dose–response curves
of at least three independent experiments; ND: not determined. See text for details of the cell lines examined; the first six cell lines listed are derived from
human solid tumors, the last five are from human leukemias.
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M. G. Ferlin et al.
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these compounds have more hydrophilic N-alkyl side chains
than other 7-PPyQs.
bly are in good agreement with the relative antiproliferative
activities of the compounds.
Of the three compounds bearing a substituent at the 1-posi-
tion, 21 and 22, with cyano and carboxyamide groups, respec-
tively, are completely inactive (GI50 >50 mm) in the solid tumor
cells, but show activity at sub-micromolar concentrations in
the leukemia lines. Compound 17, with a methyl group, has
only modest activity in the leukemia and HeLa cells. We con-
clude that a substituent at the 1-position does not abolish an-
tiproliferative activity, as do substituents at position 2.[2] This
finding indicates that suitable substituents at the 1-position of
7-PPyQs might result in compounds with high selectivity for
leukemic diseases.
In the colchicine binding studies, compounds 11 and 38
were roughly equal in activity, but are less potent than CA-4
despite their greater potency as inhibitors of assembly. Such
differences are commonly observed, and CA-4 is a particularly
potent inhibitor of colchicine binding.[9] Compounds 12 and
13 are much less active as inhibitors of colchicine binding than
are CA-4, 11, and 38, with convincing inhibition only observed
when the inhibitor concentration was increased to 50 mm, 10-
fold higher than the concentration of [3H]colchicine in the
reaction mixtures.
Finally, compound 13, with a pyridine ring in place of the
side phenyl group and an ethyl group at the N3 position, like
38, shows slightly less selectivity toward the leukemic lines
than compounds 38, 11, 12, 17, 21, and 22.
Cell-cycle analysis
The effects of various concentrations of compounds 11, 12, 13,
and 22 on cell-cycle progression were examined with Jurkat
leukemic T-lymphocytes (Figure 3). Untreated Jurkat cells
Inhibition of tubulin polymerization and colchicine binding
In a previous study,[3] we found that compound 38 and a
number of other 7-PpyQs cause cells to arrest at the G2/M
phase of the cell cycle. This finding suggests that the target of
these agents is likely to be tubulin, as this is a universal obser-
vation in cells treated with anti-tubulin agents. To determine
whether this is true, the most active compounds from the cur-
rent series, 11–13 and 22, together with an active agent from
the earlier studies (compound 38), were evaluated for their in
vitro inhibition of tubulin polymerization and for their inhibito-
ry effect on the binding of [3H]colchicine to tubulin
(Table 2).[8,9] For comparison, combretastatin A-4 (CA-4) was ex-
amined in simultaneous experiments.
In the assembly assay, compound 11 was found to be the
most active (IC50 =0.75 mm), and it is nearly twice as potent as
CA-4 (IC50 =1.2 mm). Compound 38 is also more inhibitory than
CA-4, whereas 12 and 13 are less inhibitory than CA-4. Com-
pound 22 showed only minimal inhibitory activity, with less
than 50% inhibition of tubulin assembly at the highest con-
centration (40 mm) examined. These effects on tubulin assem-
Figure 3. Effects of compounds A) 11, B) 12, C) 13, and D) 22 on cell cycle
&
*
distribution and G2/M arrest in Jurkat cells after 24 h incubation ( : G1,
:
~
G2/M, : S). Cells were treated with various concentrations of test com-
pounds (0.015–1.0 mm). The cells were then fixed and stained with propidi-
um iodide (PI) to analyze DNA content by flow cytometry. Data are ex-
pressed as the mean ÆSEM for two independent experiments.
showed a classical pattern of proliferation, with large numbers
of cells in the G1 (47.2%) and S (43.4%) phases, and fewer cells
in the G2/M (9.4%) phase. As in our earlier study,[3] the two
most active compounds, 11 and 12, showed a clear G2/M
arrest pattern in a concentration-dependent manner, with a
concomitant decrease of cells in the other phases of the cell
cycle after 24 h treatment. In particular, as shown in Figure 3,
the G2/M cell population increased from 9.4% in the control to
77% with compound 11 at 0.06 mm. The G1 cells decreased
from 47% in the control to 3%, while the S-phase cells de-
creased from 43 to 20%. Similar behavior was observed for
compound 12, but a similar arrest (65%) in G2/M phase re-
quired 0.25 mm compound 12. The other two compounds, 13
and 22, only induced G2/M arrest at yet higher concentrations,
in good agreement with their lower potency in the tubulin
Table 2. Inhibition of tubulin polymerization and colchicine binding by
compounds 11–13, 22, 38, and CA-4.
Compd
Tubulin Assembly[a]
Colchicine Binding[b]
Inhibition [%]
5 mm
IC50 [mm]
1 mm
50 mm
11
12
13
0.75Æ0.04
1.4Æ0.2
2.4Æ0.4
>40
44Æ6
ND
ND
75Æ4
30Æ4
ND
56Æ2
23Æ6
ND
65Æ0.3
ND
22
ND
38
CA-4
0.57Æ0.02
1.2Æ0.01
34Æ2
84Æ3
73Æ0.7
98Æ0.3
ND
ND
[a] Inhibition of tubulin polymerization; tubulin was present at 10 mm;
error values are ÆSD. [b] Inhibition of [3H]colchicine binding; tubulin and
colchicine were present at 1 and 5 mm, respectively, and the tested com-
pounds were present at 1, 5, or 50 mm, as indicated; ND: not determined.
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Anti-leukemic 3H-Pyrrolo[3,2-f]quinolin-9-ones
polymerization assay and their lower antiproliferative activity.
Further experiments showed that the accumulation of mitotic
cells occurs in a time-dependent manner associated with the
appearance of a hypodiploid peak (sub-G1) indicative of apop-
tosis (data not shown). This observation prompted us to inves-
tigate the possible activation of apoptotic mechanisms in the
presence of the most active derivatives.
gate the apoptotic machinery after treatment with compounds
11 and 12.
Apoptosis is mediated by mitochondrial depolarization. Mi-
tochondria play an essential role in the propagation of apopto-
sis.[12,13] It is well established that, at an early stage, apoptotic
stimuli alter the mitochondrial transmembrane potential
(DYmt). DYmt was monitored by the fluorescence of the dye
JC-1.[14] In the presence of normal cells (high DYmt), JC-1 forms
fluorescent red (l 590 nm) aggregates locally and spontane-
ously that are associated with a large shift in the emission;
upon mitochondrial membrane depolarization (low DYmt),
JC-1 forms monomers that emit at l 530 nm. Treated Jurkat
cells in the presence of derivatives 11 and 12 (0.5 and 0.1 mm)
exhibited a significant increase in the percentage of cells with
low DYmt in a concentration- and time-dependent manner rel-
ative to the control cells, indicating depolarization of the mito-
chondrial membrane potential (Figure 5A). The disruption of
DYmt is associated with the appearance of annexin V positivity
in the treated cells when they are in early apoptotic stage. In
fact, the dissipation of DYmt is characteristic of apoptosis and
has been observed with both microtubule destabilizing and
stabilizing agents in various cell types.[15]
Loss of plasma membrane asymmetry during apoptosis
To better characterize drug-induced apoptosis, we performed a
bi-parametric cytofluorimetric analysis using propidium iodide
(PI) and annexin V–FITC, which stain DNA and phosphatidylser-
ine (PS) residues, respectively.[10,11] Annexin V is a Ca2+-depen-
dent phospholipid binding protein with high affinity for PS.
Annexin V staining precedes the loss of membrane integrity
that accompanies the final stages of cell death resulting from
either apoptotic or necrotic processes. Because the externaliza-
tion of PS occurs in the earlier stages of apoptosis, annexin V
staining identifies apoptosis at an earlier stage than the ap-
pearance of sub-G1 cells. Such cells represent a later stage of
cell death involving nuclear changes such as DNA fragmenta-
tion.
Mitochondrial generation of ROS
After drug treatment at various concentrations and time in-
tervals, Jurkat cells were labeled with the two dyes, and the re-
sulting red (PI) and green (FITC) fluorescence was monitored
by flow cytometry. Compounds 11 and 12 provoked a remark-
able induction of apoptotic cells after 24 h treatment, whereas
the less active compounds 13 and 22 showed lower efficacy
(Figure 4). The percentage of annexin-V-positive cells increased
further at 48 h. These findings prompted us to further investi-
Mitochondrial membrane depolarization is associated with mi-
tochondrial production of reactive oxygen species (ROS).[16]
Therefore, we investigated whether ROS production increased
after treatment with compounds 11 and 12. We used the fluo-
rescence indicator hydroethidine (HE), the fluorescence of
which appears if ROS are generated.[17,18] HE is oxidized by the
superoxide anion into the ethidium ion, which emits red fluo-
rescence. Superoxide is produced by mitochondria due to a
shift from the normal four-electron reduction of O2 to a one-
electron reduction when cytochrome c is released from mito-
chondria. ROS generation was also measured with the dye 2,7-
dichlorodihydrofluorescein diacetate (H2-DCFDA), which is oxi-
dized to the fluorescent compound dichlorofluorescein (DCF)
by a variety of peroxides including hydrogen peroxide.[18] As
shown in Figure 5B,C, compounds 11 and 12, as expected, in-
duced the production of ROS relative to control cells, in agree-
ment with the dissipation of DYmt.
Caspase-3 activation, PARP cleavage, and Bcl-2 down-
regulation
Caspases are the central executioners of apoptosis mediated
by various inducers.[19,20] Caspases are synthesized as proen-
zymes, which are activated by cleavage. Caspases-2, -8, -9, and
-10 are termed apical and are usually the first to be stimulated
in the apoptotic process. These, in turn, activate effector cas-
pases, such as caspase-3 in particular.[21] Exposure of Jurkat
cells to compounds 11 or 12 was found to activate caspase-3,
as shown in Figure 6, in a time- and concentration-dependent
manner.
Figure 4. Flow cytometric analysis of apoptotic cells after treatment of
~
!
&
*
Jurkat cells with compounds 11 ( ), 12 ( ), 13 ( ), or 22 ( ). After treatment
for A) 24 or B) 48 h, cells were harvested and labeled with annexin V–FITC
and PI and then analyzed by flow cytometry. The data are expressed as the
mean percentage of annexin-V-positive cells ÆSEM for three independent
experiments.
Poly(ADP-ribose) polymerase (PARP) is a 116 kDa nuclear
protein that appears to be involved in apoptosis.[22] This pro-
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M. G. Ferlin et al.
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Figure 5. Assessment of mitochondrial dysfunction after treatment with compounds 11 or 12. A) Induction of loss of mitochondrial membrane potential in
Jurkat cells after 24 and 48 h incubation with 11 and 12 at 0.1 and 0.5 mm. Cells were stained with the fluorescent probe JC-1 and analyzed by flow cytome-
try. B) and C) Mitochondrial production of ROS in Jurkat cells after 24 and 48 h incubation with 11 and 12. Cells were stained with B) HE or C) H2-DCFDA and
analyzed by flow cytometry. The data are expressed as a percentage of the fluorescence intensity relative to untreated control and represent the mean
ÆSEM of three independent experiments.
tein is one of the main cleavage targets of caspase-3 both in
vitro and in vivo.[22] As illustrated in Figure 7A, immunoblot
Conclusions
analysis shows that the typical 89 kDa fragment of PARP in-
creased in treated cells with both compounds in a time-depen-
dent manner. Bcl-2 is a protein extensively investigated as a
modulating agent of apoptosis, and it plays a major role in in-
hibiting apoptosis. Bcl-2 regulates the mitochondrial mem-
brane potential and avoids the subsequent release of cyto-
chrome c to prevent caspase activation.[23,24] Therefore, we ex-
amined whether the induced apoptosis by 11 and 12 is associ-
ated with changes in Bcl-2 expression. As depicted in Fig-
ure 7B, flow cytometric analysis shows that both compounds
decrease Bcl-2 expression. Altogether, these data indicate that
the induction of apoptosis by these new derivatives is mediat-
ed through caspase-3 activation, PARP cleavage, and Bcl-2
down-regulation.
Several 7- and 8-phenylpyrroloquinolinone derivatives were
prepared and assayed for their antiproliferative activity on a
panel of solid tumor and leukemic cell lines. Depending on the
position and nature of substitutions, new SARs on PPyQs were
demonstrated that add to those already known. Among these,
the most surprising was the inactivity of the regioisomer
8-PPyQs 33–37 toward all cell lines. This indicates that the in-
teraction of the 2-phenylquinolin-4-one moiety in the colchi-
cine site of tubulin either requires the C7 phenyl group or is
abolished with a bulky C8 substituent.
Another important SAR is the finding that small hydrophilic
groups such as cyano and carboxyamide at position 1 of the 3-
ethyl-7-PPyQ 38 (compounds 21 and 22, respectively) retain
good antiproliferative activity, but only toward leukemic cells.
This should permit the future design of compounds specifically
directed against leukemias. Finally, introduction of a less hydro-
phobic side chain at the N3 or C7 positions of the PyQ nucleus
provided compounds 11–13, which maintain high activity and
1378
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ChemMedChem 2010, 5, 1373 – 1385

Anti-leukemic 3H-Pyrrolo[3,2-f]quinolin-9-ones
Experimental Section
Chemistry
Melting points were determined on a Gallenkamp MFB 595 010 M/
B capillary melting point apparatus and are not corrected. IR spec-
tra were recorded on a PerkinElmer 1760 FTIR spectrometer using
KBr pressed disks; all values are expressed in cmÀ1. UV/Vis spectra
were recorded on a PerkinElmer Lambda UV/Vis spectrometer.
1H NMR spectra were recorded on Bruker spectrometers (300 MHz)
with the indicated solvents; chemical shifts (d) are reported in
ppm downfield from (CH3)4Si as internal reference. Coupling con-
stants (J) are given in Hz. In the case of multiplets, d values were
measured by 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 Microanalyti-
cal Laboratory, Department of Pharmaceutical Sciences, University
of Padova (Italy), using a PerkinElmer elemental analyzer model
240B; results fell in the range of calculated values Æ0.4%. The ana-
lytical data are presented in detail for each final compound. Mass
spectra were obtained with a Mat 112 Varian Mat Bremen (70 eV)
mass spectrometer and an Applied Biosystems Mariner System
5220 LC–MS (nozzle potential: 250.00). Column flash chromatogra-
phy was performed on Merck silica gel (250–400 mesh ASTM); re-
actions were monitored by analytical thin-layer chromatography
(TLC) using Merck silica gel 60 F254 glass plates. Solutions were con-
centrated in a rotary evaporator under reduced pressure. Starting
materials were purchased from Aldrich Chimica and Acros, and sol-
vents from Carlo Erba, Fluka, and Lab-Scan. DMSO was obtained
anhydrous by distillation under vacuum and stored on molecular
sieves.
Figure 6. Caspase-3 activity induced by compounds 11 and 12. Jurkat cells
were incubated in the presence of A) 11 or B) 12 at 0.1 and 0.5 mm. After
treatment for 48 h, cells were harvested and stained with an anti-human
active caspase-3 fragment monoclonal antibody conjugated with FITC. Data
obtained by flow cytometry analysis are expressed as the percentage of cas-
pase-3 active fragment positive cells. Data are expressed as the mean
ÆSEM of three independent experiments.
Ethyl 3-oxo-3-(pyridin-3-yl)propanoate (1).[4] Reagent 1 was ob-
tained by reacting commercial ethyl nicotinate (2.71 mL,
19.8 mmol) with EtOAc (1.5 equiv) in the presence of tBuOK
(2 equiv) in excess benzene at 808C for 6 h. At the end of the reac-
tion, solvent was removed by evaporation, a mixture of ice and
H2O was added, and the mixture was extracted with Et2O after
neutralization with CH3COOH. The extract was washed with H2O,
dried on anhydrous Na2SO4 and evaporated under vacuum. A
light-yellow oil product was obtained that was purified by distilla-
tion. Yield 45%; Rf =0.52 (EtOAc/n-hexane 8:2); bp: 121–1238C
an antiproliferative profile similar to the reference compound
38, all of which have greater cytotoxicity against leukemia cells
than toward solid tumor cells.
In particular, compound 11 exhibits potent inhibition of tu-
bulin polymerization and is one of the most potent inhibitors
of colchicine binding (IC50 =0.76 mm for assembly, 44% inhibi-
tion of [3H]colchicine binding when equimolar with tubulin
and one fifth the concentration of colchicine in the reaction
mixture). Flow cytometry demonstrated that 11 has cellular ef-
fects typical for microtubule-interacting agents, causing accu-
mulation of cells in the G2/M phase of the cell cycle and in-
creased numbers of apoptotic sub-G1 cells. Moreover, com-
pound 11 is a potent inducer of multiple additional markers of
apoptosis in the Jurkat cell line. Apoptosis induced by multiple
antimitotic agents has been associated with alteration in a vari-
ety of cellular signaling pathways, and compound 11 had simi-
lar effects. This agent induced Bcl-2 down-regulation after 24 h
treatment. Because Bcl-2 prevents the initiation of the cellular
apoptotic program by stabilizing mitochondrial permeability,
its loss has multiple consequences. These include loss of DYmt,
which, in turn, results in an uncoupling of the respiratory chain
and mitochondrial efflux of caspase-9 and the apoptosis-induc-
ing factor (AIF). Thus, the proteolytic activation of caspase-3
occurred with subsequent PARP cleavage. Therefore, com-
pound 11, with its apparent selectivity toward leukemic cell
lines, merits further investigation as a potential chemothera-
peutic agent.
1
(0.4 mm); 1a: H NMR ([D6]DMSO): d=1.17 (t, 3H, J=7.1 Hz, CH3),
4.12 (q, 2H, J=7.1 Hz, CH2), 4.27 (s, 2H, CH2), 7.58 (ddd, 1H, J=0.8,
4.8, and 8.0 Hz, 5-H), 8.29 (dt, 1H, J=2.3 Hz, 8.01, 4-H), 8.76 (dd,
1H, J=4.7 and 6.5 Hz, 6-H), 9.12 (dd, 1H, J=0.6 and 2.3 Hz, 2-H);
1b: 6.08 (s, 1H, CH), 12.52 (s, 1H, OH).
Ethyl 3-(5-nitro-1H-indol-1-yl)propanoate (2). An amount corre-
sponding to 35 mmol NaH (60% dispersion in mineral oil) was
placed in a 100 mL round-bottomed flask and washed with toluene
(3ꢁ10 mL). A solution of commercial 5-nitroindole (12 mmol) in
dry toluene (60 mL) was dropped into the flask, and the initial
yellow color changed to red, with the formation of H2 gas. After
30 min at room temperature, a solution of ethyl acrylate (12 mmol)
in dry toluene (10 mL) was added, and the reaction mixture was
held at reflux for 4 h. Next, the reaction mixture was cooled to
08C, a few drops of absolute EtOH were added, the volatiles were
removed, and the residue was partitioned between H2O (15 mL)
and CHCl3 (60 mL). The organic extract was evaporated under
vacuum. Yellow–orange solid, yield 76%; mp: 65–668C; Rf =0.71
(eluent benzene/n-hexane/EtOAc 1:1:1); 1H NMR ([D6]DMSO): d=
1.08 (t, 3H, J=7.1 Hz, CH3), 2.88 (t, 2H, J=6.7 Hz, CH2), 3.99 (q, 2H,
J=7.1 Hz, CH2), 4.56 (t, 2H, J=6.7 Hz, CH2), 6.80 (d, 1H, J2,3
3.2 Hz, 2-H), 7.81 (m, 2H, 7-H, 3-H), 8.02 (dd, 1H, J4,6 =2.2 Hz, J6,7
=
=
ChemMedChem 2010, 5, 1373 – 1385
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M. G. Ferlin et al.
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Figure 7. Western blot analysis of PARP and flow cytometric analysis of Bcl-2 expression. A) Jurkat cells were treated with compounds 11 and 12 at the indi-
cated concentrations for 24 and 48 h. The cells were then subjected to immunoblot analysis as described in the Experimental Section. B) Cells were treated as
above, and after permeabilization were labeled with a Bcl-2 FITC-conjugated antibody. Shown are representative histograms after 24 h treatment with com-
pound 11 at the indicated concentrations. C) Bcl-2 expression in Jurkat cells after treatment with compounds 11 and 12 at the indicated concentrations for
24 and 48 h. Data are expressed as the mean ÆSEM of two independent experiments.
9.1 Hz, 6-H), 8.58 (d, 1H, J4,6 =2.2 Hz, 4-H); HRMS (ESI) calcd for
C13H15N2O4 [M+H]+ m/z 263.0987, found 263.1012.
General procedure for the synthesis of 5-aminoindole deriva-
tives 5–7.[3] A solution of a nitroindole 2–4[3] (5.87 mmol) in 400 mL
of EtOH was dropped into a suspension of 10% Pd/C (125 mg) sa-
turated with H2 in 200 mL EtOH. The mixture was stirred at room
temperature with hydrogen at atmospheric pressure for 3 h, and
the reaction was monitored by TLC analysis (EtOAc/n-hexane 1:3).
The catalyst was then removed by filtration, and the filtrate was
evaporated under reduced pressure to dryness to give the corre-
sponding aminoindole. The nearly pure residue (90% yield) was
used in the next reaction without purification.
1-(3,4,5-Trimethoxybenzyl)-5-nitro-1H-indole (3). An amount cor-
responding to 37 mmol NaH (60% dispersion in mineral oil) was
placed in
a three-necked 100 mL round-bottomed flask and
washed with toluene (3ꢁ10 mL). A solution of commercial 5-nitro-
indole (12.3 mmol) in 20–30 mL dry DMF was dropped into the
flask, and the initial yellow color changed to red, with the forma-
tion of H2 gas. After 30 min at room temperature, a solution of tri-
methoxybenzyl chloride (25 mmol) in dry DMF was added, and the
reaction mixture was left stirring for 20–48 h with monitoring of
the ongoing reaction by TLC analysis (eluent: toluene/n-hexane/
EtOAc 1:1:0.3). At the end, the solvent was evaporated, and the
residue was extracted with EtOAc. The organic phase, washed with
H2O and dried over anhydrous Na2SO4, was dried under vacuum.
Yield 98%; Rf =0.50 (benzene/n-hexane/EtOAc 1:1:1); mp: 158–
1608C; 1H NMR ([D6]DMSO): d=3.59 (s, 3H, 4’-OCH3), 3.69 (s, 6H,
3’- and 5’-OCH3), 5.41 (s, 2H, CH2), 6.65 (s, 2H, 2’- and 6’-H), 6.80 (d,
Ethyl 3-(5-amino-1H-indol-1-yl)propanoate (5). Brown dense
liquid; yield 98%; Rf =0.48 (benzene/n-hexane/EtOAc 1:1:1);
1H NMR ([D6]DMSO): d=1.12 (t, 3H, J=7.1 Hz, CH3), 2.75 (t, 2H, J=
6.7 Hz, CH2), 4.01 (q, 2H, J=7.1 Hz, CH2), 4.63 (t, 2H, J=6.7 Hz,
CH2), 5.18 (bs, 2H, NH2), 6.15 (dd, 1H, J3,2 =3.1 Hz, J3,4 =0.8 Hz, 3-H),
6.52 (dd, 1H, J6,7 =8.6 Hz, J6,4 =2.1 Hz, 6-H), 6.67 (dd, 1H, J4,6
2.1 Hz, J4,3 =0.6 Hz, 4-H), 7.16 (d, 1H, J7,6 =8.6 Hz, 7-H), 7.19 (d, 1H,
2,3 =3.0 Hz, 2-H).
=
J
1-(3,4,5-Trimethoxybenzyl)-5-amino-1H-indole (6). Reddish waxy
solid; yield 90%; Rf =0.58 (EtOAc/n-hexane 7:3); 1H NMR
([D6]DMSO): d=3.59 (s, 3H, 4’-OCH3), 3.69 (s, 6H, 3’- and 5’-OCH3),
4.40 (bs, 2H, NH2), 5.18 (s, 2H, CH2), 6.53 (s, 2H, 2’- and 6’-H), 6.64
1H, J2,3 =3.2 Hz, 2-H), 7.81 (m, 2H, 7-H and 3-H), 8.02 (dd, 1H, J4,6
=
2.2 Hz, J6,7 =9.1, 6-H), 8.58 (d, 1H, J4,6 =2.2 Hz, 4-H); HRMS (ESI)
calcd for C18H19N2O5 [M+H]+ m/z 343.1249, found 343.1364.
1380
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ChemMedChem 2010, 5, 1373 – 1385

Anti-leukemic 3H-Pyrrolo[3,2-f]quinolin-9-ones
(m, 3- and 7-H), 6.78 (d, 1H, J4,6 =2.1 Hz, 4-H), 7.22 (dd, 1H, J6,7
8.6 Hz, 6-H), 7.32 (d, 1H, J2,3 =3.2 Hz, 2-H).
=
found 361.1532; anal. calcd for C22H20N2O3: C 73.32, H 5.59, N 7.77,
found: C 73.56, H 5.42, N 7.43.
General procedure for the synthesis of ethyl 3-(1-substituted-in-
doleamino)-3-phenylacrylates (8–10). In a 50 mL round-bottomed
flask, 3–4 mmol 3-substituted aminoindole 5–7[2] in 10–20 mL abso-
lute EtOH were condensed with an equimolar quantity of commer-
cial ethyl benzoylcetate for 8 and 9 or ethyl 3-oxo-3-(pyridin-3-yl)-
propanoate for 10 with 1 mL glacial CH3COOH and 100 mg Drier-
ite. The mixture was held at reflux for ~24 h, the reaction being
monitored by TLC analysis (CH2Cl2/EtOAc 8:2). As the reaction did
not come to completion, after 24 h the mixture was cooled and fil-
tered to remove the Drierite. The resulting solution was evaporat-
ed to dryness under vacuum, and the residue was purified by flash
chromatography.
7-Phenyl-3-(3,4,5-trimethoxybenzyl)-3H-pyrrolo[3,2-f]quinolin-
9(6H)-one (12). Light-brown solid; yield 40%; mp: 249–2508C; Rf =
0.55 (eluent EtOAc/MeOH 9:1); 1H NMR (CD3OD): d=3.91 (s, 9H,
3ꢁOCH3), 5.70 (s, 2H, CH2), 6.60 (s, 2H, 2’- and 6’-H), 6.90 (s, 1H, 8-
H), 7.77 (m, 5H, ar), 7.92 (d, 1H, J1,2 =3.1 Hz, 2-H), 8.03 (m, 3H, 4-H,
2’’- and 6’’-H); 13C NMR (CD3OD): d=51.2, 56.5, 61.1, 105.1, 128.6,
130.7, 135.9, 154.8, 180.3; HRMS (ESI) calcd for C27H24N2O4 [M+H]+
m/z 441.1809, found 441.1668; anal. calcd for C27H24N2O4: C 73.62,
H 5.49, N 6.36, found: C 73.81, H 5.39, N 6.15.
3-Ethyl-7-(pyridin-3-yl)-3H-pyrrolo[3,2-f]quinolin-9(6H)-one (13).
Beige solid; yield 35%; mp: 278–2808C (dec.) (MeOH); Rf =0.46
(eluent EtOAc/MeOH 8:2); 1H NMR (CD3OD): d=1.38 (t, 3H, CH3),
4.25 (q, 2H, CH2), 6.56 (bs, 1H, 8-H), 7.35 (d, 1H, J1,2 =2.9 Hz, 1-H),
7.44 (d, 1H, J4,5 =9.0 Hz, 5-H), 7.53 (m, 2H, 2- and 5’-H), 7.79 (d, 1H,
Ethyl 3-[1-(3-ethoxy-3-oxopropyl)-1H-indol-5-ylamino]-3-phenyl-
acrylate (8). Brown dense liquid; yield 68%; Rf =0.83 (CH2Cl2/EtOAc
8:2); 1H NMR ([D6]DMSO): d=1.11 (t, 3H, J=7.1 Hz, CH3), 1.16 (t,
3H, J=7.1 Hz, CH3), 2.80 (t, 2H, J=6.7 Hz, CH2), 4.01 (q, 2H, J=
7.1 Hz, CH2), 4.13 (q, 2H, J=7.1 Hz, CH2), 4.38 (t, 2H, J=6.7 Hz,
CH2), 4.83 (s, 1H, CH), 6.20 (d, 1H, J3,2 =3.1 Hz, 3-H), 6.60 (dd, 1H,
J
4,5 =9.0 Hz, 4-H), 8.14 (m, 1H, 6’-H), 8.60 (dd, 1H, J=1.5 and
4.9 Hz, 4’-H), 8.88 (d, 1H, J=2.3 Hz, 2’-H); 13C NMR (CD3OD): d=
16.5, 42.2, 105.7, 117.5, 125.5, 129.5, 133.3, 148.7, 151.7, 182.9;
HRMS (ESI) calcd for C18H15N3O [M+H]+ m/z 290.1288, found
290.1385; anal. calcd for C18H15N3O: C 74.72, H 5.23, N 14.52,
found: C 74.95, H 5.36; 14.11
J
6,7 =8.8 Hz, J6,4 =2.1 Hz, 6-H), 6.97 (d, 1H, J4,6 =2.1 Hz, 4-H), 7.30
(m, 7H, ar), 10.25 (s, 1H, NH); HRMS (ESI) calcd for C24H27N2O4
[M+H]+ m/z 407.1926, found 407.1997.
1-Ethyl-5-nitro-1H-indole-3-carbaldehyde (14). A solution of 1-
ethyl-5-nitro-1H-indole 4[3] (1.5634 g, 8.22 mmol) in DMF was drop-
ped into a mixture of 2 mL POCl3 and 3 mL DMF cooled at 108C.
The temperature was raised to 35–408C with stirring, and the reac-
tion continued until the starting material was no longer detectable
by TLC. The volatile products were removed in vacuo, and the resi-
due was diluted with a mixture of H2O and ice, followed by prior
basification with 6% NaOH. A heavy precipitate formed, which was
removed by filtration and washed with a mixture of H2O and
EtOAc. The organic phase of the filtrate was separated and evapo-
rated to give a yellow residue (1.0052 g). The precipitate was resus-
pended in EtOAc, and the mixture was stirred overnight. It was
then filtered, and the EtOAc solution was dried under vacuum to
give another 0.512 g of product. Light-yellow solid; yield 87%; mp:
178–1808C; Rf =0.46 (EtOAc/n-hexane 7:3); 1H NMR ([D6]DMSO):
d=1.44 (t, 3H, J=7.1 Hz, CH3), 4.40 (q, 2H, J=7.1 Hz, CH2), 7.90 (d,
1H, J6,7 =9.1 Hz, 7-H), 8.20 (dd, 1H, J4,6 =2.2 and J6,7 =9.1 Hz, 6-H),
8.65 (s 1H, 2-H), 8.95 (d, 1H, J4,6 =2.1 Hz, 4-H), 10.00 (s, 1H, HCO);
HRMS (ESI) calcd for C11H11N2O3 [M+H]+ m/z 219.0725, found
219.0757.
Ethyl 3-phenyl-3-[1-(3,4,5-trimethoxybenzyl)-1H-indol-5-ylami-
no]acrylate (9). Dark-green liquid; yield 70%; Rf =0.96 (CH2Cl2/
EtOAc 8:2); 1H NMR ([D6]DMSO): d=1.23 (t, 3H, J=7.1 Hz, CH3),
3.59 (s, 3H, 4’-OCH3), 3.69 (s, 6H, 3’- and 5’-OCH3), 4.12 (q, 2H, J=
7.1 Hz, CH2), 4.83 (s,1H, CH), 5.18 (s, 2H, CH2), 6.20 (d, 1H, J3,2
=
3.1 Hz, 3-H), 6.60 (dd, 1H, J6,7 =8.8 Hz, J6,4 =2.1 Hz, 6-H), 6.53 (s, 2H,
2’- and 6’-H), 6.97 (d, 1H, J4,6 =2.1 Hz, 4-H), 7.30 (m, 7H, ar), 10.20
(s, 1H, NH); HRMS (ESI) calcd for C29H31N2O5 [M+H]+ m/z 487.2188,
found 487.2410.
Ethyl 3-(1-ethyl-1H-indol-5-ylamino)-3-(pyridin-3-yl)acrylate (10).
Brown dense liquid; yield 40%; Rf =0.75 (EtOAc/n-hexane 8:2);
1H NMR ([D6]DMSO): d=1.23 (t, 3H, J=7.3 Hz, CH3), 1.29 (t, 3H,
CH3), 4.11 (m, 4H, J=7.1 Hz, 2ꢁCH2), 4.90 (s, 1H, CH), 6.22 (dd, 1H,
J
3,2 =3.1 and J3,4 =0,6 Hz, 3-H), 6.62 (dd, 1H, J6,7 =8.8 and J6,4 =
2.01 Hz, 6-H), 6.97 (d, 1H, J4,6 =2.0 Hz, 4-H), 7.31 (m, 4H, ar), 7.40
(d, 1H, J3,2 =3.1 Hz, 2-H), 7.46 (d, 1H, J6,7 =8.8 Hz, 7-H), 10.35 (s, 1H,
NH); HRMS (ESI) calcd for C20H22N3O2 [M+H]+ m/z 336.1667, found
336.1749.
General procedure for the synthesis of 3-substituted 7-phenyl-
and 7-pyridin-3H-pyrrolo[3,2-f]quinolin-9-(6H)ones 11–13. In a
two-necked round-bottomed flask, 50 mL Ph2O was heated at boil-
ing point; 1–2 mmol of acrylates 8–10 were then added portion-
wise, and the mixture was held at reflux for 30 min. After cooling
to 608C, the separated precipitate was collected by filtration and
washed several times with Et2O. In all cases, the collected products
were purified by flash chromatography (EtOAc/MeOH 8:2) or re-
crystallized from a suitable solvent.
1-Ethyl-3-methyl-1H-indole-5-amine (15). As with the aminoin-
dole compounds 5–7, the nitroindole carbaldehyde 14 was catalyt-
ically reduced, at 40–508C for 10–14 h, to give a dense, red liquid
product. Yield 77%; Rf =0.74 (EtOAc/n-hexane 8:2); 1H NMR
([D6]DMSO): d=1.05 (t, 3H, CH3), 2.11 (s, 3H, CH3), 3.99 (q, 2H,
CH2), 4.35 (bs, 2H, NH2), 6.49 (dd, 1H, J6,7 =8.2 and J6,4 =2.3 Hz, 6-
H), 6.60 (d, 1H, J6,4 =2.3, 4-H), 6.91 (s, 1H, 2-H), 7.07 (bd, 1H, J6,7
=
8.2 Hz, 7-H).
Ethyl 3-(9-oxo-7-phenyl-6H-pyrrolo[3,2-f]quinolin-3(9H)-yl)propa-
noate (11). Yellow solid; yield 45%; mp: 202–2048C; Rf =0.60
(EtOAc/MeOH 9:1); mp: 249–2508C (dec.); 1H NMR (CD3OD): d=
1.17 (t, 3H, CH3), 2.91 (d, 2H, CH2), 4.09 (q, 2H, CH2), 4.64 (d, 2H,
CH2), 6.69 (s, 1H, 8-H), 7.46 (d, 1H, J1,2 =3.1 Hz, 1-H), 7.59 (m, 4H,
ar), 7.67 (d, 1H, J1,2 =3.1 Hz, 2-H) 7.82 (m, 2H, 2’- and 6’-H), 7.93 (d,
1H, J4,5 =8.9 Hz, 4-H); 13C NMR (CD3OD): d=14.8, 36.7, 43.5, 62.3,
105.9, 109.8, 113.6, 118.3, 126.1, 128.1, 130.4, 131.8, 136.4, 173.3,
180.2; HRMS (ESI) calcd for C22H20N2O3 [M+H]+ m/z 361.1547,
Ethyl 3-(1-ethyl-3-methyl-1H-indol-5-ylamino)-3-phenylacrylate
(16). As with the acrylate compounds 8–10, aminoindole 15 was
condensed with ethyl benzoylacetate to give a brown liquid prod-
uct. Yield 56%; Rf =0.91 (CH2Cl2/EtOAc 8:2); 1H NMR ([D6]DMSO):
d=1.23 (t, 3H, J=7.3 Hz, CH3), 1.29 (t, 3H, CH3), 2.03 (s, 3H, CH3),
4.11 (m, 4H, J=7.1 Hz, 2ꢁCH2), 4.83 (s, 1H, CH), 6.54 (dd, 1H, J6,7
=
8.7 and J6,4 =1.8 Hz, 6-H), 6.87 (d, 1H, J4,6 =1.9 Hz, 4-H), 7.16 (d, 1H,
J
7,6 =8.8 Hz, 7-H), 7.31 (m, 6H, ar), 10.25 (s, 1H, NH); HRMS (ESI)
calcd for C22H25N2O2 [M+H]+ m/z 349.1871, found 349.2023.
ChemMedChem 2010, 5, 1373 – 1385
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3-Ethyl-1-methyl-7-phenyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one
(17). As with the pyrroloquinolinone derivatives 11–13, the acrylate
16 was cyclized to give a light-brown solid product. Yield 30%;
compound 21 (0.60 mmol). The mixture was heated at 708C for
16 h. At the end (followed by TLC, EtOAc/MeOH 8:2), the reaction
was stopped by adding a mixture of H2O and ice and left at 48C
for 4 h. The formed precipitate was collected, washed with H2O,
and dried to yield a brown solid. Yield 37%; mp: 223–2258C; Rf =
0.56 (EtOAc/MeOH 9:1, light-blue fluorescent spot at l 365 nm);
1H NMR (CD3OD): d=1.53 (t, 3H, J=7.3 Hz, CH3), 4.43 (q, 2H, J=
7.3 Hz, CH2), 6.91 (s, 1H, 2-H), 7.61 (d, 1H, J4,5 =9.1 Hz, 5-H), 7.82
(m, 3H, 3’-, 4’- and 5’-H), 8.11 (d, 1H, 6-H), 8.22 (s, 1H, 8-H); HRMS
(ESI) calcd for C20H17N3O2 [M+H]+ m/z 332.1541, found 332.1444;
anal. calcd for C20H17N3O2: C 72.49, H 5.17, N 12.68, found: C 72.61,
H 4.92, N 12.29.
1
Rf =0.77 (EtOAc/MeOH 8:2); mp: 163–1658C; H NMR (CD3OD): d=
1.58 (t, 3H, J=7.3 Hz, CH3), 2.92 (s, 3H, CH3), 4.31 (q, 2H, J=7.3 Hz,
CH2), 6.73 (s, 1H, 2-H), 7.33 (bs, 1H, 8-H), 7.56 (d, 1H, J=8.9 Hz, 5-
H), 7.64 (m, 3H, 3’-, 4’- and 5’-H), 7.85 (m, 3H, 4-, 2’- and 6’-H);
13C NMR (CD3OD): d=12.3, 16.2, 42.3, 105.6, 125.6, 129.5, 148.8,
180.0; HRMS (ESI) calcd for C20H18N2O [M+H]+ m/z 303.1492, found
303.1722; anal. calcd for C20H18N2O: C 79.44, H 6.00, N 9.26, found:
C 79.61, H 6.23, N 9.01.
1-Ethyl-5-nitro-1H-indole-3-carbonitrile (18). In a round-bottomed
flask, 5-nitroindolo-3-carbaldehyde 14 (1.539 g, 7.05 mmol) was dis-
solved in 5 mL anhydrous DMF. To this was added 0.514 g
(7.4 mmol) NH2OH·HCl and 0.57 mL (7.4 mmol) anhydrous pyridine.
The reaction mixture was stirred at 808C under N2 for ~2 h, until
the starting material disappeared and another spot with higher Rf
formed (TLC, EtOAc/n-hexane 7:3). At this point, 0.821 g of SeO2
and 0.400 g of MgSO4 were added to the hot reaction mixture. Stir-
ring continued at 808C for another 2.5 h. The reaction mixture was
cooled and filtered, and the filtered solution was evaporated to
dryness under vacuum. The residue was resuspended in cold H2O,
and the suspension was re-filtered. The collected yellow solid was
washed several times with H2O and finally dried. Yield 60%; Rf =
0.53 (EtOAc/n-hexane 7:3); mp: 158–1618C; 1H NMR ([D6]DMSO):
d=1.41 (t, 3H, J=7.1 Hz, CH3), 4.38 (q, 2H, J=7.1 Hz, CH2), 7.96 (d,
1H, J6,7 =9.1 Hz, 7-H), 8.20 (dd, 1H, J4,6 =2.2 and J6,7 =9.1 Hz, 6-H),
8.51 (d, 1H, J4,6 =2.1 and J6,7 =9.1 Hz, 4-H), 8.64 (s, 1H, 2-H); HRMS
(ESI) calcd for C11H10N3O2 [M+H]+ m/z 216.0728, found 216.0783.
General procedure for the synthesis of ethyl 3-oxo-2-phenylpro-
panoate derivatives (23[7]–25). NaH (60% in oil, 4 equiv in mmol)
was added portionwise to a mixture of commercial ethyl phenyla-
cetate, m-methoxyphenylacetate or p-bromophenylacetate (3.1–
6.1 mmol) and ethyl formate (61.4–121.3 mmol). This mixture was
stirred at room temperature under N2 for 24 h (TLC, CHCl3/MeOH
9.9:0.1), then poured into a mixture of ice and H2O, acidified with
HCl, and extracted with EtOAc. The extract was dried over anhy-
drous Na2SO4 and concentrated under reduced pressure to yield
colored oils.
Ethyl 3-oxo-2-(m-methoxyphenyl)propanoate (24). Orange oil;
1
yield 87%; Rf =0.84 (EtOAc/n-hexane 8:2); H NMR ([D6]DMSO): d=
1.18 (t, 3H, J=7.1 Hz, CH3), 3.72 (s, 1H, OCH3), 4.09 (q, 2H, J=
7.1 Hz, CH2), 6.78 (dd, 1H, J=2.7 and 8.1, 4-H), 6.85 (m, 2H, 2- and
6-H), 7.21 (dd, 1H J=8.1, 5-H),7.84 (s, 1H, CH), 10.95 (bs, 1H, CHO);
HRMS (ESI) calcd for C12H15O4 [M+H]+ m/z 223.0926, found
223.0939.
5-Amino-1-ethyl-1H-indole-3-carbonitrile (19). As with the amino-
indole compounds 5–7 and 15, the nitroindole carbonitrile 18 was
catalytically reduced at 40–508C for 10–24 h to give a yellow semi-
solid product. Yield 98%; Rf =0.66 (EtOAc/n-hexane 8:2); 1H NMR
([D6]DMSO): d=1.36 (t, 3H, J=7.2 Hz, CH3), 4.15 (q, 2H, J=7.2 Hz,
CH2), 4.96 (bs, 2H, NH2), 6.65 (dd, 1H, J4,6 =1.9 and J6,7 =8.7 Hz, 6-
H), 6.72 (d, 1H, J6,4 =1.9 Hz, 4-H), 7.35 (d, 1H, J6,7 =8.7 Hz, 7-H), 8.02
(s, 1H, HC-2); HRMS (ESI) calcd for C11H12N3 [M+H]+ m/z 186.0987,
found 186.1047.
Ethyl 3-oxo-2-(p-bromophenyl)propanoate (25). Yellow oil; yield
67%; Rf =0.86 (EtOAc/n-hexane 8:2); H NMR ([D6]DMSO): d=1.18
(t, 3H, J=7.1 Hz, CH3), 4.10 (q, 2H, J=7.1 Hz, CH2), 7.26 (d, 1H, J=
8.4 Hz, 5- and 6-H), 7.49 (d, 1H, J=8.6 Hz, 2- and 3-H), 7.87 (s, 1H,
CH), 11.194 (bs, 1H, CHO); HRMS (ESI) calcd for C11H11O3Br [M+H]+
m/z 272.9949, found 270.9987 and 272.9994.
1
General procedure for the synthesis of 3-(1H-indol-5-ylamino-2-
phenylpropanoate derivatives (28–32). A solution of aminoindole
(7,[2] 26,[3] or 27[3]) (1.9–2.2 mmol) in 20 mL absolute EtOH was
added to aldehyde compounds (23,[7] 24, or 25) in equimolar ratio
dissolved in a minimal quantity of EtOH. Drierite (100 mg) was
added to the mixture, and the mixture was stirred at room temper-
ature for 10–15 h. At the end (TLC, CHCl3/MeOH 9:1) the reaction
mixture was filtered, and the filtrate was concentrated under
vacuum to give semi-solid products.
Ethyl
3-(3-cyano-1-ethyl-1H-indol-5-ylamino)-3-phenylacrylate
(20). As with the acrylate compounds 8–10 and 16, aminoindole
19 was condensed with benzoylacetate to give a dense yellow
liquid. Yield 52%; Rf =0.84 (EtOAc/n-hexane 8:2); 1H NMR
([D6]DMSO): d=1.23 (t, 3H, J=7.3 Hz, CH3), 1.29 (t, 3H, CH3), 4.11
(m, 4H, J=7.1 Hz, 2ꢁCH2), 4.92 (s, 1H, CH), 6.67 (dd, 1H, J6,7 =8.7
and J6,4 =2.0 Hz, 6-H), 6.95 (d, 1H, J4,6 =1.9 Hz, 4-H), 7.35 (d, 1H,
J
7,6 =8.7 Hz, 7-H), 7.31 (m, 6H, ar), 10.25 (s, 1H, NH).
Ethyl 3-(1H-indol-5-ylimino)-2-phenylpropanoate (28). Dark oil;
yield 57%; Rf =0.46 and 0.83 (EtOAc/n-hexane 8:2);
1H NMR([D6]DMSO): d=1.28 (t, 3H, J=7.2 Hz, CH3), 4.14 (q, 2H, J=
7.2 Hz, CH2), 7.44–7.18 (m, 10H, ar), 7.64 (d, 1H, J=13.0 Hz, =CH),
8.05 (d, 1H, J=13.9 Hz, CH), 8.43 (d, 1H, J=13.6 Hz, CH), 10.34 (d,
1H, J=13.0 Hz, =CH), 11.04 (bs, 1H, NH); HRMS (ESI) calcd for
C19H19N2O2 [M+H]+ m/z 307.1402, found 307.1444.
3-Ethyl-9-oxo-7-phenyl-6,9-dihydro-3H-pyrrolo[3,2-f]quinoline-1-
carbonitrile (21). As with the pyrroloquinolinone derivatives 8–10
and 17, the acrylate 20 was cyclized to give a brown solid. Yield
24%; mp: 2098C; Rf =0.33 (EtOAc/MeOH 9:1, light-blue fluorescent
spot at l 365 nm); 1H NMR (CD3OD): d=1.63 (t, 3H, J=7.3 Hz,
CH3), 4.55 (q, 2H, J=7.3 Hz, CH2), 6.76 (s, 1H, 2-H), 7.70 (m, 3H, 3’-,
4’- and 5’-H), 7.84 (d, 1H, J4,5 =8.9 Hz, 4-H), 7.93 (m, 2H, 2’- and 6’-
H), 8.11 (d, 1H, J4,5 =9.1 Hz, 5-H), 8.31 (s, 1H, 2-H); 13C NMR
(CD3OD): d=15.9 (CH3), 43.4 (CH2), 89.9, 118.8 (CN), 128.8 (C-2’,
C-3’, C-5’, C-6’); HRMS (ESI) calcd for C20H15N3O2 [M+H]+ m/z
314.1288, found 314.1301; anal. calcd for C20H15N3O: C 76.66, H
4.82, N 13.41, found: 76.94, H 4.53, N 13.11.
Ethyl 3-(1-ethyl-1H-indol-5-ylimino)-2-phenylpropanoate (29).
Black oil; yield 74%; Rf =0.30 and 0.85 (EtOAc/n-hexane 8:2);
1H NMR ([D6]DMSO): d=1.20 (m, 6H, J=7.2 Hz, 2ꢁCH3), 4.14 (m,
6H, J=7.2 Hz, 2ꢁCH2), 6.34 (t, 2H, J=2.5 Hz, 1- and 2-H), 7.27–
7.45 (m, 8H, ar), 7.65 (d, 1H, J=12.9 Hz, =CH), 8.05 (d, 1H, J=
13.5 Hz, NH), 8.45 (d, 1H, J=13.5 Hz, NH), 10.35 (d, 1H, J=12.9 Hz,
=CH); HRMS (ESI) calcd for C21H23N2O2 [M+H]+ m/z 335.1715,
found 335.1707.
3-Ethyl-9-oxo-7-phenyl-6,9-dihydro-3H-pyrrolo[3,2-f]quinoline-1-
caboxamide (22). CH3COOH, H2SO4, and H2O (2 mL each) were
added in sequence to a 100 mL flask containing 0.188 g cyano
1382
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ChemMedChem 2010, 5, 1373 – 1385

Anti-leukemic 3H-Pyrrolo[3,2-f]quinolin-9-ones
Ethyl 3-(1-cyclopropylmethyl-1H-indol-5-ylimino)-2-phenylpropa-
noate (30). Black liquid; yield 95%; Rf =0.58 and 0.92 (EtOAc/
n-hexane 8:2); H NMR ([D6]DMSO): d=0.33 (m, 4H, 2ꢁCH2), 0.47
(m, 4H, 2ꢁCH2), 1.30–1.90 (m, 12H, 2ꢁCH), 4.15–4.41 (m, 6H, 2ꢁ
CH2), 7.14–7.47 (m, 10H, ar), 8.06 (d, 1H, J=13.0 Hz, =CH), 8.40 (d,
1H, J=13.7 Hz, NH), 8.60 (d, 1H, J=13.7 Hz, NH), 10.33 (d, 1H, J=
13.0 Hz, =CH).
7.62 (dd, 1H, J=3.6 Hz, 1-H), 7.78 (d, 2H, J=1.3 Hz, 5’ and 3’-H),
7.91 (d, 1H, J=8.8 Hz, 4-H), 8.07 (d, 1H, J=6.3 Hz, 7-H), 12.0 (d,
1H, J=6.10 Hz, NH); 13C NMR ([D6]DMSO): d=4.1 (2ꢁCH2), 12.2
(CH), 50.2 (CH2), 100.4, 104.1, 110.1, 114.8, 115.9, 118.9, 120.6, 124.0,
126.4, 128.1, 128.5, 128.9, 128.9, 130.4, 135.5, 137.3, 175.9 (CO);
HRMS (ESI) calcd for C21H20N2O [M+H]+ m/z 315.1445, found
315.1422; anal. calcd for C21H19N2O: C 80.23, H 5.77, N 8.91, found:
C 79.91, H 5.46, N 8.81.
1
Ethyl 3-(1-ethyl-1H-indol-5-ylimino)-2-(3-methoxyphenyl)propa-
noate (31). Black liquid; yield 75%; mp: 249–2508C; Rf =0.64 and
0.85 (EtOAc/n-hexane 9:1); 1H NMR ([D6]DMSO): d=1.33 (m, 6H,
2ꢁCH3), 4.14 (m, 4H, 2ꢁCH2), 6.82–7.45 (m, 9H, ar), 7.66 (d, 1H,
J=13.2 Hz, =CH), 8.04 (d, 1H, J=13.6 H z, CH), 8.45 (d, 1H, J=
13.9 Hz, CH), 10.36 (d, 1H, J=13.2 Hz, =CH); HRMS (ESI) calcd for
C22H25N2O3 [M+H]+ m/z 365.1820, found 365.1796.
3-Ethyl-8-(3-methoxyphenyl)-3H-pyrrolo[3,2-f]quinolin-9(6H)-one
(36). Light-yellow solid; yield 60%; mp: 262–2648C (MeOH); Rf =
0.65 (EtOAc/n-hexane 9:1, violet fluorescent spot at l 365 nm);
1H NMR ([D6]DMSO): d=1.40 (t, 3H, J=7.2 Hz, CH3), 3.8 (s, 3H,
OCH3), 4.32 (q, 2H, J=7.2 Hz, CH2), 6.85 (m, 1H, J=1.5 Hz, 4’-H),
7.28 (d, 1H, J=8.3 Hz, 5-H), 7.33 (d, 1H, J=1.9 Hz, 6’-H), 7.36 (d,
1H, J=1.9 Hz, 5’-H), 7.43 (m, 1H, J=1.5 Hz, 2’-H), 7.45 (d, 1H, J=
2.9 Hz, 1-H), 7.61 (d, 1H, J=2.9 Hz, 2-H), 7.88 (dd, 1H, J=8.3 and
0.8 Hz, 4-H), 8.09 (s, 1H, 7-H), 12.00 (bs, 1H, NH); 13C NMR
([D6]DMSO): d=16.26 (CH3), 40.9 (CH2), 55.4 (OCH3), 104.1, 111.9,
111.9, 114.6, 115.8, 120.3, 121.2, 124.0, 129.0, 129.1, 131.7, 135.4,
135.8, 138.6, 159.2, 175.8 (CO); HRMS (ESI) calcd for C20H19N2O2
[M+H]+ m/z 333.1511, found 333.1516; anal. calcd for C20H18N2O2:
C 75.45, H 5.70, N 8.80, found: C 75.18, H 5.35, N 8.71.
Ethyl
2-(4-Bromophenyl)-3-(1-ethyl-1H-indol-5-ylimino)propa-
noate (32). Black oil; yield 73.93%; Rf =0.52 and 0.92 (EtOAc/
1
n-hexane 8:2); H NMR ([D6]DMSO): d=1.30 (m, 6H, 2ꢁCH3), 4.17
(m, 4H, 2ꢁCH2), 7.57–7.07 (m, 9H, ar), 7.69 (d, 1H, J=13.2 Hz, =
CH), 8.05 (d, 1H, J=13.6 Hz, NH), 8.65 (d, 1H, J=13.6 Hz, NH),
10.38 (d, 1H, J=13.2 Hz, =CH); HRMS (ESI) calcd for C21H22N2O2Br
[M+H]+ m/z 414.0766, found 413.0823 and 415.0799.
General procedure for the synthesis of 8-phenyl-pyrrolo[3,2-
f]quinolinones (33–37). In a two-necked round-bottomed flask,
Ph2O (30 mL) was heated at boiling point; 1–2 mmol of propa-
noates 28–32 were then added in portions, and the mixture was
held at reflux for 15 min. After cooling to 308C, the light precipi-
tate slowly separated and was then collected by filtration and
washed several times with Et2O. In all cases, the collected products
were purified by recrystallization from a suitable solvent.
8-(4-Bromophenyl)-3-ethyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one
(37). Grey solid; yield 50%; mp: 216–2188C (MeOH/EtOH 80:20);
Rf =0.65 (EtOAc/n-hexane 9:1, violet fluorescent spot at l 365 nm);
1H NMR ([D6]DMSO): d=1.40 (t, 3H, J=7.2 Hz, CH3), 4.32 (q, 2H, J=
7.2 Hz, CH2), 7.34 (d, 1H, J=3.2 Hz, 1-H), 7.36 (d, 1H, J=3.1 Hz,
2-H), 7.56 (dd, 2H, J=8.6 and J=2.0 Hz, 2’- and 6’-H), 7.60 (d, 1H,
J
5,4 =8.4 Hz, 5-H), 7.81 (dd, 2H, J=8.6 and 1.97 Hz, 3’and 5’-H), 7.89
(dd, 1H, J5,4 =8.4 and J4,1 =0.6 Hz, 4-H), 8.14 (s, 1H, 7-H), 12.07 (bs,
1H, NH); 13C NMR ([D6]DMSO): d=16.3 (CH3), 40.9 (CH2), 100.3,
110.3, 114.9, 118.9, 119.2, 119.3, 123.8, 128.5, 130.4, 130.8, 130.9,
132.3, 136.4, 153.8, 175.7 (CO); HRMS (ESI) calcd for C19H16BrN2O
[M+H]+ m/z 383.0538, found 332.0870; anal. calcd for C19H15BrN2O:
C 62.14, H 4.12, N 7.63, found: C 62.01, H 3.89, N 7.51.
8-Phenyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one (33). Light-brown
solid; yield 87.98%; mp: 178–1908C (MeOH); Rf =0.28 (EtOAc/
n-hexane 8:1, violet fluorescent spot at l 365 nm); 1H NMR
([D6]DMSO): d=7.25 (dd, 1H, J=7.4 Hz, 4’-H), 7.29 (dd, 1H, J=
2.7 Hz, 1-H), 7.39 (d, 2H, J=7.4 Hz, 2’- and 6’-H), 7.45 (dd, 1H, J=
2.7 Hz, 2-H), 7.61 (dd, 2H, J=8.4 Hz, 4-H and 5-H), 7.76 (d, 2H, J=
7.4 Hz, 3’-H and 5’-H), 8.06 (d, 1H, J=6.1 Hz, 7-H), 11.50 (bs, 1H,
NH), 11.98 (d, 1H, J=5.7 Hz, NH); 13C NMR ([D6]DMSO): d=104.8,
111.9, 117.5, 119.2, 120.5, 123.5, 125.6, 126.3, 128.0, 128.9, 131.8,
135.5, 135.6, 137.3, 175 (CO); HRMS (ESI) calcd for C17H13N2O
[M+H]+ m/z 261.0975, found 261.1272; anal. calcd for C17H12N2O: C
78.44, H 4.65, N 10.76, found: C 78.31, H 4.52, N 10.56.
Biology
Growth inhibitory activity. Human T-leukemia (Jurkat), promyelo-
cytic leukemia (HL-60), chronic myelogenous leukemia (K562), mye-
loid leukemia (ML-2), and acute lymphoblastic leukemia (RS 4;11)
cells, the latter with a t(4;11) translocation, were grown in RPMI-
1640 medium (Gibco Milan, Italy). Human breast adenocarcinoma
(MCF-7), cervix carcinoma (HeLa), ovarian cancer (IGROV), anaplas-
tic thyroid (ARO), hepatoma (HepG2), and colon adenocarcinoma
(HT-29) cells were grown in Dulbecco’s modified Eagle’s medium
(DMEM; Gibco) supplemented with penicillin G (115 UmLÀ1; Gibco),
streptomycin (115 mgmLÀ1; Invitrogen, Milan, Italy) and 10% fetal
bovine serum (Invitrogen). Individual wells of a 96-well tissue cul-
ture microtiter plate were inoculated with 100 mL complete
medium containing 8ꢁ103 cells. The plates were incubated at
378C in a humidified incubator containing 5% CO2 for 18 h. At this
time, the initial medium was removed from each well, and drug
solutions (100 mL each), dissolved in complete medium, at various
concentrations, was added to each well and incubated at 378C for
72 h. Cell viability was assayed by the [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide] (MTT) test as previously de-
scribed.[25] The GI50 value is defined as the compound concentra-
tion required to inhibit cell proliferation by 50%.
3-Ethyl-8-phenyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one (34). Light-
brown solid; yield (93%); mp: 248–2508C (EtOH); Rf =0.45 (EtOAc/
n-hexane 8:1, violet fluorescent spot at l 365 nm); 1H NMR
([D6]DMSO): d=1.40 (t, 3H, J=7.3 Hz, CH3), 4.32 (q, 2H, J=7.1 Hz,
CH2), 7.27 (dd, 1H, J=7.4 Hz, 4’-H), 7.36 (dd, 1H, J=8.5 Hz, 5-H),
7.40 (dd, 2H, J=7.6 Hz, 2’-H and 6’-H), 7.51 (d, 1H, J=2.7 Hz, 1-H),
7.60 (d, 1H, J=2.7 Hz, 2-H), 7.78 (d, 2H, J=7.8 Hz, 3’- and 5’-H),
7.88 (d, 1H, J=8.5 and 0.6 Hz, 4-H), 8.07 (s, 1H, 7-H), 12.02 (bs, 1H,
NH); 13C NMR ([D6]DMSO): d=16.3 (CH3), 40.9 (CH2), 104.2, 112.1,
115.7, 119.3, 120.6, 124.1, 126.4, 128.1, 128.3, 128.8, 131.1, 135.58,
135.7, 137.2, 175.8 (CO); HRMS (ESI) calcd for C19H17N2O [M+H]+
m/z 289.1288, found 289.1266; anal. calcd for C19H16N2O: C 79.14, H
5.59, N 9.72, found: C 78.94, H 5.34, N 9.60.
3-Methylcyclopropyl-8-phenyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-
one (35). Grey solid; Yield 60%; mp: 197–1988C (70% EtOH); Rf =
0.57 (EtOAc/n-hexane 8:2, violet fluorescent spot at l 365 nm);
1H NMR ([D6]DMSO): d=0.38 (m, 2H, CH2), 0.51 (m, 2H, CH2), 1.24
(m, 2H, CH), 3.98 (d, 2H, J=6.87 Hz, CH2), 7.27 (dd, 1H, J=7.3 Hz,
4’-H), 7.38 (m, 3H, 2’-, 6’- and 5-H), 7.55 (dd, 1H, J=2.9 Hz, 2-H),
Effects on tubulin polymerization and on colchicine binding to
tubulin. Bovine brain tubulin was purified as described previously.
To evaluate the effect of the compounds on tubulin assembly in
ChemMedChem 2010, 5, 1373 – 1385
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1383

M. G. Ferlin et al.
MED
vitro, various concentrations were pre-incubated with 10 mm tubu-
lin in glutamate buffer at 308C and then cooled to 08C. After addi-
tion of GTP, the mixtures were transferred to 08C cuvettes in a re-
cording spectrophotometer and warmed to 308C, and the assem-
bly of tubulin was observed turbidimetrically. The IC50 value is de-
fined as the compound concentration required to inhibit the
extent of assembly by 50% after incubation for 20 min. The capaci-
ty of the test compounds to inhibit colchicine binding to tubulin
was measured as described,[8,9] except that the reaction mixtures
contained 1 mm tubulin, 5 mm [3H]colchicine, and 1 mm test com-
pound.
collected by centrifugation and permeabilized using a Fix&Perm
cell permeabilization kit (Caltag Laboratories, Burlingame, CA,
USA). The cells were then labeled with Bcl-2 antibody, washed, and
analyzed by flow cytometry.
Western blot analysis. Jurkat cells were incubated in the presence
of test compounds and, at various time points, were collected, cen-
trifuged, and washed twice with ice-cold PBS. The pellet was then
resuspended in lysis buffer. After the cells were lysed on ice for
30 min, lysates were centrifuged at 15000 g at 48C for 10 min. The
protein concentration in the supernatant was determined using
BCA protein assay reagents (Pierce, Italy). Equal amounts of protein
(20 mg) were resolved by SDS-PAGE (12% acrylamide) and trans-
ferred to PVDF Hybond-p membrane (GE Healthcare). Membranes
were blocked with I-block (Tropix) overnight, under rotation at
48C. Membranes were then incubated with a primary antibody
against the cleaved fragment of PARP (rabbit, 1:1000, Cell Signal-
ing), or b-actin (mouse, 1:10000, Sigma) for 2 h at room tempera-
ture. Membranes were next incubated with peroxidase-labeled
goat anti-rabbit IgG (1:100000, Sigma) or peroxidase-labeled goat
anti-mouse IgG (1:100000, Sigma) for 60 min. All membranes were
visualized using ECL Advance (GE Healthcare) and exposed to
Hyper film MP (GE Healthcare). To ensure equal protein loading,
each membrane was stripped and re-probed with anti-b-actin anti-
body.
Flow cytometric analysis of cell-cycle distribution and apoptosis.
For flow cytometric analysis of DNA content, 5ꢁ105 Jurkat cells in
exponential growth were treated with various concentrations of
the test compounds for 24 and 48 h. After an incubation period,
the cells were collected, centrifuged, and fixed with ice-cold etha-
nol (70%). The cells were then treated with lysis buffer containing
RNase A and Triton X-100 (0.1%), and then stained with PI. Sam-
ples were analyzed on a Cytomic FC500 flow cytometer (Beckman
Coulter). DNA histograms were analyzed using MultiCycleꢂ for Win-
dows (Phoenix Flow Systems, CA, USA).
Annexin V assay. Surface exposure of PS by apoptotic cells was
measured by flow cytometry with a Coulter Cytomics FC500 (Beck-
man Coulter, USA) by adding annexin V–FITC to cells according to
the manufacturer’s instructions (annexin V Fluos, Roche Diagnos-
tics). The cells were simultaneously stained with PI. Excitation was
set at l 488 nm, and the emission filters were set at l 525 and
585 nm.
Acknowledgements
This work was supported by grants from the Italian Ministry of
Education, University and Research (MIUR).
Assessment of mitochondrial changes. The mitochondrial mem-
brane potential was measured with the lipophilic cation 5,5’,6,6’-
tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine
iodide
Keywords: anticancer agents
apoptosis · pyrroloquinolinones · tubulin
· antiproliferative activity ·
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10 min at 378C, centrifuged, and resuspended in HBSS. The pro-
duction of ROS was measured by flow cytometry using HE (Molec-
ular Probes) and H2-DCFDA (Molecular Probes).
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Received: April 28, 2010
Revised: June 4, 2010
Published online on July 13, 2010
ChemMedChem 2010, 5, 1373 – 1385
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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