Synthesis and biological evaluation of pyrroloiminoquinone derivatives

Article abstract of DOI:10.1016/j.bmc.2007.11.063

Synthesis of 10 pyrroloiminoquinone derivatives is presented. The strategy is based around the elaboration of a common intermediate by reaction with primary amines. All the compounds obtained have been subjected to antiproliferative activity with three different cell lines (NCI-H460, HeLa, and HL-60). The capacity of 4 selected compounds to affect the enzymatic activity of the nuclear enzyme DNA topoisomerase II and to form the typical DNA fragmentation which occurs in the apoptotic process is discussed here.

Full text of DOI:10.1016/j.bmc.2007.11.063

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2431–2438
Synthesis and biological evaluation of
pyrroloiminoquinone derivatives
Daniele Passarella,a,* Francesca Belinghieri,a Michele Scarpellini,a Graziella Pratesi,b
Franco Zunino,b Ornella Maria Gia,c Lisa Dalla Via,c
Giuseppe Santoroa and Bruno Danielia
a
`
Dipartimento di Chimica Organica e Industriale, Universita degli Studi di Milano, Via Venezian 21, 20133 Milano, Italy
bIstituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1, 20133 Milano, Italy
`
Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Padova, Via F. Marzolo, 5-35131 Padova, Italy
c
Received 12 September 2007; revised 14 November 2007; accepted 21 November 2007
Available online 28 November 2007
Abstract—Synthesis of 10 pyrroloiminoquinone derivatives is presented. The strategy is based around the elaboration of a common
intermediate by reaction with primary amines. All the compounds obtained have been subjected to antiproliferative activity with
three different cell lines (NCI-H460, HeLa, and HL-60). The capacity of 4 selected compounds to affect the enzymatic activity of
the nuclear enzyme DNA topoisomerase II and to form the typical DNA fragmentation which occurs in the apoptotic process is
discussed here.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
normal conditions, they are present at low equilibrium
levels and are tolerated by the cell. However, conditions
that significantly increase the concentration of cleavage
complexes generate permanent DNA strand breaks that
trigger illegitimate recombination, chromosomal aberra-
tions, sister chromatid exchange, and cell death path-
ways.12–16 All topoisomerase II-directed agents are
able to interfere with at least one step of the catalytic cy-
cle. Agents able to stabilize the covalent DNA topoiso-
Drugs that inhibit or poison the function of topoisomer-
ase enzymes are one of the mainstays of cancer chemo-
therapy. Two classes of inhibitors are known that
include some of the most widely used anticancer drugs,
topo I, exemplified by the camptothecins, and topo II,
exemplified by etoposide and anthracyclines.1,2 A major
effort has been directed toward the synthesis of ana-
logues designed to improve on the deficiencies of these
established agents. At the same time, new types of struc-
tures are also being explored and developed. Topoiso-
merase II modulates the topological state of DNA by
generating transient double-stranded breaks in the back-
bone of the genetic material.3–8 To maintain genomic
integrity during this cleavage event, the enzyme forms
covalent bonds between active site tyrosyl residues and
the 50-DNA termini created by scission of the double he-
lix.9–11 These covalent topoisomerase II cleaved DNA
intermediates are known as cleavable complexes. Under
merase
II
complex
are
traditionally
called
topoisomerase II poisons, while agents acting on any
of the other steps in the catalytic cycle are called cata-
lytic inhibitors. Recently a class of marine compounds
characterized by the presence of the pyrroloiminoqui-
none17 structural motif was discovered to be cytotoxic,
potentially by multiple mechanisms but with a proven
activity toward the catalytic cycle of topoisomerase II
(Fig. 1).18 As a consequence several syntheses of pyirro-
loiminoquinone derivatives with interesting pharmaco-
logical results have been reported in the literature.19–23
In this paper, we report experimental results concerning
the synthesis and the biological evaluation of some new
compounds characterized by the presence of the pyrro-
loiminoquinone framework with a methyl group at posi-
tion 1.
Keywords: Pyrroloiminoquinone derivatives; Antiproliferative activity;
Topoisomerase II; DNA fragmentation.
*
Corresponding author. Tel.: +39 02 50314080; fax: +39 02
50314078; e-mail: Daniele.Passarella@unimi.it
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.063

2432
D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 2431–2438
H3C
H3C
Br
N
N
HO
O
O
HO
N
H
N
S
N
N
H3C
H
N1
makaluvamine E
makaluvamine F
O
H3C
N
N
H3C
SMe
N1-methyl-
pyrroloiminoquinone
structural motif
O
N
O
HN
N
Br
O
H2N
N
S
discorhabdin Q
isobatzlline B
Figure 1. Representative structures of marine compounds characterized by the presence of the N1-methyl-pyrroloiminoquinone structural motif.
2. Results and discussion
solution was directly treated with a large excess of
NiCl2/NaBH4 to give 3 (yield from 1: 79%). Treatment
of compound 3 with a 1:1 Ac2O/HCOOH mixture for
60 h resulted in the formation of 4 (yield 36%) accompa-
nied by compound 5 (yield 10%) as an inseparable mix-
ture of diastereoisomers. We studied the possibility to
convert byproduct 5 to main compound 4 by a formal
elimination of methanol. To our surprise compound 5
proved to be unreactive even by treatment with
HCOOH at reflux. The formyl groups of compound 4
could be manipulated selectively on the basis of the dif-
ferent reactivity of the indole and aniline nitrogens.
Treatment of 4 in MeOH/CH2Cl2 (1:1) with a saturated
solution of Na2CO3 for 8 h at room temperature gave
selectively compound 6 and this immediately converted
to 7 by reaction with NaH and MeI (yield from 4:
97%). Treatment of 7 with 20% NaOH permitted the re-
moval of the formyl group from the quinoline nitrogen.
Finally the oxidation to quinine-imine 926–30 (yield from
7: 57%) was realized with CAN as described by Alvarez
and Joule. The reaction of 9 with different primary
amines in EtOH at reflux allowed us to prepare 9 deriv-
atives (10–17, yields 18–91%) (Table 1).23 Reaction of 9
with mercaptoethylamine resulted in the formation of
compound 18 (yield 40%) by a formal thiol substitution
of the methoxy group, a subsequent addition reaction of
the primary amine, and aromatization.
2.1. Synthesis
The preparation of the reported derivatives was realized
according to the synthetic Scheme 1 taking advantage
from the possibility to substitute the methoxyl group
of the intermediate 9 by a conventional addition-elimi-
nation reaction.
The preparation of 9 was achieved using nitro-aldehyde
124,25 as described by Alvarez and Joule24 (Scheme 1).
The subsequent formation of the dimethoxyacetal and
reduction to obtain tetrahydroisoquinoline 3 was stud-
ied in order to simplify the purification. To this end
the formation of the acetal was conducted in MeOH
in the presence of acidic Amberlite IR-120 resin in order
to overcome the problem of the removal of HCl. When
the reaction was complete the resin was removed and the
NH2 CH(OCH3)2
NO2
R
MeO
MeO
MeO
MeO
b
N
N
H
3
1
2
R = CHO
R = CH(OCH3)2
a
c
(ref. 35)
R1
H3C
N
N
MeO
MeO
O
The structure of compound 1431–35,9 offered us the
opportunity to approach a carbon framework with a
grater structural complexity and rigidity. In fact, treat-
ment of 14 with PIFA36 gave the spirodienone 19 (yield
47%) that presents a complex pentacyclic system struc-
turally related to discorhabdins (Scheme 2).37
g
(refs. 26-30)
N
MeO
N
R2
9
4
6
7
8
R1 = CHO, R2 = CHO
R1 = H, R2 = CHO
d
e
f
h
R
R
1 = CH3, R2 = CHO
1 = CH3, R2 = H
2.2. Biological activity
10 - 18 (Table 1)
OHC
OMe
The biological activity of the compounds was evaluated
on three different types of human tumor cell lines repre-
senting solid and hematological malignancies. A preli-
minary evaluation of the antiproliferative activity was
performed against the NCI-H460 nonsmall cell lung car-
cinoma cell line and then, for the most active com-
pounds, against the HeLa cervix adenocarcinoma and
HL-60 promyelocytic leukemia cell lines. In order to
investigate the mechanism of action responsible for the
N
MeO
MeO
N
CHO
5
Scheme 1. General synthetic procedure for the preparation of
compounds 10–18. Reagents: (a) Amberlite IR-120/MeOH; (b) NiCl4,
NaBH4; (c) Ac2O, HCOOH; (d) Na2CO3; (e) NaH, Mel; (f) NaOH
20%; (g) CAN (from 8); (h) R-NH2.

D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 2431–2438
2433
Table 1. Structures of compounds 10–18
Me
N
O
Me
Me
N
N
R-NH2
Product
Product
number
O
O
N
H
N
PIFA
H3C
HN
N
HN
N
Br
O
N
N
O
10
O
S
N
H
14
N
H
OH
19
NH2
N
H
N
N
Discorhabdin Q
F
F
Scheme 2. Preparation of compound 19. Comparison with discorhab-
din Q.
H3C
O
11
N
H
N
H
N
H
NH2
2.3. Antiproliferative activity
OMe
The in vitro cytotoxic potency of the newly synthesized
pyrroloiminoquinones 4-19 was preliminarily evaluated
by a growth inhibition assay against the NCI-H460 hu-
man nonsmall cell lung carcinoma cell line. The results,
expressed as IC50 values, are shown in Table 2 and indi-
cated that many compounds of the series possessed a
good antiproliferative activity, with a cytotoxic potency
lower than 11 lM. Compound 18, that is characterized
by the thiomorpholine subunit, showed the highest po-
tency of the series. The comparison of the IC50 values
of compounds 4–9 with those of compounds 10–19 high-
lights the importance of the iminoquinone system. The
synthetic intermediate 5, that is characterized by a ben-
zopyrrolidine framework, is an exception in this behav-
ior. For the most active derivatives 5, 9, 10, 11, 14, 18,
and 19, the antiproliferative function was further inves-
tigated on HeLa human cervix adenocarcinoma and
HL-60 human promyelocytic leukemia cell lines (Table
2). The majority of derivatives, 10, 14, 18, and 19, main-
tained a significant antiproliferative effect and, as for in
the NCI-H460 cell line, compound 18 exerted the high-
est capability to inhibit cell growth, showing the lowest
OMe
H3C
N
N
O
12
N
H
N
H
N
H
N
N
NH2
OH
OH
H3C
O
13
N
H
N
H
N
H
NH2
H3C
N
HO
O
1431–35,9
HO
NH2
N
N
H
H3C
N
MeO
O
MeO
15
NH2
N
N
H
H3C
N
Table 2. Antiproliferative activity of derivatives, 4–19, after 72 h of
drug exposure
O
NH2
16
N
N
a
Compound
IC50 (lM)
H
NCI-H460
HeLa
HL-60
H3C
4
>60
7.7
—
>20
—
>20
N
H
5
H3C
O
H
6
>60
>60
>60
9
—
—
—
—
H3C
17
NH2
7
N
H
N
8
—
>20
—
>20
9
10
2.12
5.50
8.8
4.1 0.3
>20
7.8 0.4
>20
H3C
N
11
12
O
—
—
—
—
NH2
18
HS
13
14
>40
10.8
>40
>40
23
S
N
4.9 0.4
—
6.9 0.5
—
NH
15
16
—
—
—
—
17
18
0.96
6.2
1.9 0.1
5.5 0.4
—
2.7 0.2
3.1 0.3
—
19
Doxorubicin
m-Amsacrine
antiproliferative activity, we assayed the capacity of the
new compounds to affect the enzymatic activity of the
nuclear enzyme DNA topoisomerase II. Moreover, the
ability of the compounds to induce apoptosis was inves-
tigated in the HeLa cells, by DNA fragmentation assay.
0.028
—
0.012 0.002
0.0051 0.0011
a IC50: concentration required to reduce cell number to 50% of control
culture.

2434
D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 2431–2438
Figure 2. Effect of derivatives 10, 14, 18, and 19 on the relaxation of supercoiled plasmid DNA by human recombinant topoisomerase II. Lane a:
pBR322 DNA, lane b: same as lane a in the presence of 1U topoisomerase II, lanes c–e: same as lane b in the presence of 10, 25, and 50 lM test
compound, respectively; lane f: same as lane b in the presence of 8 lM m-amsacrine; lane g: same as lane b in the presence of solvent alone.
IC50 value in the series. The other derivatives were un-
able to induce 50% growth inhibition up to 20 lM
concentration.
nificant level of inhibition, although lower than that of
10.
2.3.2. DNA fragmentation. During the apoptotic pro-
cess, activated endonucleases degrade the high order
chromatin of DNA into mono- and oligonucleosomal
DNA-fragments, giving rise to a ‘ladder’ of nucleo-
somal-sized multimers. To establish the cell death
modality that follows the treatment, DNA was extracted
from HeLa cells and treated with the test derivatives or
with cisplatin (taken as positive control). Figure 3 shows
the gel electrophoresis of the DNA extracted from con-
trol cells and from cells after treatment with cisplatin or
compounds 10, 14, and 19. The results indicate the abil-
ity of the compounds to form the typical DNA fragmen-
tation which occurs in the apoptotic process.
2.3.1. Inhibition of topoisomerase II enzymatic activity.
In order to investigate the mechanism of action account-
able for the antiproliferative activity exerted by 10, 14,
18, and 19, we assayed their capability to inhibit the
activity of the nuclear enzyme DNA topoisomerase II.
This enzyme catalyzes the conversion of pBR322 plas-
mid DNA from the supercoiled to the fully relaxed con-
formation and Figure 2 shows the effect of the new
derivative on topoisomerase II relaxation ability. In de-
tail, compound 10 shows the strongest capability to in-
hibit the enzyme, whose activity is completely
abolished at 50 lM concentration. Derivatives 14 and
19 exerted a quite weak and similar inhibitory effect at
test concentrations, while compound 18 displayed a sig-
3. Conclusions
The discovery that natural marine compounds charac-
terized by the presence of pyrroloiminoquinone system
are able to interfere with the catalytic cycle of DNA
topoisomerase II has stimulated the interest for new
compounds presenting such a structural motif. In this
paper, we report an improvement in the synthesis of
compound 9 which is pivotal for the preparation of pyr-
roloiminoquinone derivatives. A quality collection of
compounds was synthesized and evaluated for growth
inhibitory properties in a small panel of human tumor
cell lines. The results of the present study indicated that
the insertion of arylamino side chains to the pyrroloimi-
noquinone system leads to new derivatives which are
able to induce a good antiproliferative effect on human
tumor cells. In particular, both indole and phenol termi-
nal groups were demonstrated to be the most effective
chemical moieties (compounds 10 and 14). Also, the
condensation of one (18) or two (19) rings to the tricy-
clic pyrroloiminoquinone moiety yielded derivatives en-
Figure 3. Agarose gel electrophoresis of DNA extracted from HeLa
cells. Lane a: untreated control, lane b: 50 lM cisplatin, lanes c–e:
100 lM of compounds 10, 14, and 19, respectively; lane f: solvent
alone.

D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 2431–2438
2435
dowed with an interesting antiproliferative activity. Pre-
liminary studies of the mechanism of action of the most
active compounds suggested an inhibitory effect on
DNA topoisomerase II activity, in accord with results
from the natural compounds. The analysis of struc-
ture-activity relationships obtained in the series of com-
pounds investigated may represent the basis for the
design of more active molecules.
4.1.3.
1,5-Diformyl-1,3,4,5-tetrahydro-7,8-dimethoxy-
2,3-dihydropyrrolo[4,3,2-de]quinoline (4). Compound 3
was added to a mixture of HCOOH (2.2 ml) and Ac2O
(4.4 ml) at 0 ꢁC and the solution was stirred for 60 h
at room temperature. The solvent was then evaporated
under reduced pressure to give a crude product, which
was purified by column chromatography (EtOAc/Hex-
ane 7:3) to give the 4 (245.6 mg, 36%) as a white amor-
phous solid and compound 5 (68.2 mg, 10%) as a yellow
amorphous solid. 4: 1H NMR (300 MHz, CDCl3) d 1.52
(ddd, J = 5.5, 12.7 25.4 Hz, 1H), 2.38–2.53 (m, 1H), 3.44
(dt, J = 4.3, 12.7 Hz, 1H), 3.85 (s, 3H), 3.90 (s, 3H), 4.05
(dd, J = 5.5, 13.8 Hz, 1H), 5.76 (s, 1H), 6.92 e 7.85 (2s,
1H), 8.43 e 9.12 (2s, 1H), 9.27 (s, 1H). 13C NMR
(50 MHz, CDCl3) d 22.1 e 23.2 (2t), 40.4 e 47.4 (2t);
57.6 e 58.2 (2q); 60.9 (q); 94.1 (d); 99.7 (s); 108.8 e
109.3 (2s); 116.8 e 117.5 (2d); 125.8 e 126.8 (2s); 127.8
e 128.3 (2s); 131.7 e 132.4 (2s); 147.9 e 148.5 (2s);
160.2 e 161.7 (2d); EI-MS: 274; Compound 5: 1H
NMR (300 MHz, CDCl3, 50 ꢁC) d 2.95 (m, 2H), 3.5
(s, 3H,), 3.90 (s, 3H), 3.93 (s, 3H), 3.96 (m, 1H), 4.09
(m, 2H), 5.9 (d, J = 6.1 Hz, 1H), 7.45 and 7.90 (2s,
1H), 8.45 and 9.71 (2s, 1H), 8.9 (s, 1H); 13C NMR
(50 MHz, CDCl3) 22.1 and 23.2 (2t), 40.4 and 47.4
(2t); 57.6 and 58.2 (2q); 60.9 (q); 94.1 (d); 99.7 (s);
108.8 and 109.3 (2s); 116.8 and 117.5 (2d); 125.8 and
126.8 (2s); 127.8 and 128.3 (2s); 131.7 and 132.4 (2s);
147.9 and 148.5 (2s); 160.2 and 161.7 (2d); EI-MS:
306. Anal. Calcd for C15H18N2O5: C, 58.82; H, 5.92;
N, 9.15; Found: C, 58.78; H, 5.96; N, 9.19.
4. Experimental
4.1. General
Thin-layer chromatography (TLC) was performed on
Merck precoated 60F254 plates. Reactions were moni-
tored by TLC on silica gel, with detection by UV light
(254 nm) or by charring with sulfuric acid. Flash chro-
matography was performed using Silica gel (240–400
mesh, Merck). 1H NMR spectra were recorded with
Brucker 200, 300, and 400 MHz spectrometers using
chloroform-d (CDCl3) and methanol-d4 (CD3OD).
Chemical shifts are reported in parts per million (d)
downfield from tetramethylsilane (TMS) as internal
standard. EI mass spectra were recorded at an ionizing
voltage of 6 keV on a VG 70-70 EQ. ESI mass spectra
were recorded on FT-ICR APEXII (Bruker Daltonics).
All reactions were carried out in dry solvents.
4.1.1. 4-(Dimethoxymethyl)-6,7-dimethoxy-5-nitroquino-
line (2). In HCl/MeOH: A solution of 4-formyl-6,7-dime-
thoxy-5-nitroquinoline (1, 2.5 g, 9.73 mmol) in MeOH
(300 mL) saturated with HCl was refluxed for 24 h, then
diluted with EtOAc and washed with small portions of
NaHCO3 (5%). Compound 2 (2.8 g, 94%) was obtained
as an amorphous solid. With resin: To a solution of 4-for-
myl-6,7-dimethoxy-5-nitroquinoline (2.5 g, 9.73 mmol)
in MeOH (40 mL) Amberlite IR-120 (1.02 g) was added.
After stirring under reflux for 72 h, the resin was sepa-
rated by filtration and the solvent was evaporated. Com-
pound 2 (2.5 g, 84%) was obtained as an amorphous solid.
1H NMR (300 MHz, CDCl3) d 3.40 (s, 6H), 4.12 (s, 3H),
4.15 (s, 3H), 5.60 (s, 1H), 7.65 (s, 1H), 7.80 (d, J = 4.9 Hz),
8.80 (d, J = 4.9 Hz, 1H). 13C NMR (75 MHz, CDCl3): d
54.3 (q); 56.8 (q);63.2 (q); 100.5 (d); 112.9 (d); 113.5 (s);
119.6(d); 140.9 (s); 144.4(s); 146.7 (s); 150.5 (d); 153.9 (s).
4.1.4. 5-Formyl-1,3,4,5-tetrahydro-7,8-dimethoxy-2,3-
dihydropyrrolo[4,3,2-de]quinoline (6). A mixture of 4
(114.8 mg, 0.42 mmoli), MeOH (2 mL), CH2Cl2
(2 mL), and Na2CO3 (5%, 1 mL) was stirred for 8 h at
room temperature. The reaction mixture was then di-
luted with H2O and extracted with CH2Cl2. Compound
1
6 (101.8 mg, 99%) was obtained as a yellowish solid. H
NMR (200 MHz, CDCl3): d 2.94 e 3.12 (2t, J = 5.7 Hz,
2H), 3.93 e 4.00 (2t, J = 5.8 Hz, 2H), 3.90 (s, 3H), 3.97
(s, 3H), 6.60 e 7.60 (2s, 1H), 6.76 (s, 1H), 8.12 (br s,
1H), 8.35 e 8.68 (2s, 1H); 13C NMR (50 MHz, CDCl3):
d 23.2 e 23.6 (2t), 39.2 e 46.4 (2t), 57.8 e 58.6 (2q), 60.2
(q,), 94.5 (d), 99.1 (s), 107.7 e 108.9 (2s), 116.3 e 117.0
(2d), 125.1 e 126.1 (2s), 127.4 e 128.0 (2s), 131.0 e
132.1 (2s), 147.5 e 148.6 (2s), 160.5 e 161.6 (2d).
4.1.2. 5-Amino-1,2,3,4-tetrahydro-6,7-dimethoxy-4-
(dimethoxymethyl)quinoline (3). To a solution of 2
(1.32 g, 4.3 mmol) in MeOH (135 mL) was added Ni-
Cl2Æ6H2O (12.24 g, 51.5 mmol). After stirring for
5 min. at room temperature the solution was cooled to
ꢀ20 ꢁC and NaBH4 (11.4 g, 300.5 mmol) was added in
small portions. The reaction mixture was then diluted
with H2O (200 mL), filtered, and the water-phase was
extracted with EtOAc. Compound 3 (1.34 g, 94%) was
4.1.5. 5-Formyl-1,3,4,5-tetrahydro-7,8-dimethoxy-1-
methylpyrrolo[4,3,2-de]-quinoline (7). A solution of 6
(101.8 mg, 0.42 mmol) in dry THF (4 mL) was added
to a suspension of NaH (19.9 mg, 0.83 mmol) in dry
THF (4 mL) at room temperature. The mixture was stir-
red for 30 min. at room temperature, MeI (0.9 mL,
12.6 mmol) was added, and the resulting mixture was
stirred for 20 h. Thereafter, H2O was added, the solution
was concentrated under reduced pressure, and extracted
with CH2Cl2. Compound 7 (105.6 mg, 98%) was ob-
1
obtained as a reddish oil. H NMR (300 MHz, CDCl3)
1
d 1.55–1.60 (m, 1H), 2.09–2.20 (m, 1H), 3.00–3.16 (m,
1H), 3.37 (s, 3H), 3.20–3.50 (m,, 2H), 3.67 (s, 3H),
3.77 (s, 3H), 3.82 (s, 3H), 4.51 (d, J = 8.6 Hz, 1H),
5.659 (s, 1H) 13C NMR (75 MHz, CDCl3) d 20.0 (q),
26.2 (d), 28.0 (t), 36.8 (t), 55.5 (q), 61.2 (q), 89.2 (d),
105.7 (s), 129.1 (s), 138.9 (s), 140.0 (s), 153.2 (s).
tained as a white solid. H NMR (300 MHz, CDCl3) d
2.96 e 3.05 (2t, J = 5.7 Hz, 2H), 3.90 (s, 3H), 3.94 (s,
3H), 3.95 (s, 3H), 3.85 e 4.08 (2t, J = 5.7 Hz, 2H), 6.60
(s, 1H), 8.38 e 8.85 (2s, 1H). 13C NMR (75 MHz,
CDCl3) d 21.3 e 22.9 (2 t), 34.8 (q), 40.1 e 46.6 (2 t);
57.1 e 57.8 (2q), 61.9 (q); 94.5 e 99.4 (2d); 106.8 e

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D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 2431–2438
107.9 (2s); 118.0 (s); 122.0 e 122.7 (2d); 124.8 e 127.0
(2s); 127.8 e 128.2 (2s); 132.2 e 133.1 (2s) 148.1 e 148.9
(2s), 158.0 e 161.0 (2d).
154.0, 157.8, 159.0, 160.1; ESI positive MS Anal. Calcd
for C21H19N4OF+H+ 363.16157. Found: 363.16160.
4.2.3. 1,3,4,8-Tetrahydro-7-(5-methoxy-3-(2-amino-
ethyl)indolyl)-1-methyl-8-oxopyrrolo [4,3,2-de]-quinoline
(12). Red oil (15 mg, 57%) (EtOAc/MeOH, 7:3 with
Et3N 3%); 1H NMR (400 MHz, CD3OD) d 2.78 (t,
J = 7.6 Hz, 2H), 3.08 (t, J = 7.0 Hz, 2H), 3.60 (t,
J = 7.0 Hz, 2H), 3.75 (t, J = 7.6 Hz, 2H), 3.78 (s, 3H),
3.80(s, 3H), 5.25 (s, 1H), 6.65–6.80 (m), 6.92 (s,1H),
6.95–7.05 (m), 7.08 (s, 1H), 7.15–7.28 (m); 13C NMR
(100 MHz, CD3OD) (APT) d 20.4, 26.1, 37.2, 40.5,
45.4; 46.4, 87.1, 104.4, 113.6, 112.4, 113.8, 120.5,
125.1, 125.7, 132.7, 134.1, 136.0, 146.0, 152.3, 155.0,
160.0, 171.0; ESI positive MS Anal. Calcd for
C22H22N4O2+H+ 375.1815. Found: 375.1818.
4.1.6. 1,3,4,5-Tetrahydro-7,8-dimethoxy-1-methylpyrrol-
o[4,3,2-de]-quinoline (8). A solution of 7 (105.6 mg,
0.41 mmol) in NaOH (20%, 8 mL) was stirred under re-
flux for 24 h. The solution was extracted with CH2Cl2.
Compound 8 (73.1 mg, 78%) was obtained as a brown
solid. 1H NMR (300 MHz, CDCl3)
d 2.90 (t,
J = 5.5 Hz, 2H), 3.40 (t, J = 5.5 Hz, 2H), 3.80 (s, 3H),
3.89 (s, 3H), 3.91 (s, 3H), 5.90 (s, 1H), 6.40 (s, 1H);
13C NMR (75 MHz, CDCl3) d 22.0 (t), 34.8 (q),
43.4(t); 57.7 (q), 62.1 (q); 89.0 (d); 109.8 (s); 114.6 (s);
120.0 (d),128.6 (s),128.8(s), 136.5(s), 149.0 (s).
4.1.7. 1,3,4,8-Tetrahydro-7-methoxy-1-methyl-8-oxopyr-
rolo[4,3,2-de]-quinoline (9). A mixture of 8 (73.1 mg,
0.32 mmol), MeCN (5 mL), CAN (ammonium ceriu-
m(IV) nitrate) (349 mg, 0.64 mmol), and H2O (9 mL)
was stirred for 10 min. The solution was then diluted
with H2O and extracted with CH2Cl2. Compound 9
(49.2 mg, 73%) was obtained as an amorphous orange
4.2.4. 1,3,4,8-Tetrahydro-7-(5-hydroxy-3-(2-amino-
ethyl)indolyl)-1-methyl-8-oxopyrrolo-[4,3,2-de]-quinoline
(13). Red oil (14 mg, 39%) (EtOAc/MeOH, 8:2 with
Et3N 3%); 1H NMR (400 MHz, CD3OD) d 2.86 (t,
J = 7.6 Hz, 2 H), 3.08 (t, J = 7.0 Hz, 2H), 3.60 (t,
J = 7.0 Hz, 2H), 3.78 (t, J = 7.6 Hz, 2H), 3.92 (s, 3H),
5.33 (s, 1H), 6.68 (dd, J = 8.7, 2.3 Hz, 1H), 6.94 (d,
J = 2.3 Hz, 1H), 7.00 (s, 1H), 7.08 (s, 1H), 7.18 (d,
J = 8.6 Hz, 1H); 13C NMR (100 MHz, CD3OD) (APT)
d 20.4, 26.1, 37.2, 45.4, 46.4, 87.1, 104.4, 113.6, 112.4,
113.8, 120.5, 125.1, 125.7, 132.7, 134.1, 136.0, 146.0,
152.3, 155.0, 160.0, 171.0; ESI positive MS Anal. Calcd
for C21H20N4O2+H+ 361.1659. Found: 361.1660.
solid, 1H NMR (300 MHz, CDCl3)
d 3.05 (t,
J = 8.1 Hz, 2H), 3.99 (s, 3H), 4.00 (s, 3H), 4.14 (t,
J = 8.1 Hz, 2H), 6.60 (s, 1H), 6.85 (s, 1H); 13C NMR
(75 MHz, CDCl3) d 18.6 (t), 36.6 (q), 39.8 (t); 58.5 (q),
97.9 (d); 117.8 (s); 119.3 (s); 124.7 (s), 128.2 (d), 159.9
(s), 162.6 (s), 168.0 (s).
4.2. General procedure for the preparation of compounds
10–18
4.2.5. 1,3,4,8-Tetrahydro-7-(4-hydroxyphenethylamino)-
1-methyl-8-oxopyrrolo[4,3,2-de]-quinoline (14). Red oil
1
(73 mg, 52%) (EtOAc/MeOH, 8:2 with Et3N 3%); H
A solution of 9 (23 mg, 0.11 mmol) and primary amine
(0.28 mmol) in EtOH (10 mL) was refluxed for 4 h.
The solvent was removed to give a crude product, which
was purified by column chromatography.
NMR (400 MHz, CD3OD) dH 2.70 (t, J = 7.6 Hz, 2H),
2.90 (t, J = 7.6 Hz, 2H), 3.45 (t, J = 7.6 Hz, 2H),
3.85(t, J = 7.6 Hz, 2H), 3.95 (s, 3H), 5.40 (s, 1H), 6.75
(d, J = 8.5 Hz, 2H), 7.05 (d, J = 8.5 Hz, 2H), 7.10
(s,1H); 13C NMR (100 MHz, CD3OD) d 18.0 (t), 33.0
(t), 34.7 (q), 44.7 (t), 45.2 (t), 88.2 (d), 115.0 (d), 120.2
(s), 123.9 (s), 125.4 (s), 127.2 (s), 129.4 (d), 129.6 (d),
154.9 (s), 157.4 (s), 159.6 (s), 168.4 (s). MS (APCI); m/
z = 322 (M+H)+. ESI positive MS Anal. Calcd for
C19H19N3O2+H+ 322.1550. Found: 322.1553.
4.2.1. 1,3,4,8-Tetrahydro-7-(3-(2-aminoethyl)indolyl)-1-
methyl-8-oxopyrrolo[4,3,2-de]-quinoline (10). Red oil
(11 mg, 30%) (EtOAc/MeOH 8:2 with Et3N 3%); 1H
NMR (400 MHz, CD3OD) d 2.93 (t, J = 8.0 Hz, 2H),
3.12 (t, J = 6.9 Hz, 2H), 3.60 (t, J = 7.0, 2H), 3.73 (t,
J = 8.0 Hz, 2H), 3.85 (s, 3H), 5.36 (s, 1H), 7.00–7.18
(m, 4H), 7.30–7.40 (m, 1H), 7.58 (d, J = 8 Hz, 1H), 8.0
(s, 1H); 13C NMR (100 MHz, CD3OD) (APT) d 18.6,
24.2, 35.6, 43.0, 44.8, 84.5, 110.7, 111.3, 118.3, 119.1,
123.9, 127.7, 129.2, 131.1, 137.4, 139.5, 153.9,
157.9,168.1, 174.4; ESI positive MS Anal. Calcd for
C21H20N4O+H+ 345.1710. Found: 345.1709 [M+H]+.
4.2.6. 1,3,4,8-Tetrahydro-7-(3-methoxyphenethylamino)-
1-methyl-8-oxopyrrolo[4,3,2-de]-quinoline (15). Red oil
(3 mg, 18%) (EtOAc/MeOH, 8:2 with Et3N 2%); 1H
NMR (400 MHz, CDCl3) d 2.65–2.80 (m, 2H), 2.85–
3.05 (m, 2H), 3.35–3.45 (m, 2H), 3.80 (s, 3H), 3.95 (s,
3H), 4.00–4.15 (m, 2H), 5.68 (s, 1H), 6.65–6.85 (m,
3H), 7.15–7.25 (m, 1H), 7.28 (s, 1H); ESI MS: 336.4
(M+1); ESI positive MS Anal. Calcd for
C20H21N3O2+H+ 336.17065. Found: 336.17071.
4.2.2. 1,3,4,8-Tetrahydro-7-(5-fluoro-3-(2-amino-
ethyl)indolyl)-1-methyl-8-oxopyrrolo[4,3,2-de]-quinoline
(11). red oil (17 mg, 47%) (EtOAc/MeOH, 7:3 with Et3N
3%) 1H NMR (400 MHz, CD3OD) d 2.83 (t, J = 7.6 Hz,
2H), 3.08 (t, J = 7.2 Hz, 2H), 3.60 (t, J = 7.2 Hz, 2H),
3.80 (t, J = 7.6 Hz, 2H), 3.90 (s, 3H), 5.35 (s, 1H), 6.85
(ddd, J = 11.6, 9.2, 2.4 Hz, 1H), 7.00 (s, 1H), 7.18
(s,1H), 7.22 (dd, J = 10.0, 2.4 Hz, 1H), 7.30 (dd,
J = 8.8, 4.4 Hz, 1H). 13C NMR (100 MHz, CD3OD) d
19.4, 25.0, 36.3, 44.6, 45.5, 86.4, 103.9, 110.86, 111.5,
113.3, 118.0, 119.0, 131.7, 126.1, 129.0, 135.0, 143.0,
4.2.7.
1,3,4,8-Tetrahydro-7-(benzylamino)-1-methyl-8-
oxopyrrolo[4,3,2-de]-quinoline (16). Red oil (25 mg,
86%) (EtOAc/MeOH, 8:2 with Et3N 2%); 1H NMR
(400 MHz, DMSO) d 2.15–2.25 (m, 2H), 3.78–3.88 (m,
2H), 3.90 (s, 3H), 4.45 (s, 2H), 5.78 (s, 1H), 7.20 (s,
1H), 7.20–7.50 (m, 5H); ESI positive MS Anal. Calcd
for C18H17N3O+H+ 292.1444. Found: 292.1449.

D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 2431–2438
2437
4.2.8. 1,3,4,8-Tetrahydro-7-S(-)-(1-phenylethylamino)-1-
methyl-8-oxopyrrolo[4,3,2-de]-quinoline (17). Red oil
(25 mg, 91%) (EtOAc/MeOH, 8:2 with Et3N 2%);
8 · 104 cells/well were seeded, and the final counting
was performed with a Coulter counter model ZM. For
HeLa and HL-60, 4 · 104 cells/well were seeded in dupli-
cate into 24-well plates and a Trypan blue assay was per-
formed to determine cell viability. Cytotoxicity data are
expressed as IC50 values, that is, the concentration (in
lM) of the test agent inducing 50% reduction in cell
number compared with untreated control cultures. All
compounds are insoluble in water and were dissolved
in DMSO prior to dilution into the biological assay,
being the final concentration of DMSO at a maximum
of 1%.
1
[a]D11.2 (c 1, CHCl3); H NMR (400 MHz, CDCl3) d
2.70 (t, J = 7.6 Hz, 2 H), 3.95 (t, J = 7.6 Hz, 2H), 4.10
(s, 3H), 4.15 (s, 3H), 4.55–4.65 (m, 1H), 6.65 (s, 1H),
7.20–7.40 (m, 7H). MS (FAB); m/z = 306 (M+H)+;
ESI positive MS Anal. Calcd for C19H19N3O+H+
306.1601. Found: 306.1600.
4.2.9. 5-Methyl-3,5,9,8,10-tetrahydro-2H,8H-7-thia-
1,5,10-triaza-acephenantrylen-6-one (18). Red amor-
phous solid (22 mg, 40%), polarity gradient from
(EtOAc/Hexane 1.1) to (EtOAc/MeOH, 8:2 with Et3N
4.4.1. DNA topoisomerase II relaxation assay. DNA
topoisomerase II activity was assayed by the relaxation
of a supercoiled DNA substrate. In detail, 0.25 lg
pBR322 plasmid DNA (Fermentas Life Sciences) was
incubated with 1U topoisomerase II (USB) and test
compound as indicated for 60 min at 37 ꢁC in 20 lL
reaction buffer consisting of 10 mM Tris–HCl
(pH = 7.9), 50 mM NaCl, 50 mM KCl, 5 mM MgCl2,
0.1 mM EDTA, 15 lg/mL BSA and 1 mM ATP.
2%); 1H NMR (400 MHz, CD3OD)
d 2.70 (t,
J = 7.7 Hz, 2H), 2.92 (t, J = 4.7 Hz, 2H); 3.70 (t,
4.7 Hz, 2H),3.98 (s, 3H), 4.10 (t, 2H), 5.50 (s, 1H),
6.51 (s, 1H); ESI positive MS Anal. Calcd for
C13H13N3OS+H+ 260.0852. Found: 260.0847.
4.2.10. 1,3,4,8-Tetrahydro-20H-spiro[cyclohexa[2.5]diene-
1-methyl-8-oxopyrrolo[4,3,2-de]1,7phen-antrolyne-12,30-
dione (19). To a solution of 14 (73 mg, 0.23 mmol) in
CF3CH2OH (4 mL) a solution of PIFA (phenyliodine
Reaction was stopped by adding 4 lL stop buffer (5%
SDS, 0.125% bromophenol blue, and 25% glycerol),
50 lg/mL proteinase K (Sigma Chemical) and incubat-
ing for further 30 min at 37 ꢁC. The samples were sepa-
rated by electrophoresis on a 1% agarose gel. The gel
was stained with ethidium bromide 1 lg/ml in TAE
buffer, transilluminated by UV light and fluorescence
emission visualized using a CCD camera coupled to a
Bio-Rad Gel Doc XR apparatus.
bis(trifluoroacetate)
(147 mg,
0.34 mmol)
in
CF3CH2OH (4 mL) was added at 0 ꢁC. The mixture
was stirred for 2 h and then treated with Na2CO3
(10%). The mixture was extracted with CH2Cl2. Purifica-
tion by flash chromatography (AcOEt/MeOH 8:2 with
Et3N 3%) gave 19 (35 mg, 47%) as an amorphous brown
1
solid; H NMR (400 MHz, CD3OD) d 1.92–1.98 (m,
2 H), 2.73 (t, J = 7.3 Hz, 2H), 3.60–3.70 (m, 2H), 3.67–
3.73 (m, 2H), 3.95 (s, 3H), 6.25 (d, J = 10.0 Hz, 2H),
7.10 (s, 1H), 7.20 (d, J = 10.0 Hz, 2H); 13C NMR
(100 MHz, CD3OD) (APT): d 20.0, 36.5, 36.9, 40.5,
48.0, 48.3, 97.1, 120.3, 122.9, 123.5, 130.1, 130.2,
131.1, 131.2, 133.0, 151.0, 154.1, 165.1, 181.0; MS
(APCI): m/z: 320.2 [M+H]+; ESI positive MS Anal.
Calcd for C19H17N3O2+H+ 320.13935. Found:
320.13940.
4.4.2. DNA isolation and electrophoresis. HeLa (5 · 105)
cells were incubated in standard conditions for 24 h at
37 ꢁC, then the medium was replaced with an equal vol-
ume of fresh medium, and the test agent was added at
the indicated concentration. Control cells were grown
under the same conditions with the addition of the sol-
vent alone. After 24 h of incubation, DNA from control
and treated cells was extracted according to the proce-
dure described by Sambrook et al.38 The isolated
DNA was dissolved in TE buffer (Tris 10 mM, pH = 8,
EDTA 1 mM) and analysed by agarose (1%) gel electro-
phoresis. The gel was stained with ethidium bromide
solution (1 lg/mL), transilluminated by UV light, and
fluorescence emission visualized using a CCD camera
coupled to a Bio-Rad Gel Doc XR apparatus.
4.3. Cell cultures
The NCI-H460 human large cell lung carcinoma cell line
(ATCC HTB 177) was cultured in RPMI-1640 (Sigma
Chemical Co.) containing 10% fetal calf serum (FCS,
Biological Industries). The HeLa human cervical carci-
noma (ATCC CCL2) cells were grown in Nutrient Mix-
ture F-12 Ham’s (Sigma Chemical Co.) with 10% heat-
inactivated FCS. HL-60 human promyelocytic leukemia
cells were cultured in RPMI 1640 with 15% heat-inacti-
vated FCS. 100 U/mL penicillin, 100 lg/mL streptomy-
Acknowledgments
cin, and 0.25 lg/mL amphotericin
Chemical Co.) were added to media for HeLa and
HL-60 cell lines.
B
(all Sigma
This work was supported by MIUR F.I.R.B Project
(‘Disegno e sintesi di composti per l’inibizione di enzimi
coinvolti in meccanismi specifici di controllo della prolifer-
azione delle cellule tumorali’). This research has been
developed under the umbrella of D28 COST Action
‘Natural Products as a Source for Discovery, Synthesis,
and Application of New Pharmaceuticals’ WG 0008/03.
This research was partially supported by Ministry of
Sciences and Technology (Spain)-FEDER (Project
CTQ2006-02390/BQU) and the DURSI, Generalitat de
Catalunya (Grant 2005SGR-0603).
4.4. Inhibition growth assays
For cell proliferation assay, cells in the logarithmic
phase of growth were harvested and seeded in duplicate
into six-well plates. Twenty-four hours after seeding,
cells were exposed to the test compound and harvested
72 h after exposure and counted. For NCI-H460,

2438
D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 2431–2438
20. Tohma, H.; Harayama, Y.; Hashizume, M.; Iwata, M.;
Kiyono, Y.; Egi, M.; Kita, Y. J. Am. Chem. Soc. 2003,
Supplementary data
125, 11235.
21. LaBarbera, D. V.; Skibo, E. B. Bioorg. Med. Chem. 2005,
13, 387.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2007.11.063.
´ ´
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