Synthesis of pyrrolo[3,2-h]quinolinones with good photochemotherapeutic activity and no DNA damage

Article abstract of DOI:10.1016/j.bmc.2010.04.080

In the search for new photochemotherapeutic agents, a series of derivatives of the ring system pyrrolo[3,2-h]quinoline - bioisosters of the angular furocoumarin angelicin - were synthesized through a four-step synthetic approach, in reasonable overall yields. Eight of the synthesized derivatives showed a remarkable phototoxicity against a panel of four human tumor cell lines and a great dose UV-A dependence, reaching IC₅₀ values at submicromolar level. The mode of cellular death photoinduced by pyrrolo[3,2-h]quinolines was evaluated through a series of flow cytometric analysis and other tests were performed to clarify their mechanism of action.

Full text of DOI:10.1016/j.bmc.2010.04.080

Bioorganic & Medicinal Chemistry 18 (2010) 4830–4843
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of pyrrolo[3,2-h]quinolinones with good photochemotherapeutic
activity and no DNA damage
Paola Barraja a, Libero Caracausi a, Patrizia Diana a, Anna Carbone a, Alessandra Montalbano a,
b
b
b
c
Girolamo Cirrincione a, , Paola Brun , Giorgio Palù , Ignazio Castagliuolo , Francesco Dall’Acqua ,
*
Daniela Vedaldi c, Alessia Salvador c
a Dipartimento Farmacochimico, Tossicologico e Biologico, Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy
b Dipartimento di Istologia, Microbiologia e Biotecnologie Mediche, Università degli Studi di Padova, via Gabelli 63, 35121 Padova, Italy
c Dipartimento di Scienze Farmaceutiche, Università degli Studi di Padova, Via Marzolo 5, 35131 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In the search for new photochemotherapeutic agents, a series of derivatives of the ring system pyrrol-
Received 11 January 2010
Revised 26 April 2010
Accepted 26 April 2010
Available online 29 April 2010
o[3,2-h]quinoline—bioisosters of the angular furocoumarin angelicin—were synthesized through
a
four-step synthetic approach, in reasonable overall yields. Eight of the synthesized derivatives showed
a remarkable phototoxicity against a panel of four human tumor cell lines and a great dose UV-A depen-
dence, reaching IC50 values at submicromolar level. The mode of cellular death photoinduced by pyrrol-
o[3,2-h]quinolines was evaluated through a series of flow cytometric analysis and other tests were
performed to clarify their mechanism of action.
Keywords:
Pyrrolo[3,2-h]quinolinones
Angelicin heteroanalogues
Photochemotherapy
Phototoxicity
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
bind to DNA pyrimidine bases. DNA adducts are formed by a [2+2]-
photocycloaddition between one of the two photoreactive sites of
Psoralen 1 and its derivatives are furocoumarins, either syn-
thetic or naturally occurring, used in the treatment of human skin
diseases with hyperproliferative and/or autoimmune character in
combination with UV-A light, commonly referred as PUVA therapy.
The photochemotherapy of psoriasis was introduced in clinical use
in the early seventies at the Harvard Medical School of the Massa-
chusetts General Hospital by oral administration of 8-methoxyp-
soralen (8-MOP) and subsequent exposure of the skin to UV-A
irradiation.1 Nowadays, the PUVA therapy is commonly used in
dermatology for the treatment of diseases such as vitiligo, psoria-
sis, plaque parapsoriasis, atopic dermatitis, generalized lichen pla-
nus, pigmentous urticaria, alopecia areata and T-cell lymphoma.2,3
In 1987, Edelson et al. developed the extracorporeal photoche-
motherapy (ECP), called photopheresis, using 8-MOP for the treat-
ment of T-cell cutaneous lymphoma.4 Thanks to photopheresis,
which consists of the reinfusion of UV-A irradiated autologous leu-
kocytes collected by apheresis and extracorporeally incubated with
8-MOP, the photochemotherapy of furocoumarins was used for the
treatment of tumors.5 Furocoumarins are tricyclic aromatic com-
pounds whose planar structure allows the intercalation between
nucleic acid base pairs. Upon UV-A irradiation, they can covalently
psoralen (40,50 double bond of furan ring or 3,4 double bond of pyr-
an ring) and the 5,6 double bond of thymine. The psoralen 40,50-
monoadducts can further photochemically react with a pyrimidine
base on the complementary strand of the DNA, thus leading to
interstrand cross links (ICL).6 Such ICL proved to be the primary
cause of many side effects. Such adverse effects can limit the use
of PUVA and can be divided into short-term side effects that com-
prise nausea, skin phototoxic reactions and immune depression
and long-term ones such as premature skin aging, degeneration
of the dermic collagen and elastic tissues, cataract formation and
also mutagenicity and increased risk of neoplastic diseases.7
Many researchers managed to obtain new derivatives devoid of
side effects but able to maintain the psoralen efficacy. In particular,
the risk of skin neoplastic diseases could be avoided by using
angelicin derivatives, since their geometrical structure makes ICL
impossible.8 The synthesis of heteroanalogues of psoralen 1 and
angelicin
2 represented another promising approach against
undesired side effects (Chart 1). Sulfur and nitrogen isosters, such
as thioangelicins, thienocoumarins, pyrrolocoumarins and furo-
quinolinones, were studied and some of them showed improved
interaction with DNA both in the dark and under UV-A irradiation
if compared to the lead compounds.9 More recently, we reported
the synthesis of the new ring systems pyrrolo[2,3-h]quinolinones
3 and thiopyrano[2,3-e]indolones 4 (Chart 1), which are diaza
* Corresponding author. Tel.: +39 916161606; fax: +39 916169999.
E-mail address: gcirrinc@unipa.it (G. Cirrincione).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.080

P. Barraja et al. / Bioorg. Med. Chem. 18 (2010) 4830–4843
4831
O
The N-sulfonyl derivative 6a, upon heating in basic medium, al-
lowed the isolation of the N-unsubstituted tetrahydroindol-7-one
6c, which, as well as the ethoxycarbonyl derivative 6b, can under-
go nucleophilic reactions with alkyl or aralkyl halides to give N-
substituted derivatives. Thus, reaction in tetrahydrofuran or
dimethylformamide with methyl iodide or benzyl or substituted
benzyl chlorides in the presence of sodium hydride gave deriva-
tives 6d,e,g–l in 70–90% yields. Instead, the N-phenyl derivative
6f was obtained in 66% yield by a modified Ulmann cross-coupling
reaction.14 The variously substituted tetrahydroindoles 6a,b,d–l
were reacted with the Bredereck reagent, tert-butoxy-bis-(dimeth-
O
O
O
O
O
1
2
O
O
H
N
S
ylamino)methane (TBDMAM),15 to give the
a-enaminoketons
7a,b,d–l in excellent yields (80–90%). Compounds 7g,h,l were
unstable and were utilized as crude products for the successive
step. Once obtained the suitable substrates 7 to undergo the final
cyclization, it was planned to react them with phenylsulfonyl-
acetonitrile or cyanoacetamide as a C–C–N 1,3-dinucleophile to
get the tricyclic derivatives 9. This choice resulted from the fact
that in the pyrroloquinolinones of type 3 the presence of the phen-
ylsulfonyl group in position 3 of the pyridone moiety was neces-
sary to obtain compounds with good photoantiproliferative
activity. Therefore, reaction of the dimethylamino substituted
derivatives 7a,b,d–l with phenylsulfonylacetonitrile in refluxing
ethanol, under nitrogen atmosphere, gave the desired angelicin
heteroanalogues 9a,d–m in acceptable yields (35–65%). In the case
of derivative 7a, when the reaction was performed at room tem-
perature, a complex mixture was formed and it was possible to iso-
late the uncyclized intermediate 8, obtained in 40% yield. As the
latter still possesses a cyano group, it strongly suggests that the
reaction initiates with the nucleophilic attack of the methylene
of the phenylsulfonylacetonitrile on the enaminone carbon, the
most electrophilic site of compounds 7. Prolonged refluxing brings
about the conversion of the nitrile moiety to carboxamide, which—
by nucleophilic attack to the ring carbonyl—cyclizes to the pyri-
done ring of the tricyclic system. Basic hydrolysis of the N-phenyl-
sulfonyl derivative 9a led, in excellent yield, to the corresponding
N-unsubstituted derivative 9b. The reaction of 7a with cyanoacet-
amide led to a very complex reaction mixture from which the 7-cy-
ano substituted pyrroloquinolinone 9c was isolated in very poor
yield (13%). Reaction of 7d with the same methylene active com-
pound did not lead to the isolation of the corresponding tricyclic
derivative. This behavior was similar to that observed in the series
of compounds 3 when the corresponding enaminones were reacted
with cyanoacetamide.10a,10b
X
X
N
N
R
X
3
X
4
R
X-X= CH2-CH2 or CH=CH
Chart 1. Structures of psoralen 1, angelicin 2, pyrrolo[2,3-h]quinolinones 3 and
thiopyrano[2,3-e]indolones 4.
and thio-aza analogues of angelicin 2, respectively.10 All deriva-
tives of the latter ring systems showed photoantiproliferative
activity in human tumor cell lines (MCF-7, Jurkat, K-562, LoVo)
and keratinocytes (NCTC-2544), with IC50 values at micromolar
and submicromolar level (3, IC50 0.4–16.4 lM; 4, 0.2–14.8 lM)
and a significant UV-A dose-dependent cytotoxicity. Several deriv-
atives showed a biological activity higher than that of 8-MOP, 5-
methoxypsoralen (5-MOP) and angelicin, used as reference drugs.
However, linear dichroism (LD) studies strongly suggested that
the new derivatives did not efficaciously interact with DNA, thus
indicating a different mechanism from that of the lead compound.
Taking into account the previous satisfactory results on pyrrolo-
quinolinones, and in light of the potent antitumor activity of com-
pounds incorporating the quinoline-2-one portion,11 it was
decided to search for new angelicin heteroanalogues by focusing
on the synthesis of the pyrrolo[3,2-h]quinoline ring system of type
9. The aim was to evaluate the effect of the different condensation
of the pyrrole nucleus to the quinoline moiety on the photobiolog-
ical activity. Herein the paper discusses the synthesis of such a ring
system, along with the results of its remarkable photobiological
activity and the studies aimed at explaining the mode of action.
2. Results and discussion
2.1. Chemistry
2.2. Biology
2.2.1. Physico-chemical properties
The synthetic pathway optimized for pyrroloquinolinones of
type 3, starting from the corresponding tetrahydroindole-4-ones,
has already pointed out the great versatility of the key enaminone
intermediates which could be utilized, as reliable intermediates,
for the desired cyclization due to their electronic features.10a,10b
Tetrahydro-7H-indol-7-ones of type 6, in which it is possible to
introduce the suitable functionality in a position adjacent to car-
bonyl, would therefore represent the ideal building blocks for our
synthesis. Such compounds can be prepared through known proce-
dures. One of them, apparently convenient, led to tetrahydro-7H-
indol-7-one 6d, from 1,2-dioxocyclohexane and a commercially
available acetal, but in low yield.12 Instead, a more convenient
multistep approach involved the annelation of the cyclohexanone
moiety on the pyrrole ring.13 Thus, compounds 5a,b were obtained
in excellent yields from the corresponding pyrrole derivative by an
acylation with succinic anhydride followed by reduction. Cycliza-
tion in trifluoroacetic anhydride gave the tetrahydroindol-7-ones
6a,b in 80–90% yield (Scheme 1).
The absorption and emission spectra of pyrrolo[3,2-h]quino-
lines were measured in DMSO. All compounds absorbed solar radi-
ation spectrum and presented bands in UV-A region; this fact is
fundamental for a photosensitizer. Absorption maxima wavelengths
(see Table 1) exhibited a remarkable bathochromic shift compared
to angelicin (Ang) due to the conjugation with the substituents in
1 and 7 positions. The partition coefficients of the test compounds
were calculated using the computational method as described by
Ghose and Crippen.16 Most pyrrolo[3,2-h]quinolines were highly
hydrophobic, reaching c log P values of 6.01 in the case of 9l.
2.2.2. Photodegradation/photostability
When irradiated in solution, furocoumarins are subjected to
photolysis.17 Thus, absorption spectra of 20
lM pyrrolo[3,2-h]quin-
olinones in DMSO were recorded after increasing UV-A doses. All
spectra were significantly modified by increasing UV-A doses, indi-
cating that all compounds underwent a certain degree of photodeg-
radation (see Fig. 1 of Supplementary data). Figure 1 reports the

4832
P. Barraja et al. / Bioorg. Med. Chem. 18 (2010) 4830–4843
R
N
R
N
O
R1
R1
O
OH
i
6a,b
5a,b
a R=SO2Ph; R1=H
b R=H, R1=CO2Et
c R=R1=H
ii
iv
a,b
b,c
iii
O
R
O
R
R1
N
Me2NHC
N
R1
iv
6d-l
7a,b,d-l
d R=Me, R1=H; e R=Bn, R1=H; f R=Ph, R1=H; g R=BnpMe, R1=H;
h R=BnpOMe, R1=H; i R=Me, R1=CO2Et; j R=Bn, R1=CO2Et;
k R=BnpMe, R1=CO2Et; l R=BnpOMe, R1=CO2Et
v
v
a
rt
O
SO2Ph
CN
2
R2
H
1
3
HC
HC
N
O
SO2Ph
R
N
9
4
N
vi
R1
8
5
7
6
8
9a,c-m
a
vii
9b
9: a R=R2=SO2Ph, R1=H; b R=R1=H, R2=SO2Ph; c R=SO2Ph,
R1=H, R2=CN; d R=Me, R1=H, R2=SO2Ph; e R=Bn, R1=H,
R2=SO2Ph; f R=Ph, R1=H, R2=SO2Ph; g R=BnpMe, R1=H,
R2=SO2Ph; h R=BnpOMe, R1=H, R2=SO2Ph; i R=H, R1=CO2Et,
R2=SO2Ph; j R=Me, R1=CO2Et, R2=SO2Ph; k R=Bn, R1=CO2Et,
R2=SO2Ph; l R= BnpMe, R1=CO2Et, R2=SO2Ph; m R= BnpOMe,
R1=CO2Et, R2=SO2Ph
Scheme 1. Reagents and conditions: (i) trifluoroacetic anhydride, rt, 24 h, 80–90%; (ii) KOH (10%), EtOH, reflux, 2 h, 88%; (iii) for 6d, e, g–l: NaH, THF or DMF, 0 °C to rt, 1 h
then alkyl or aralkylhalide at 0 °C to rt or reflux 2–3 h, 70–90%, for 6f: K2CO3, N-methylpyrrolidone, N2, rt, 1 h, then CuBr, rt 1 h, then PhI, reflux 4 h, 66%; (iv) TBDMAM,
toluene, N2, reflux, 1–48 h, 80–90%; (v) PhSO2CH2CN or H2NCOCH2CN (for 8, yield 40%), EtOH, N2, reflux 1–48 h, 13–65%; (vi) EtOH, reflux, 4 h, 45%; (vii) KOH, EtOH, reflux,
1 h, 90%.
photodegradation of 9c and 9e as an example. The changes are due to
a simple reduction of maxima absorbance, as well as the onset of
new bands and the shift of peak in wavelength. In most spectra,
the presence of isosbestic points can be detected. An isosbestic point
indicates that two absorbing species, the non-degraded compound
and a photoproduct, are in equilibrium in solution.
possible cytotoxicity, and with two different UV-A doses (2.5 and
3.75 J/cm2), using Wood’s lamps emitting principally at 365 nm.
UV-A doses are compatible with those used in PUVA therapy3a
and were chosen to avoid cellular death in irradiated controls.
For phototoxicity experiments, cells were incubated with com-
pounds 9 or Ang for 30 min prior to irradiation. Cellular survival
was monitored by MTT (3-[4,5-dimethylthiazol-2yl] 2,5-diphenyl
tetrazolium bromide) reduction test18 after 72 h of incubation with
compounds in the case of cytotoxicity tests or after 72 h from irra-
diation in the phototoxicity ones. The incubation of these cells with
compounds 9 without irradiation did not induce any change in cel-
lular survival in respect to control (not treated cells). Table 2 shows
IC50 72 h after irradiation. Among pyrrolo[3,2-h]quinolinones,
some compounds were non-phototoxic at the employed concen-
2.2.3. Cellular phototoxicity
The phototoxicity tests were carried out in different human tu-
mor cell lines: K-562 (chronic myeloid leukemia), Jurkat (T-cell
leukemia), LoVo (intestinal adenocarcinoma) and MCF-7 (breast
adenocarcinoma). The same tests were performed in an immortal-
ized cell line of human keratinocytes, NCTC-2544. Cellular survival
experiments were carried out without irradiation to evaluate a

P. Barraja et al. / Bioorg. Med. Chem. 18 (2010) 4830–4843
4833
Table 1
10 lM When detected, the photoantiproliferative activity of pyr-
Physico-chemical properties of pyrrolo[3,2-h]quinolinones
rolo[3,2-h]quinolinones was dependent on UV-A dose and on com-
pound concentration. The most phototoxic compounds, 9j, 9k and
9m, presented micromolar–submicromolar IC50 values that were
similar or even lower (in cell lines derived from solid tumors) than
those of the reference compound, angelicin. A relationship be-
tween structure and activity can be hypothesized starting from
the structure of the most phototoxic compounds 9j, 9k, 9m. The
latter compounds present lipophilic groups (Me; Bn; BnpOMe) in
position 1 and an ethoxycarbonyl moiety in position 2. The pres-
ence of more lipophilic groups (BnpMe, 9l) or the absence of a sub-
stituent at position 1 (9i) reduces the activity. The comparison
between the most active compounds and their analogues devoid
of the ester group in position 2 (9a–h) is very interesting: indeed,
the latter molecules showed a partial or total inactivity. The pres-
ence of the ethoxycarbonyl substituent in position 2 is therefore
extremely important for the phototoxicity of compounds 9.
Compound kmax absa (nm)
e
kmax emissa
(nm)
c log Pb
a
(Mꢀ1 cmꢀ1
)
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
Ang
375
430
442
375
375
430
375
376
439
381
381
381
382
306
42,432
37,138
13,565
37,148
33,182
26,707
40,531
48,050
44,091
41,576
38,202
37,088
35,577
14,602
415
456
479
416
415
413
413
415
461
406
406
405
406
413
2.98 0.66
1.42 0.40
1.97 0.66
1.92 0.40
3.44 0.45
3,79 0.42
3.90 0.45
3.36 0.47
3.43 0.64
4.03 0.64
5.55 0.68
6.01 0.68
5.47 0.69
2.01 0.34
abs, absorbance; emiss, emission; Ang, angelicin.
a
Measured in dimethyl sulfoxide.
Calculated as described in Ref. 16.
b
2.2.4. Externalization of phosphatidylserine
The apoptotic loss of plasma membrane asymmetry with the
exposure of phosphatidylserine (PS) was used to evaluate the
mode of cell death. In fact, this early event of apoptosis was mon-
itored by flow cytometry, using a PS-binding protein, Annexin V,
trations (9a, 9b, 9c, 9d and 9f), whereas others showed high photo-
toxicity9(e, 9g, 9h, 9i, 9j, 9k, 9l and 9m) with IC50 values lower than
Figure 1. Assessment of phosphatidylserine surface exposure. (a) Expression of phosphatidylserine and analysis of propidium iodide staining after 30 min, 1 and 2 h from the
irradiation (2.5 J/cm2) of Jurkat cells in the presence of 1
M 9j, 9k and 9m. Cells were stained with Annexin V-FITC (FL1) and of PI (FL3) and analyzed by flow cytometry. IC,
irradiated control; NIC, non-irradiated control. (b) Percentages of cells detected by Annexin V/PI staining as mean of three different experiments.
l

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P. Barraja et al. / Bioorg. Med. Chem. 18 (2010) 4830–4843
Table 2
Photocytotoxicity of pyrrolo[3,2-h]quinolinones in a panel of human cell lines
a
Compound
IC50
(lM)
Jurkat
K-562
LoVo
MCF-7
NCTC
2.5b
3.75
2.5
3.75
2.5
3.75
2.5
3.75
2.5
3.75
9a
9b
9c
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
9d
9e
9f
>20
1.3 0.2
>20
>20
1.1 0.1
>20
>20
4.8 0.5
>20
>20
3.3 0.5
>20
>20
2.6 0.2
>20
>20
2.2 0.2
>20
>20
4.0 0.6
>20
>20
1.7 0.7
>20
>20
7.5 0.8
>20
>20
5.5 0.6
>20
9g
9h
9i
9j
9k
9l
1.6 0.1
1.6 0.2
3.5 0.2
0.7 0.1
0.8 0.1
1.5 0.1
0.8 0.1
1.0 0.2
1.2 0.2
1.2 0.1
2.5 0.2
0.5 0.1
0.6 0.1
1.2 0.1
0.7 0.1
0.9 0.1
2.7 0.2
3.1 0.3
6.1 0.2
1.0 0.1
0.9 0.1
1.3 0.1
1.0 0.1
1.2 0.1
2.4 0.1
2.5 0.2
5.3 0.3
0.9 0.1
0.6 0.1
1.0 0.1
0.9 0.1
1.0 0.1
2.7 0.1
2.8 0.3
5.5 0.2
1.1 0.1
0.9 0.1
1.6 0.2
1.1 0.1
3.6 0.4
2.3 0.2
2.0 0.2
4.6 0.4
1.0 0.1
0.8 0.1
1.0 0.1
1.0 0.1
1.5 0.3
3.8 0.5
4.2 0.3
6.1 0.2
1.2 0.1
0.9 0.1
1.7 0.1
1.6 0.2
4.4 0.5
2.4 0.2
1.8 0.1
4.3 0.3
1.0 0.1
0.8 0.1
1.4 0.1
1.3 0.1
1.5 0.2
3.3 0.3
4.3 0.3
9.3 0.6
1.2 0.1
1.1 0.1
2.7 0.2
1.9 0.1
4.2 0.5
2.6 0.5
2.7 0.2
7.2 0.7
1.1 0.1
1.0 0.1
2.2 0.2
1.6 0.1
0.9 0.1
9m
Angc
a
b
c
Values are expressed as means SEM of at least three independent experiments.
UV-A doses expressed as J/cm2.
Ang, angelicin reference drug.
conjugated with fluorescein isothiocyanate (FITC). Another probe,
propidium iodide (PI), was added during the analysis for detecting
the integrity of plasmatic membrane. PI penetrated the cell only
with a collapsed plasmatic membrane, intercalated into DNA and
gave the typical red fluorescence. Thus, the cytofluorimetric mea-
sure discriminated between intact (FITCꢀ/PIꢀ), early apoptotic
(FITC+/PIꢀ), late apoptotic/necrotic (FITC+/PI+) or necrotic cells
(PI+).19 As reported in Figure 1, treated cells demonstrated a
time-dependent apoptotic features of cell death. Among the pyr-
rolo[3,2-h]quinolinones tested, compound 9j was the most active,
showing important apoptotic effects 30 min after incubation,
whereas the apoptotic effects of compounds 9k and 9m became
evident 2 h after treatment. A small percentage of necrotic cells
was also detected but the main mode of cell death seemed to be
apoptosis.
2.2.6. Membrane potential
The collapse of mitochondrial membrane potential (
one of the events of mitochondrial dysfunction. The determination
of wmt was carried out through a flow cytometric analysis using
JC-1 as a probe. This test was performed 2 and 4 h after irradiation
of Jurkat cells in the presence of Ang, 9j, 9k and 9m. In normal cells
Dwmt) is
D
(high
Dwmt), JC-1 accumulates selectively in mitochondria and, as
a consequence of the electrochemical gradient, forms red fluores-
cence aggregates which are associated with emission at 590 nm,
whereas JC-1 forms monomers which emit at 530 nm if the mito-
chondrial membrane is depolarized (low
Dw
mt).21 As reported in
Figure 2, a time-dependent increase of percentage of cells with col-
lapsed mitochondrial potential was detected after irradiation in
the presence of Ang, and 9k. For 9m a rapid involvement of mito-
chondria in cell death induction can be hypothesized as a high per-
centage of cells JC-1 monomers can be detected even after only 2 h
from irradiation.
2.2.5. Cell cycle analysis
Conversely, compound 9j did not induce mitochondrial dys-
This test is based on the fact that DNA content is different in
each phase of the cell cycle. Thus, after fixation in ethanol, cells
were stained with PI which made it possible to do rapid determi-
nations of DNA per cell.20 From the data in Table 3, the most impor-
tant modification was the onset of a subG1 peak, 24 h after
treatment. The subG1 phase represents apoptotic cells and the rise
in fluorescence is a consequence of the ordered degradation of DNA
during cell death. This phenomenon is considered to be a later
event than PS externalization, thus producing results that are con-
sistent with the findings of the previous experiment. In contrast
with Ang, which induced a S phase block, the other compounds
produced a decrease of all phase percentages (G1, S and G2-M)
as most cells were detected in subG1 phase.
function. Thus, since the loss of Dwmt precedes the cell death, com-
pounds 9k and 9m induced apoptosis through mitochondrial
depolarization.
2.2.7. ROS production
The mitochondrial membrane depolarization was associated
with mitochondrial production of reactive oxygen species
(ROS).22 The determination of ROS generation during apoptosis
was performed using the fluorescence indicators hydroethidine
(HE) and 20,70-dichlorodihydrofluorescein diacetate (DCFDA),
whose fluorescence appeared if reactive oxygen species were pro-
duced.23 HE was oxidized by superoxide anion into ethidium bro-
mide, which emitted red fluorescence. DCFDA was oxidized to
the fluorescent compound 20,70-dichlorofluorescein (DCF) by a vari-
ety of peroxides including hydrogen peroxide. Results are shown in
Figure 3 and in Figure 2 of Supplementary data. There was a signif-
icant production of ROS for compounds Ang, 9k and 9m, as evi-
denced by the high percentage of HE- and DCF-positive cells,
while there was minor ROS production in the case of 9j. The data
are consistent with mitochondrial membrane depolarization.
Table 3
Cell percentages in each cell cycle phase after 24 h from irradiation (25 J/cm2)
G1
S
G2-M
subG1
NIC
9j
9k
9m
Ang
50.0
16.1
16.1
12.6
12.3
16.5
6.6
8.2
6.1
46.7
25.0
14.0
7.2
6.6
10.2
8.5
63.3
68.5
74.7
30.8
2.2.8. Lysosomal stability assessment
Flow cytometric analysis was performed, by using the fluores-
cent dye acridine orange (AO), in order to investigate the integrity
of lysosomes after irradiation with pyrroloquinolinones. AO is a
NIC, non-irradiated control.

P. Barraja et al. / Bioorg. Med. Chem. 18 (2010) 4830–4843
4835
80
60
40
20
0
100
HE
DCFDA
2 H
4 H
80
60
40
20
0
IC
Ang
9j
9k
9m
IC
Ang
9j
9k
9m
Figure 3. Assessment of mitochondrial dysfunction. Production of ROS in Jurkat
cells 6 h after irradiation (2.5 J/cm2) with 2.5
M Ang or 1 M 9j, 9k and 9m. Cells
l
l
were stained with HE or DCFDA and analyzed by FACS. Values are expressed as
means SD of three independent experiments.
2.2.9. Linear dichroism
A potential interaction with DNA was investigated, as this bio-
molecule represents an important target for antiproliferative activ-
ity of PUVA. In order to understand the binding nature between the
test compounds and DNA, linear dichroism (LD) measurements
were performed on solutions with salmon testes DNA (st-DNA)
and the title compounds at various molar ratios. Indeed, linear
dichroism proved to be an efficient tool to evaluate the binding
mode between a dye and nucleic acids.25 The sign and the extent
of LD signal is related to the orientation of the compound with re-
spect to DNA helix axis. Ang is a DNA intercalant as can be observed
by its LD spectrum: a significant increase of the values of LD of the
DNA band at 260 nm for these drug–DNA complexes was observed,
indicating that the DNA became better oriented due to a stiffening
of the helix upon binding of the drug. The negative LD signals in the
long-wavelength absorption (300–360 nm) of Ang revealed that it
was oriented perpendicularly to the flow field. The investigation
of the LD spectra of three derivatives (9j, 9k and 9m) in the presence
of st-DNA revealed that they gave no LD bands in the 300–450 nm
region despite a strong absorption ( Fig. 5). These observations
strongly suggested that the new derivatives did not interact effica-
ciously without irradiation with the macromolecule, as indeed
demonstrated with natural furocoumarins.
2.2.10. pBR322 DNA strand breaks
Even if pyrrolo[3,2-h]quinolinones did not display a high affin-
ity for DNA, further experiments were carried out to determine
whether the new derivatives were able to photosensitize DNA
strand break activity. Their ability to photodamage DNA was eval-
uated by using supercoiled circular DNA, as it allowed the detec-
tion of structural alterations such as strand breaks. Double-
stranded supercoiled (SC or I form) plasmid is sensitive to damage
by a variety of photosensitizers.26,27 Cleavage of one strand pro-
duces a relaxed, but still double-stranded, open circular (OC or II
form) DNA. Further cleavage of the other strand within a short dis-
tance of the first site of cleavage generates a linear (L or III form)
DNA. In addition to frank strand breaks, the oxidative damage of
purine and/or pyrimidine bases was also evaluated by using the
base excision repair enzymes Formamido pyrimidine glycosilase
(Fpg) and Endonuclease III (Endo III), respectively. Damaged base
recognition was followed by an elimination step, resulting in
DNA breakage. The irradiation of supercoiled plasmid pBR322
was conducted in the presence of Ang, 9j, 9k and 9m ([DNA]/[Com-
pound] = 1:3). After the irradiation, pBR322 aliquots were incu-
bated with Fpg or Endo III at 37 °C for 30 min. DNA samples
were resolved by an electrophoretic run in agarose 1% gel thanks
Figure 2. Assessment of mitochondrial dysfunction. Upper panel: induction of loss
of mitochondrial membrane potential 2 and 4 h after irradiation (2.5 J/cm2) of
Jurkat cells in the presence of 2.5 lM Ang or 1 lM 9j, 9k and 9m. Cells were stained
with JC-1 and analyzed by FACS. Values are expressed as means SD of at least
three independent experiments. Lower panels: examples of flow cytometry analysis
of mitochondrial membrane potential.
lysosomotropic base as well as a metachromatic fluorochrome
exhibiting red fluorescence when highly concentrated, as in the
case of intact lysosomes in which it is retained its protonated form.
When lysosomes are damaged, AO relocates to the cytosol where it
is predominantly in the deprotonated form and it shows green
fluorescence.24 The percentage of cells with intact lysosomes was
evaluated by assessing red fluorescence after AO staining. A
time-dependent increase of lysosomal disruption was assessed
after irradiation in the presence of Ang, 9j, 9k and 9m (Fig. 4), indi-
cating that lysosomes were significantly involved in cell death.

4836
P. Barraja et al. / Bioorg. Med. Chem. 18 (2010) 4830–4843
70
60
50
40
30
20
10
0
0.01
a
2 H
4 H
0.00
-0.01
-0.02
-0.03
-0.04
DNA
[Ang]/[DNA]= 0.02
[Ang]/[DNA]= 0.04
240
260
280
300
320
340
360
380
400
Wavelenght (nm)
IC
Ang
9j
9k
9m
0.01
0.00
b
-0.01
-0.02
-0.03
DNA
[9j]/[DNA]=0.02
[9j]/[DNA]=0.04
250
300
350
400
450
Wavelength (nm)
0.01
0.00
c
-0.01
-0.02
-0.03
DNA
[9k]/[DNA]=0.02
[9k]/[DNA]=0.04
250
300
350
400
450
Wavelength (nm)
0.01
0.00
Figure 4. Assessment of lysosomial dysfunction. Upper panel: percentages of cells
d
stained with AO (acridine orange) were reported and analyzed by flow cytometry 2
and 4 h after irradiation (2.5 J/cm2) in the presence of 2.5
lM Ang or 1 lM 9j, 9k
and 9m. Values are expressed as means SD of at least three independent
experiments. Lower panels: examples of flow cytometry analysis of Jurkat cells
stained with AO.
-0.01
-0.02
-0.03
to the different hydrodynamic properties of the plasmid forms
(Fig. 3 of Supplementary data). The percentages of OC form were
calculated as suggested by Ciulla et al.27 and are summarized in
Figure 6. The results pointed out that Ang and the new compounds
did not generate any formation of open circular or linear bands,
indicating that they did not sensitize single strand breaks. Even
after the treatment with the two base excision enzymes, no DNA
modification was detected for compounds 9 whereas a significant
increase of OC band was detected in the case of Ang thus indicating
an important induced DNA oxidative damage.
DNA
[9m]/[DNA]=0.02
[9m]/[DNA]=0.04
250
300
350
400
450
Wavelength (nm)
Figure 5. Linear dichroism (LD) spectra of mixtures of salmon testes DNA and
compounds Ang (a), 9j (b), 9k (c) and 9m (d) at different [Drug]/[DNA] ratio (0, 0.02
and 0.04).
of oxidative modifications was measured by monitoring the car-
bonyl content, an index of photodamage of proteins.28 Results
are reported in Figure 4 of Supplementary data and demonstrate
that the carbonyl content of BSA and RNAse significantly increased
after irradiation in all three test compounds and with a greater ex-
tent than the Ang induced one.
2.2.11. Protein photodamage
In order to investigate the photosensitizing properties of the ti-
tle compounds towards proteins, solutions containing bovine ser-
um albumin (BSA) or ribonuclease A (RNAseA) as models and the
drug in phosphate buffer were irradiated several times. The degree

P. Barraja et al. / Bioorg. Med. Chem. 18 (2010) 4830–4843
4837
100
80
60
40
20
0
file after UV-A irradiation in the presence of pyrroloquinolinones is
due to the onset of a subG1 peak, indicating a distribution of cells
with a low content in DNA: this is a typical feature of apoptotic
cells in which a nuclear DNA fragmentation occurs. As mitochon-
drion was found to be a key effector of apoptosis and many reports
evidenced a mitochondrial dysfunction brought about by PUVA
therapy,34 a possible involvement of this organelle in photoin-
duced cell death was studied. A clear and rapid involvement of
mitochondria was observed in 9k and 9m apoptosis but a scarce
one in 9j cell death. The involvement of another organelle, such
as lysosome, in inducing apoptosis was examined. Indeed, in addi-
tion to necrosis, the lysosomal destabilization can be associated
with apoptosis: for example, the release of lysosomal hydrolytic
enzymes was found to lead the activation of caspase cascade or
of other lytic cytosol enzymes.24 Lysosomes proved to be involved
in pyrroloquinolinones cell death.
Studies directed to elucidate the molecular target, demon-
strated that pyrroloquinolinones photoinduce oxidation in model
proteins and lipid peroxidation in cell membrane significantly in-
volved in their phototoxicity. Moreover, a potential interaction
with DNA was investigated, as this biomolecule represents an
important target for the antiproliferative activity of PUVA.
Pyrroloquinolinones did not interact efficiently with DNA (no
intercalation); and above all these latter did not induce DNA
photodamage (no DNA strand breaks and no DNA oxidative dam-
ages). This finding represents a significant advantage for a possible
introduction in therapy of pyrroloquinolinones, as the main side
effects of classical psoralen administration are genotoxicity, muta-
genicity and skin cancer.
no enz
+ Fpg
+ Endo III
pBR
Ang
9j
9k
9m
Figure 6. DNA base modifications photoinduced by pyrrolo[3,2-h]quinolinones.
Percentage of open circular form (OC) after irradiation (3.75 J/cm2) of supercoiled
pBR322 form (SC) in the presence of Ang (b), 9j (a), 9k (a) and 9m (a) ([DNA]/
[Drug] = 1:3) and treated with base excision repair enzymes Formamido pyrimidine
glycosilase (Fpg) and Endonuclease III (Endo III). Values are expressed as
means SD of at least three independent experiments.
2.2.12. Lipid peroxidation
Membrane lipids may be a central site of photodamage if
sensitizing agents localize in the membrane bilayer.29 Being
hydrophobic, as their partition coefficients indicated (see Table
1), pyrrolo[3,2-h]quinolinones may be expected to be localized
mainly in plasma and/or in subcellular membranes, making these
structures particularly sensitive to photodamage. The thiobarbitu-
ric assay (TBA test) was used in order to determine a potential lipid
peroxidation after irradiation of Jurkat cells in the presence of test
compounds. The TBA test was performed as described in Section 4.
Briefly, this assay used the reaction of one molecule of malondial-
dehyde (MDA), which is a secondary product of lipid peroxidation,
with two molecules of TBA, forming a pink chromogen, which was
monitored by fluorescence at 553 nm.30 Figure 5 of Supplementary
data shows the results for compounds Ang, 9j, 9k and 9m. Thiobar-
bituric reactive substances (TBARS) were significantly produced in
a concentration-dependent manner when cells were exposed to
the compounds and UV-A. A good correlation was observed be-
tween the extent of lipid peroxidation and the hydrophobic charac-
ter of pyrrolo[3,2-h]quinolinones.
4. Experimental
4.1. Chemistry
All melting points were taken on a Buchi-Tottoli capillary appa-
ratus and were uncorrected; IR spectra were determined, in CHBr3,
with a Jasco FT/IR 5300 spectrophotometer; 1H and 13C NMR
spectra were measured in DMSO-d6 solutions, unless otherwise
specified (TMS as internal reference), at 200 and 50.3 MHz, respec-
tively, using a Bruker Avance II series 200 MHz spectrometer. Col-
umn chromatography was performed with Merck silica gel 230–
400 Mesh ASTM or with a SEPACORE chromatography apparatus
BÜCHI. Elemental analyses (C, H, N) were within 0.4% of the the-
oretical values.
3. Conclusion
1-(Phenylsulfonyl)-1,4,5,6-tetrahydro-7H-indole-7-one
(6a),
ethyl 1,4,5,6,-tetrahydro-7H-indole-7-one-2-carboxylate (6b) and
1,4,5,6-tetrahydro-7H-indole-7-one (6c) were prepared as previ-
ously described and the white solids obtained showed spectro-
scopic data identical to those reported in the literature (6a13a
yield 85%; mp: 115–116 °C; 6b13b yield 82%; mp: 96–97 °C; 6c13a
yield 85%; mp: 90–92 °C).
In this report, the synthesis of pyrrolo[3,2-h]quinolinones 9 new
photochemotherapeutic derivatives for the treatment of hyperpro-
liferative diseases is described. These compounds were synthesised
with the aim of modulating the long-term side effects related to
the PUVA therapy, in particular the DNA photodamage. Most of
the pyrrolo[3,2-h]quinolinones were phototoxic and from a struc-
ture–activity relationship point of view, the presence of a lipophilic
group in position 1 seemed to be important as well as an ethoxy-
carbonyl moiety in position 2, a group needed for the phototoxicity
of other analogues of angelicin.31 It is worthy to observe that all
these compounds are not fully unsaturated as in the case of angel-
icin; on the contrary, they are dihydro-derivatives, such as the
most phototoxic compounds of series 3.10a,10b
The evaluation of the photoinduced cellular death by pyrrolo-
quinolinones was performed. Cell cycle analysis of cells irradiated
in the presence of compounds 9 were carried out. It is reported that
significant modifications on cell cycle after PUVA treatment de-
pends on the type of cells. For example, a G1 phase arrest was ob-
served in NCTC,32 whereas a G2-M phase block was detected in
Karpas 299 T-lymphoma cells.33 The main change in cell cycle pro-
4.1.1. Preparation of 1-substituted 1,4,5,6-tetrahydro-7H-
indole-7-ones (6d,e,g–l)
To a solution of the suitable ketone 6b or 6c (15 mmol) dis-
solved in anhydrous THF or DMF (20 mL), NaH (0.64 g, 16 mmol)
was added at 0 °C and the reaction mixture was stirred at rt. After
1 h the suitable alkyl or aralkyl halide (16 mmol) was added at 0 °C
and the reaction mixture was stirred at rt or refluxed for 2–4 h.
Then, the reaction mixture was poured onto crushed ice and the
precipitate was filtered off. In absence of precipitate the aqueous
solution was extracted with dichloromethane (DCM, 3ꢁ 50 mL)
and the organic layers were separated, dried over sodium sulfate
and the solvent removed in vacuo. Column chromatography of
the residue, using DCM as eluent, gave the expected product.

4838
P. Barraja et al. / Bioorg. Med. Chem. 18 (2010) 4830–4843
4.1.2. 1-Methyl-1,4,5,6-tetrahydro-7H-indole-7-one (6d)
This compound was obtained from the reaction of 6c with iodo-
85 °C; IR: t ;
1709 (CO), 1655 (CO) cmꢀ1 1H NMR: d 1.21 (3H, t,
J = 7.1 Hz, CH3), 1.93–2.05 (2H, m, CH2), 2.48 (2H, t, J = 6.0 Hz,
CH2), 2.74 (2H, t, J = 6.0 Hz, CH2), 4.20 (2H, q, J = 7.1 Hz, CH2),
6.01 (2H, s, CH2), 6.84 (1H, s, H-3), 6.93–7.31 (5H, m, Ar); 13C
NMR: d 13.9 (q), 22.9 (t), 24.1 (t), 39.6 (t), 48.7 (t), 60.5 (t), 99.4
(s), 115.1 (d), 126.0 (d), 126.8 (d), 128.3 (d), 130.6 (s), 135.9 (s),
139.5 (s), 160.5 (CO), 190.0 (CO). Anal calcd for C18H19NO3: C,
72.71; H, 6.44; N, 4.71. Found: C, 72.45; H, 6.69; N, 4.75.
methane in THF after 3 h at rt: brown oil; yield 90%; IR:
t 1635
(CO) cmꢀ1 1H NMR: d 1.89–2.01 (2H, m, CH2), 2.35 (2H, t,
;
J = 6.0 Hz, CH2), 2.65 (2H, t, J = 6.0 Hz, CH2), 3.82 (3H, s, CH3),
5.96 (1H, d, J = 2.5 Hz, H-3), 7.03 (1H, d, J = 2.5 Hz, H-2); 13C
NMR: d 23.3 (t), 24.8 (t), 35.9 (q), 38.7 (t), 106.3 (d), 126.2 (s),
130.7 (d), 136.8 (s), 187.7 (CO). Anal calcd for C9H11NO: C, 72.46;
H, 7.43; N, 9.39. Found: C, 72.56; H, 7.69; N, 9.30.
4.1.8. Ethyl 1-(p-methyl-benzyl)-1,4,5,6-tetrahydro-7H-indole-
7-one-2-carboxylate (6k)
4.1.3. 1-Benzyl-1,4,5,6-tetrahydro-7H-indole-7-one (6e)
This compound was obtained from the reaction of 6c with ben-
This compound was obtained from the reaction of 6b with p-
methyl-benzylchloride in DMF after 2 h at rt: white solid; yield
zylchloride in DMF after 2 h at rt: brown oil; yield 90%; IR:
t 1637
(CO) cmꢀ1 1H NMR: d 1.88–2.01 (2H, m, CH2), 2.35 (2H, t,
;
73%; mp: 65–66 °C; IR: t ;
1710 (CO), 1651 (CO) cmꢀ1 1H NMR: d
J = 6.0 Hz, CH2), 2.68 (2H, t, J = 6.0 Hz, CH2), 5.49 (2H, s, CH2), 6.05
(1H, d, J = 2.5, H-3), 7.12–7.34 (6H, m, Ar and H-2); 13C NMR: d
23.4 (t), 24.8 (t), 38.8 (t), 50.8 (t), 107.2 (d), 125.5 (s), 126.9 (d),
127.2 (d), 128.4 (d), 130.4 (d), 137.5 (s), 138.9 (s), 187.7 (CO). Anal
calcd for C15H15NO: C, 79.97; H, 6.71; N, 6.22. Found: C, 80.30; H,
7.00; N, 6.03.
1.22 (3H, t, J = 7.1 Hz, CH3), 1.92–2.04 (2H, m, CH2), 2.22 (3H, s,
CH3), 2.47 (2H, t, J = 6.0 Hz, CH2), 2.72 (2H, t, J = 6.0 Hz, CH2), 4.20
(2H, q, J = 7.1 Hz, CH2), 5.96 (2H, s, CH2), 6.82 (1H, s, H-3), 6.85
(2H, d, J = 8.0 Hz, H-300 and H-500), 7.06 (2H, d, J = 8.0 Hz, H-200 and
H-600); 13C NMR: d 14.0 (q), 20.5 (q), 22.9 (t), 24.1 (t), 39.6 (t),
48.4 (t), 60.5 (t), 115.0 (d), 126.0 (d), 127.1 (s), 128.8 (d), 129.7
(s), 135.4 (s), 135.8 (s), 135.9 (s), 160.2 (CO), 190.0 (CO). Anal calcd
for C19H21NO3: C, 73.29; H, 6.80; N, 4.50. Found: C, 73.45; H, 6.70;
N, 4.24.
4.1.4. 1-(p-Methyl-benzyl)-1,4,5,6-tetrahydro-7H-indole-7-one
(6g)
This compound was obtained from the reaction of 6c with p-
methyl-benzylchloride in DMF after 2 h at rt: white solid; yield
75%; mp: 80–82 °C; IR:
t ;
1637 (CO) cmꢀ1 1H NMR: d 1.88–2.00
4.1.9. Ethyl 1-(p-methoxy-benzyl)-1,4,5,6-tetrahydro-7H-
indole-7-one-2-carboxylate (6l)
(2H, m, CH2), 2.24 (3H, s, CH3), 2.35 (2H, t, J = 6.3 Hz, CH2), 2.67
(2H, t, J = 6.3 Hz, CH2), 5.43 (2H, s, CH2), 6.03 (1H, d, J = 2.5 Hz, H-
3), 7.05 (2H, d, J = 7.5 Hz, H-300 and H-500), 7.10 (2H, d, J = 7.5 Hz,
H-200 and H-600), 7.20 (1H, d, J = 2.5 Hz, H-2); 13C NMR: d 20.6 (q),
23.4 (t), 24.8 (t), 38.8 (t), 50.6 (t), 107.1 (d), 125.4 (s), 127.1 (d),
128.9 (d), 130.2 (d), 135.8 (s), 136.4 (s), 137.4 (s), 187.7 (CO). Anal
calcd for C16H17NO: C, 80.30; H, 7.16; N, 5.85. Found: C, 80.60; H,
7.06; N, 5.50.
This compound was obtained from the reaction of 6b with p-
methoxy-benzylchloride in DMF after 2 h at rt: yellow oil; yield
75%; IR:
t ;
1711 (CO), 1651 (CO) cmꢀ1 1H NMR: d 1.23 (3H, t,
J = 7.1 Hz, CH3), 1.95–2.04 (2H, m, CH2), 2.47 (2H, t, J = 5.9 Hz,
CH2), 2.72 (2H, t, J = 5.9 Hz, CH2), 3.69 (1H, s, CH3), 4.22 (2H, q,
J = 7.1 Hz, CH2), 5.92 (2H, s, CH2), 6.77–6.97 (5H, m, Ar and H-3);
13C NMR: d 14.0 (q), 22.9 (t), 24.1 (t), 39.6 (t), 47.9 (t), 54.9 (q),
60.5 (t), 113.7 (d), 115.1 (d), 127.1 (s), 127.6 (d), 129.6 (s), 130.7
(s), 135.4 (s), 158.2 (s), 160.3 (CO), 190.0 (CO). Anal calcd for
C19H21NO4: C, 69.71; H, 6.47; N, 4.28. Found: C, 70.02; H, 6.70; N,
4.14.
4.1.5. 1-(p-Methoxy-benzyl)-1,4,5,6-tetrahydro-7H-indole-7-
one (6h)
This compound was obtained from the reaction of 6c with p-
methoxy-benzylchloride in DMF after 2 h at rt: white solid; yield
75%; mp: 82–84 °C; IR:
t ;
1637 (CO) cmꢀ1 1H NMR: d 1.90–2.00
(2H, m, CH2), 2.36 (2H, t, J = 6.0 Hz, CH2), 2.66 (2H, t, J = 6.0 Hz,
CH2), 3.70 (3H, s, CH3), 5.40 (2H, s, CH2), 6.02 (1H, d, J = 2.5 Hz,
H-3), 6.85 (2H, d, J = 7.5 Hz, H-300 and H-500), 7.16 (2H, d,
J = 7.5 Hz, H-200 and H-600), 7.20 (1H, d, J = 2.5 Hz, H-2); 13C NMR:
d 23.4 (t), 24.8 (t), 38.8 (t), 50.2 (t), 54.9 (q), 107.1 (d), 113.7 (d),
125.4 (s), 128.6 (d), 130.1 (d), 130.8 (s), 137.4 (s), 158.5 (s), 187.7
(CO). Anal calcd for C16H17NO2: C, 75.27; H, 6.71; N, 5.49. Found:
C, 75.18; H, 7.02; N, 5.68.
4.1.10. Preparation of 1-phenyl-1,4,5,6-tetrahydro-7H-indole-7-
one (6f)
To a solution of 6c (1.3 g, 9.63 mmol) in N-methyl-pyrrolidone
(19 mL), K2CO3 (2.0 g, 14.45 mmol) was added under nitrogen
atmosphere and the reaction mixture was stirred at rt for 1 h. Then
CuBr (2.8 g, 19.26 mmol) was added and the reaction stirred for 1 h
and finally iodobenzene (4.0 mL, 35.63 mmol) was added and the
reaction refluxed for 4 h. After cooling, HCl (5%, 17 mL) was added
to the reaction mixture and stirred for 1 h, then AcOEt (25 mL) was
added and stirred for further 30 min. The reaction mixture was fil-
tered through celite and washed with AcOEt (25 mL). The organic
layer was shaken for 1 h with ice and NaCl, separated, dried over
sodium sulfate and the solvent removed in vacuo to give a brown
4.1.6. Ethyl 1-methyl-1,4,5,6-tetrahydro-7H-indole-7-one-2-car-
boxylate (6i)
This compound was obtained from the reaction of 6b with iodo-
methane in DMF after 2 h under reflux: brown solid; yield 88%;
t ;
1707 (CO), 1651 (CO) cmꢀ1 1H NMR: d 1.29
solid; yield 66%; mp: 66–68 °C; IR:
t ;
1650 (CO) cmꢀ1 1H NMR: d
mp: 51–52 °C; IR:
(3H, t, J = 7.1 Hz, CH3), 1.91–2.03 (2H, m, CH2), 2.47 (2H, t,
J = 6.1 Hz, CH2), 2.68 (2H, t, J = 6.1 Hz, CH2), 4.13 (3H, s, CH3),
4.26 (2H, q, J = 7.1 Hz, CH2), 6.71 (1H, s, H-3); 13C NMR: d 14.1
(q), 22.8 (t), 24.1 (t), 34.1 (q), 39.6 (t), 60.4 (t), 114.0 (d), 127.5
(s), 129.9 (s), 134.6 (s), 160.4 (CO), 190.1 (CO). Anal calcd for
C12H15NO3: C, 65.14; H, 6.83; N, 6.33. Found: C, 64.90; H, 7.10;
N, 6.68.
1.96–2.09 (2H, m, CH2), 2.39 (2H, t, J = 6.1 Hz, CH2), 2.76 (2H, t,
J = 6.1 Hz, CH2), 6.23 (1H, d, J = 2.6 Hz, H-3), 7.23–7.45 (6H, m, Ar
and H-2); 13C NMR: d 23.5 (t), 24.5 (t), 38.9 (t), 108.6 (d), 125.5
(d), 126.1 (s), 127.0 (d), 128.4 (d), 131.1 (d), 130.1 (s), 139.7 (s),
186.3 (CO). Anal calcd for C14H13NO: C, 79.59; H, 6.20; N, 6.63.
Found: C, 79.30; H, 6.00; N, 6.80.
4.1.11. Preparation of 1-substituted 6-[(dimethylamino)
methylene]-1,4,5,6-tetrahydro-7H-indole-7-one (7a,b,d–l)
To a solution of 6a,b,d–l (5.3 mmol) in anhydrous toluene
(10 mL) TBDMAM (3.31 mL, 16 mmol) was added under nitrogen
4.1.7. Ethyl 1-benzyl-1,4,5,6-tetrahydro-7H-indole-7-one-2-car-
boxylate (6j)
This compound was obtained from the reaction of 6b with ben-
zylchloride in DMF after 3 h at rt: brown solid; yield 70%; mp: 84–

P. Barraja et al. / Bioorg. Med. Chem. 18 (2010) 4830–4843
4839
atmosphere and the reaction mixture was refluxed. After cooling,
the precipitate was filtered and shaken in diethyl ether (25 mL), fil-
tered and air dried.
indole-7-one (7h), ethyl 1-(p-methoxybenzyl)-6-[(dimethyl-
amino)methylene]-1,4,5,6-tetrahydro-7H-indole-7-one-2-car-
boxylate (7l). These compounds, obtained from
6g, 6h, 6l,
respectively, after 3 h reflux as brown oils, were unstable and were
utilized for the successive step without purification.
4.1.11.1. 6-[(Dimethylamino)methylene]-1-(phenylsulfonyl)-
1,4,5,6-tetrahydro-7H-indole-7-one (7a). This product was ob-
tained from 6a after 4 h reflux and precipitated as yellow solid;
4.1.11.7. Ethyl 6-[(dimethylamino)methylene]-1-methyl-1,4,
5,6-tetrahydro-7H-indole-7-one-2-carboxylate (7i). This product
was obtained from 6i after 3 h reflux and precipitated as brown so-
yield 90%; mp: 116–118 °C; IR: t
1637 (CO) cmꢀ1; 1H NMR (CDCl3):
d 2.58 (2H, t, J = 6.5 Hz, CH2), 2.84 (2H, t, J = 6.5 Hz, CH2), 3.00 (6H,
s, 2ꢁ CH3), 6.16 (1H, d, J = 2.7 Hz, H-3), 7.27 (1H, s, CH), 7.37–7.52
(3H, m, Ar), 7.61 (1H, d, J = 2.7 Hz, H-2), 8.05 (2H, d, J = 7.2 Hz, H-20
and H-60); 13C NMR (CDCl3): d 23.5 (t), 24.2 (t), 43.4 (q), 102.9 (s),
109.8 (d), 127.8 (d), 127.9 (d), 128.5 (d), 130.3 (s), 133.1 (d), 138.1
(s), 139.6 (s), 148.6 (d), 176.6 (CO). Anal calcd for C17H18N2O3S: C,
61.80; H, 5.49; N, 8.48. Found: C, 62.10; H, 5.26; N, 8.20.
lid; yield 80%; mp: 50–52 °C; IR:
t ;
1707 (CO), 1631 (CO) cmꢀ1 1H
NMR: d 1.27 (3H, t, J = 7.1 Hz, CH3), 2.54 (2H, t, J = 7.3 Hz, CH2), 2.76
(2H, t, J = 7.3 Hz, CH2), 3.08 (6H, s, 2ꢁ CH3), 4.17 (3H, s, CH3), 4.23
(2H, q, J = 7.1 Hz, CH2), 6.66 (1H, s, H-3), 7.39 (1H, s, CH); 13C NMR:
d 14.2 (q), 22.4 (t), 24.1 (t), 33.9 (q), 43.3 (q), 59.9 (t), 103.7 (s),
113.6 (d), 125.4 (s), 129.6 (s), 132.2 (s), 148.8 (d), 160.5 (CO),
178.8 (CO). Anal calcd for C15H20N2O3: C, 65.20; H, 7.30; N,
10.14. Found: C, 65.54; H, 7.12; N, 10.05.
4.1.11.2. Ethyl 6-[(dimethylamino)methylene]-1,4,5,6-tetrahy-
dro-7H-indole-7-one-2-carboxylate (7b). This product was ob-
tained from 6b after 2 h reflux and precipitated as yellow solid;
4.1.11.8. Ethyl 1-benzyl-6-[(dimethylamino)methylene]-1,4,5,6-
tetrahydro-7H-indole-7-one-2-carboxylate (7j). This product
was obtained from 6j after 1 h reflux and precipitated as yellow so-
yield 80%; mp: 209–211 °C; IR:
t 3421 (NH), 1703 (CO), 1635
(CO) cmꢀ1 1H NMR: d 1.27 (3H, t, J = 7.1 Hz, CH3), 2.58 (2H, t,
;
J = 6.7 Hz, CH2), 2.88 (2H, t, J = 6.7 Hz, CH2), 3.07 (6H, s, 2ꢁ CH3),
4.21 (2H, q, J = 7.1 Hz, CH2), 6.60 (1H, s, H-3), 7.36 (1H, s, CH),
12.01 (1H, s, NH); 13C NMR: d 14.2 (q), 22.1 (t), 24.2 (t), 43.2 (q),
59.9 (t), 102.7 (s), 112.6 (d), 125.1 (s), 129.8 (s), 132.8 (s), 148.2
(d), 160.3 (CO), 177.7 (CO). Anal calcd for C14H18N2O3: C, 64.10;
H, 6.92; N, 10.68. Found: C, 64.46; H, 7.26; N, 10.30.
lid; yield 85%; mp: 101–103 °C; IR: t ;
1703 (CO), 1635 (CO) cmꢀ1
1H NMR: d 1.20 (3H, t, J = 7.0 Hz, CH3), 2.61 (2H, t, J = 6.1 Hz,
CH2), 2.83 (2H, t, J = 6.1 Hz, CH2), 3.06 (6H, s, 2ꢁ CH3), 4.16 (2H,
q, J = 7.0 Hz, CH2), 6.13 (2H, s, CH2), 6.78 (1H, s, H-3), 6.92–7.25
(5H, m, Ar), 7.38 (1H, s, CH); 13C NMR: d 14.1 (q), 22.3 (t), 24.0
(t), 43.3 (q), 48.2 (t), 59.9 (t), 103.5 (s), 114.7 (d), 125.0 (s), 126.0
(d), 126.5 (d), 128.2 (d), 130.2 (s), 131.9 (s), 139.7 (s), 149.1 (d),
160.3 (CO), 178.6 (CO). Anal calcd for C21H24N2O3: C, 71.57; H,
6.86; N, 7.95. Found: C, 71.84; H, 7.01; N, 7.63.
4.1.11.3. 6-[(Dimethylamino)methylene]-1-methyl-1,4,5,6-tet-
rahydro-7H-indole-7-one (7d). This product was obtained from
6d after 4 h reflux and precipitated as brown solid; yield 90%;
mp: 49–51 °C; IR:
t
1633 (CO) cmꢀ1
;
1H NMR: d 2.55 (2H, t,
4.1.11.9. Ethyl 1-(p-methyl-benzyl)-6-[(dimethylamino)methy-
lene]-1,4,5,6-tetrahydro-7H-indole-7-one-2-carboxylate (7k). This
product was obtained from 6k after 4 h reflux and precipitated
as yellow solid and purified on a silica pad (DCM); yield 80%;
J = 6.4 Hz, CH2), 2.83 (2H, t, J = 6.4 Hz, CH2), 3.02 (6H, s, 2ꢁ CH3),
3.83 (1H, s, CH3), 5.87 (1H, d, J = 2.5 Hz, H-3), 6.86 (1H, d,
J = 2.5 Hz, H-2), 7.23 (1H, s, CH); 13C NMR: d 23.0 (t), 24.7 (t),
35.8 (q), 43.1 (q), 103.5 (s), 105.3 (d), 127.3 (s), 128.6 (d), 131.8
(s), 146.6 (d), 178.4 (CO). Anal calcd for C12H16N2O: C, 70.56; H,
7.90; N, 13.71. Found: C, 70.30; H, 7.66; N, 13.42.
mp: 84–86 °C; IR:
t ;
1709 (CO), 1631 (CO) cmꢀ1 1H NMR: d 1.22
(3H, t, J = 7.1 Hz, CH3), 2.56–2.68 (4H, m, 2ꢁ CH2), 2.64 (3H, s,
CH3), 3.04 (6H, s, 2ꢁ CH3), 4.22 (2H, q, J = 7.1 Hz, CH2), 6.04 (2H,
s, CH2), 6.82 (2H, d, J = 7.9 Hz, Ar), 6.87 (1H, s, H-3), 7.05 (2H, d,
J = 7.9 Hz, Ar), 7.61 (1H, s, CH); 13C NMR: d 14.0 (q), 21.6 (q), 21.8
(t), 43.2 (q), 48.2 (t), 60.3 (t), 113.5 (s), 115.0 (d), 126.0 (d), 128.8
(d), 131.0 (s), 133.1 (s), 135.8 (s), 136.2 (s), 152.6 (d), 160.2 (CO),
180.01 (CO). Anal calcd for C22H26N2O3: C, 72.11; H, 7.15; N,
7.64. Found: C, 72.40; H, 7.38; N, 7.30.
4.1.11.4. 1-Benzyl-6-[(dimethylamino)methylene]-1,4,5,6-tetra-
hydro-7H-indole-7-one (7e). This product was obtained from 6e
after 48 h reflux and precipitated as brown solid; yield 80%; mp:
116–118 °C; IR:
t ;
1633 (CO) cmꢀ1 1H NMR: d 2.57 (2H, t,
J = 6.8 Hz, CH2), 2.83 (2H, t, J = 6.8 Hz, CH2), 3.02 (6H, s, 2ꢁ CH3),
5.56 (2H, s, CH2), 5.94 (1H, d, J = 2.5 Hz, H-3), 7.02 (1H, d,
J = 2.5 Hz, H-2), 7.12–7.28 (6H, m, Ar and CH); 13C NMR: d 23.0
(t), 24.6 (t), 43.1 (q), 50.5 (t), 103.4 (s), 106.2 (d), 126.7 (s), 126.9
(d), 127.0 (d), 128.0 (d), 128.2 (d), 132.3 (s), 139.6 (s), 147.0 (d),
178.3 (CO). Anal calcd for C18H20N2O: C, 77.11; H, 7.19; N, 9.99.
Found: C, 77.00; H, 6.94; N, 10.15.
4.1.12. Preparation of 1,7-disubstituted 1,4,5,9-tetrahydro-8H-
pyrrolo[3,2-h]quinoline-8-one (9a–m)
To a suspension of 7a,b,d–l (4 mmol) in anhydrous ethanol
(50 mL), the suitable cyanomethylene compound (6 mmol) in
anhydrous ethanol (60 mL) was added dropwise under nitrogen
atmosphere. After the addition the reaction mixture was refluxed.
Upon cooling, the precipitate formed was filtered and purified by
recrystallization or by column chromatography (Sepacore Büchi)
using DCM/AcOEt 9:1 as eluent.
4.1.11.5. 6-[(Dimethylamino)methylene]-1-phenyl-1,4,5,6-tet-
rahydro-7H-indole-7-one (7f). This product was obtained from
6f after 24 h reflux and precipitated as brown solid; yield 80%;
mp: 129–131 °C; IR:
t ;
1637 (CO) cmꢀ1 1H NMR: d 2.63 (2H, t,
J = 6.4 Hz, CH2), 2.91 (2H, t, J = 6.4 Hz, CH2), 3.02 (6H, s, 2ꢁ CH3),
6.13 (1H, d, J = 2.7 Hz, H-3), 7.08 (1H, d, J = 2.7 Hz, H-2), 7.18 (1H,
s, CH), 7.24–7.42 (5H, m, Ar); 13C NMR: d 23.2 (t), 24.2 (t), 43.1
(q), 102.9 (s), 107.6 (d), 125.0 (d), 126.3 (d), 127.7 (s), 128.2 (d),
128.9 (d), 134.2 (s), 140.3 (s), 147.0 (d), 176.6 (CO). Anal calcd
for C17H18N2O: C, 76.66; H, 6.81; N, 10.52. Found: C, 77.00; H,
7.05; N, 10.17.
4.1.12.1. 1,7-bis(Phenylsulfonyl)-1,4,5,9-tetrahydro-8H-pyrrol-
o[3,2-h]quinolin-8-one (9a). This product was obtained from the
reaction of 7a with phenylsulfonylacetonitrile after 48 h reflux.
The yellow solid precipitated was recrystallized from ethanol;
yield 65%; mp: 349–351 °C; IR:
t ;
3218 (NH), 1635 (CO) cmꢀ1 1H
NMR: d 2.70–2.86 (4H, m, 2ꢁ CH2), 6.83 (1H, d, J = 2.0 Hz, H-3),
7.54–7.71 (7H, m, Ar and H-2), 7.91–7.98 (4H, m, Ar), 8.21 (1H, s,
H-6), 11.90 (1H, s, NH); 13C NMR: d 20.4 (t), 25.1 (t), 99.5 (d),
111.1 (s), 113.5 (d), 123.6 (s), 124.9 (s), 126.6 (d), 127.9 (d),
128.4 (s), 128.8 (d), 129.8 (d), 131.8 (s), 133.2 (d), 133.7 (d),
140.4 (s), 141.3 (s), 143.9 (d), 156.3 (CO). Anal calcd for
4.1.11.6. 6-[(Dimethylamino)methylene]-1-(p-methylbenzyl)-
1,4,5,6-tetrahydro-7H-indole-7-one (7g), 6-[(dimethylamino)
methylene]-1-(p-methoxybenzyl)-1,4,5,6-tetrahydro-7H-

4840
P. Barraja et al. / Bioorg. Med. Chem. 18 (2010) 4830–4843
C23H18N2O5S2: C, 59.21; H, 3.89; N, 6.00. Found: C, 59.00; H, 4.01;
N, 5.89. When the same reaction was carried out at rt was obtained
a complex mixture which, after column chromatography (eluent
DCM/EtOAc 9:1), furnished intermediate 8 as brown oil.
yield 55%; mp: 251–252 °C; IR: t ;
3359 (NH), 1654 (CO) cmꢀ1 1H
NMR: d 2.68 (2H, t, J = 6.7 Hz, CH2), 2.79 (2H, t, J = 6.7 Hz, CH2),
6.34 (1H, d, J = 2.7 Hz, H-3), 7.29 (1H, d, J = 2.7 Hz, H-2), 7.32–
7.67 (8H, m, Ar), 7.90–7.96 (2H, d, J = 8.0 Hz, H-20 and H-60), 8.15
(1H, s, H-6), 10.10 (1H, s, NH); 13C NMR: d 22.0 (t), 26.5 (t), 99.5
(d), 108.6 (s), 109.5 (d), 112.1 (s), 119.0 (s), 121.6 (s), 124.5 (d),
127.4 (d), 127.9 (d), 128.7 (d), 129.3 (d), 130.9 (d), 133.0 (d),
133.6 (s), 138.6 (s), 140.9 (s), 156.4 (CO). Anal calcd for
C23H18N2O3S: C, 68.64; H, 4.51; N, 6.96. Found: C, 68.40; H, 4.80;
N, 7.06.
4.1.12.2. 1-Phenylsulfonyl-6-[2-(phenylsulfonyl)-propanenitrile]-
1,4,5,6-tetrahydro-7H-indole-7-one (8). Yield 40%; IR:
t 2362
(CN), 1693 (CO) cmꢀ1
;
1H NMR: d 2.53–2.72 (4H, m, 2ꢁ CH2),
5.86 (1H, d, J = 10.7 Hz, CH), 6.02 (1H, d, J = 10.7 Hz, CH), 6.36
(1H, d, J = 2.6 Hz, H-3), 7.15–8.08 (11H, m, Ar and H-2); 13C NMR:
d 23.7 (t), 34.3 (t), 111.0 (d), 125.5 (d), 128.1 (d), 128.4 (d), 128.5
(d), 128.8 (s), 129.2 (s), 129.3 (d), 131.6 (d), 133.3 (d), 134.4 (s),
134.6 (d), 136.8 (d), 137.6 (s), 147.7 (s), 143.7 (s), 162.8 (CO). Anal
calcd for C23H18N2O5S2: C, 59.21; H, 3.89; N, 6.00. Found: C, 59.04;
H, 4.12; N, 6.12.
4.1.12.7. 1-(p-Methyl-benzyl)-7-(phenylsulfonyl)-1,4,5,9-tetrahy
dro-8H-pyrrolo[3,2-h]quinolin-8-one (9g). This product was ob-
tained from the reaction of 7g with phenylsulfonylacetonitrile after
32 h reflux. The crude precipitate was purified by column chroma-
tography (Sepacore Büchi) using DCM/AcOEt 9:1 as eluent. Yellow
By refluxing in ethanol this intermediate, the cyclized com-
pound 9a, in 45% yield, was obtained.
solid; yield 40%; mp: 178–180 °C; IR: t ;
3217 (NH), 1626 (CO) cmꢀ1
1H NMR: d 2.19 (3H, s, CH3), 2.64 (2H, t, J = 7.5 Hz, CH2), 2.80
(2H, t, J = 7.5 Hz, CH2), 5.71 (2H, s, CH2), 6.04 (1H, d, J = 2.5 Hz, H-
3), 7.02–7.10 (5H, m, Ar and H-2), 7.55–7.67 (3H, m, Ar), 7.90–
7.95 (2H, m, Ar), 8.00 (1H, s, H-6), 11.56 (1H, s, NH); 13C NMR: d
20.6 (q), 21.6 (t), 50.7 (t), 99.5 (d), 105.2 (d), 107.2 (d), 124.6 (s),
126.9 (d), 127.7 (d), 128.8 (d), 128.9 (d), 129.8 (s), 133.1 (d),
136.2 (s), 137.2 (s), 140.5 (s), 141.2 (s), 152.5 (s), 158.0 (s), 174.0
(CO). Anal calcd for C25H22N2O3S: C, 69.75; H, 5.15; N, 6.51. Found:
C, 69.55; H, 5.02; N, 6.64.
4.1.12.3. 1-(Phenylsulfonyl)-1,4,5,9-tetrahydro-8H-pyrrolo[3,2-
h]quinolin-7-carbonitrile-8-one (9c). This product was obtained
from the reaction of 7a with cyanoacetamide after 32 h reflux.
The yellow solid precipitated was recrystallized from ethanol;
yield 13%; mp: 309–311 °C; IR:
t 3309 (NH), 2220 (CN), 1653
(CO) cmꢀ1
;
1H NMR: d 2.69–2.92 (4H, m, 2ꢁ CH2), 6.85 (1H, s, H-
3), 7.61–7.77 (4H, m, Ar and H-2), 7.96 (2H, d, J = 8.0 Hz, H-20
and H-60), 8.04 (1H, s, H-6), 12.06 (1H, s, NH); 13C NMR: d 20.3
(t), 25.0 (t), 99.5 (d), 112.2 (s), 113.5 (d), 117.0 (s), 125.0 (s),
126.7 (d), 128.3 (s), 129.8 (d), 131.9 (s), 133.8 (d), 138.8 (s),
141.3 (s), 148.4 (d), 159.5 (CO). Anal calcd for C18H13N3O3S: C,
61.53; H, 3.73; N, 11.96. Found: C, 61.32; H, 3.60; N, 12.20.
4.1.12.8. 1-(p-Methoxy-benzyl)-7-(phenylsulfonyl)-1,4,5,9-tetra
hydro-8H-pyrrolo[3,2-h]quinolin-8-one (9h). This product was
obtained from the reaction of 7h with phenylsulfonylacetonitrile
after 32 h reflux. The crude precipitate was purified by column
chromatography (Sepacore Büchi) using DCM/AcOEt 9:1 as eluent.
4.1.12.4.
1-Methyl-7-(phenylsulfonyl)-1,4,5,9-tetrahydro-8H-
pyrrolo[3,2-h]quinolin-8-one (9d). This product was obtained
from the reaction of 7d with phenylsulfonylacetonitrile after 24 h
reflux. The yellow solid precipitated was recrystallized from etha-
Yellow solid; yield 45%; mp: 154–156 °C; IR:
t 3315 (NH), 1635
(CO) cmꢀ1 1H NMR: d 2.64 (2H, t, J = 7.5 Hz, CH2), 2.78 (2H, t,
;
J = 7.5 Hz, CH2), 3.34 (3H, s, CH3), 5.68 (2H, s, CH2), 6.04 (1H, d,
J = 2.5 Hz, H-3), 6.80 (2H, d, J = 8.0 Hz, H-300 and H-500), 7.08 (1H,
d, J = 2.5 Hz, H-2), 7.12 (2H, d, J = 8.0 Hz, H-200 and H-600), 7.56–
7.68 (3H, m, Ar), 7.93 (2H, d, J = 8.6 Hz, H-20 and H-60), 8.00 (1H,
s, H-6), 11.59 (1H, s, NH); 13C NMR: d 21.6 (t), 27.5 (t), 50.3 (t),
54.9 (q), 99.5 (d), 107.2 (d), 113.6 (d), 114.4 (s), 121.1 (s), 126.7
(d), 128.4 (d), 128.9 (d), 129.6 (s), 131.1 (s), 133.1 (d), 133.7 (d),
136.8 (s), 141.2 (s), 145.0 (s), 158.0 (s), 158.4 (CO). Anal calcd for
C25H22N2O4S: C, 67.25; H, 4.97; N, 6.27. Found: C, 67.05; H, 5.18;
N, 6.00.
nol; yield 55%; mp: 208–209 °C; IR:
t ;
3320 (NH), 1635 (CO) cmꢀ1
1H NMR: d 2.51 (2H, t, J = 6.9 Hz, CH2), 2.82 (2H, t, J = 6.9 Hz, CH2),
3.94 (3H, s, CH3), 6.00 (1H, d, J = 2.4 Hz, H-3), 6.93 (1H, d, J = 2.4 Hz,
H-2), 7.54–7.72 (3H, m, Ar), 7.92 (2H, d, J = 8.0 Hz, H-20 and H-60),
8.03 (1H, s, H-6), 11.47 (1H, s, NH); 13C NMR: d 21.6 (t), 27.4 (t),
36.2 (q), 99.5 (d), 102.3 (s), 106.6 (d), 117.4 (s), 124.6 (s), 127.6
(d), 128.0 (d), 128.8 (d), 129.3 (s), 133.0 (d), 141.2, (s), 141.3 (s),
157.9 (CO). Anal calcd for C18H16N2O3S: C, 63.51; H, 4.74; N, 8.23.
Found: C, 63.72; H, 4.95; N, 8.02.
4.1.12.5. 1-Benzyl-7-(phenylsulfonyl)-1,4,5,9-tetrahydro-8H-pyr
rolo[3,2-h]quinolin-8-one (9e). This product was obtained from
the reaction of 7e with phenylsulfonylacetonitrile after 24 h reflux.
The solid precipitated was purified by column chromatography
(Sepacore Büchi) using DCM/AcOEt 9:1 as eluent. Brown solid;
4.1.12.9. Ethyl 7-(phenylsulfonyl)-1,4,5,9-tetrahydro-8H-pyrrol-
o[3,2-h]quinolin-8-one-2-carboxylate (9i). This product was ob-
tained from the reaction of 7b with phenylsulfonylacetonitrile
after 32 h reflux. The yellow solid precipitated was recrystallized
from ethanol; yield 60%; mp: 322–323 °C; IR:
t 3280 (NH), 3246
yield 45%; mp: 213–214 °C; IR:
t
: 3214 (NH), 1631 (CO) cmꢀ1
;
(NH), 1711 (CO), 1651 (CO) cmꢀ1 1H NMR: d 1.30 (3H, t,
;
1H NMR: d 2.65 (2H, t, J = 7.0 Hz, CH2), 2.83 (2H, t, J = 7.0 Hz,
CH2), 5.79 (2H, s, CH2), 6.06 (1H, d, J = 2.5 Hz, H-3), 7.09 (1H, d,
J = 2.5 Hz, H-2), 7.12–7.28 (5H, m, Ar), 7.54–7.71 (3H, m, Ar),
7.90–7.95 (2H, m, Ar), 8.00 (1H, s, H-6), 11.61 (1H, s, NH); 13C
NMR: d 21.6 (t), 27.5 (t), 50.8 (t), 99.5 (d), 107.3 (d), 114.7 (s),
120.9 (s), 124.4 (s), 126.9 (d), 127.1 (d), 127.7 (d), 128.3 (d),
128.9 (d), 129.7 (s), 133.1 (d), 137.2 (s), 139.3 (s), 141.2 (d),
150.9 (s), 158.1 (CO). Anal calcd for C24H20N2O3S: C, 69.21; H,
4.84; N, 6.73. Found: C, 69.46; H, 4.63; N, 6.50.
J = 7.1 Hz, CH3), 2.67–2.88 (4H, m, 2ꢁ CH2), 4.29 (2H, q, J = 7.1 Hz,
CH2), 6.73 (1H, s, H-3), 7.55–7.72 (3H, m, Ar), 7.98 (2H, d,
J = 8.0 Hz, H-20 and H-60), 8.21 (1H, s, H-6), 12.06 (2H, bs, 2ꢁ
NH); 13C NMR: d 14.2 (q), 20.5 (t), 25.4 (t), 60.3 (t), 110.8 (s),
113.4 (d), 122.8 (s), 123.9 (s), 126.1 (s), 127.9 (d), 128.4 (s), 128.7
(d), 133.1 (d), 140.6 (2ꢁ s), 143.8 (d), 156.5 (CO), 159.8 (CO). Anal
calcd for C20H18N2O5S: C, 60.29; H, 4.55; N, 7.03. Found: C, 59.98;
H, 4.68; N, 6.85.
4.1.12.10. Ethyl 1-methyl-7-(phenylsulfonyl)-1,4,5,9-tetrahy-
4.1.12.6. 1-Phenyl-7-(phenylsulfonyl)-1,4,5,9-tetrahydro-8H-pyr
rolo[3,2-h]quinolin-8-one (9f). This product was obtained from
the reaction of 7f with phenylsulfonylacetonitrile after 32 h reflux.
The crude precipitate was purified by column chromatography
(Sepacore Büchi) using DCM/AcOEt 9:1 as eluent. Yellow solid;
dro-8H-pyrrolo[3,2-h]quinolin-8-one-2-carboxylate
(9j). This
product was obtained from the reaction of 7i with phenylsulfonyl-
acetonitrile after 28 h reflux. The crude precipitate was purified
by column chromatography (Sepacore Büchi) using DCM/AcOEt
9:1 as eluent. Yellow solid; yield 52%; mp: 214–216 °C; IR:
t

P. Barraja et al. / Bioorg. Med. Chem. 18 (2010) 4830–4843
4841
3120 (NH), 1703 (CO), 1643 (CO) cmꢀ1
;
1H NMR: d 1.27 (3H, t,
4.1.12.14.
o[3,2-h]quinolin-8-one (9b). To
7-(Phenylsulfonyl)-1,4,5,9-tetrahydro-8H-pyrrol-
suspension of 9a (70 mg,
J = 7.1 Hz, CH3), 2.66 (2H, t, J = 6.5 Hz, CH2), 2.87 (2H, t,
J = 6.5 Hz, CH2), 4.22 (2H, q, J = 7.1 Hz, CH2), 4.28 (1H, s, CH3),
6.78 (1H, s, H-3), 7.57–7.74 (3H, m, Ar), 7.98 (2H, d, J = 8.0 Hz,
H-20 and H-60), 8.17 (1H, s, H-6), 11.96 (1H, s, NH); 13C NMR: d
14.2 (q), 20.9 (t), 27.2 (t), 34.8 (q), 59.8 (t), 99.9 (d), 114.7 (d),
117.4 (s), 125.7 (s), 126.8 (s), 127.8 (d), 129.0 (d), 130.8 (s),
133.3 (d), 138.3 (s), 140.7 (s), 157.9 (s), 160.3 (CO), 170.3 (CO).
Anal calcd for C21H20N2O5S: C, 61.15; H, 4.89; N, 6.79. Found: C,
61.52; H, 4.62; N, 6.85.
a
0.145 mmol) in ethanol (20 mL), KOH (0.06 g, 1 mmol) was added
and the reaction mixture was refluxed for 1 h. After cooling, the
reaction mixture was made acid with 6 N HCl. The yellow solid
formed was filtered, air dried and purified by column chromatog-
raphy (Sepacore Büchi) using DCM/AcOEt 9:1 as eluent. Yield
90%; mp: 410 °C; IR:
t ;
3365 (NH), 3255 (NH), 1647 (CO) cmꢀ1
1H NMR: d 2.64–2.83 (4H, m, 2ꢁ CH2), 6.13 (1H, t, J = 2.4 Hz, H-
3), 7.15 (1H, d, J = 2.4 Hz, H-2), 7.52–7.69 (3H, m, Ar), 7.96 (2H, d,
J = 8.0 Hz, H-20 and H-60), 8.08 (1H, s, H-6), 11.28 (1H, s, NH),
11.97 (1H, s, NH); 13C NMR: d 21.2 (t), 25.9 (t), 99.5 (d), 108.8
(d), 118.8 (s), 119.9 (s), 125.6 (d), 127.6 (d), 128.6 (d), 129.6 (s),
132.8 (d), 141.2 (s), 142.4 (s), 142.9 (s), 156.8 (CO). Anal calcd for
C17H14N2O3S: C, 62.56; H, 4.32; N, 8.58. Found: C, 62.74; H, 4.15;
N, 8.40.
4.1.12.11. Ethyl 1-benzyl-7-(phenylsulfonyl)-1,4,5,9-tetrahydro-
8H-pyrrolo[3,2-h]quinolin-8-one-2-carboxylate (9k). This prod-
uct was obtained from the reaction of 7j with phenylsulfonylaceto-
nitrile after 24 h reflux. The crude precipitate was purified by
column chromatography (Sepacore Büchi) using DCM/AcOEt 9:1
as eluent. Brown solid; yield 42%; mp: 167–169 °C; IR:
t 3309
(NH), 1701 (CO), 1637 (CO) cmꢀ1 1H NMR: d 1.21 (3H, t,
;
4.2. Biology
J = 7.1 Hz, CH3), 2.70 (2H, t, J = 7.3 Hz, CH2), 2.90 (2H, t, J = 7.3 Hz,
CH2), 4.17 (2H, q, J = 7.1 Hz, CH2), 6.43 (2H, s, CH2), 6.88 (1H, s,
H-3), 6.91 (2H, d, J = 7.2 Hz, H-200 and H-600), 7.09–7.23 (3H, m,
Ar), 7.55–7.72 (3H, m, Ar), 7.95 (2H, d, J = 7.8 Hz, H-20 and H-60),
8.14 (1H, s, H-6), 11.93 (1H, s, NH); 13C NMR: d 14.1 (q), 20.8 (t),
27.2 (t), 48.4 (t), 59.9 (t), 115.9 (d), 117.3 (s), 123.9 (s), 125.6 (s),
126.0 (d), 126.6 (d), 127.5 (s), 127.8 (d), 128.2 (d), 129.0 (d),
130.7 (s), 133.4 (d), 138.1 (d), 139.4 (s), 140.7 (s), 150.4 (s), 157.9
(CO), 160.1 (CO). Anal calcd for C27H24N2O5S: C, 66.38; H, 4.95;
N, 5.73. Found: C, 66.18; H, 5.12; N, 5.60.
4.2.1. Chemicals
Plasmid pBR322 was bought from Fermentas (Burlington, Can-
ada). Base excision repair enzymes (BER), Formamido pyrimidine
glycosilase (Fpg) and Endonuclease III (Endo III), were a generous
gift from Dr. S. Boiteux (CEA, Fontenay aux Roses, France). If not
otherwise indicated, all the chemicals were purchased from Sig-
ma–Aldrich (Milano, Italy).
Irradiation procedure. HPW 125 Philips lamps (Wood’s lamps),
mainly emitting at 365 nm, were used for irradiation experiments.
The spectral irradiance of the source was 4.0 mW cmꢀ2 as mea-
sured, at the sample level, by a Cole-Parmer Instrument Company
radiometer (Niles, IL), equipped with a 365-CX sensor.
4.1.12.12. Ethyl 1-(p-methyl-benzyl)-7-(phenylsulfonyl)-1,4,
5,9-tetrahydro-8H-pyrrolo[3,2-h]
quinolin-8-one-2-carboxylate
(9l). This product was obtained from the reaction of 7k with phen-
ylsulfonylacetonitrile after 24 h reflux. The crude precipitate was
purified by column chromatography (Sepacore Büchi) using
DCM/AcOEt 9:1 as eluent. Brown solid; yield 35%; mp: 98–
4.2.2. Spectrophotometric measurement
All spectrophotometric measures were performed using a Per-
kin-Elmer Lambda 15 spectrophotometer and emission spectra
were recorded on a Perkin-Elmer LS-50B fluorimeter.
100 °C; IR:
t ;
3299 (NH), 1701 (CO), 1664 (CO) cmꢀ1 1H NMR: d
1.22 (3H, t, J = 7.1 Hz, CH3), 2.16 (3H, s, CH3), 2.69 (2H, t,
J = 7.4 Hz, CH2), 2.85 (2H, t, J = 7.4.Hz, CH2), 4.19 (2H, q, J = 7.1 Hz,
CH2), 6.37 (2H, s, CH2), 6.78 (2H, d, J = 7.9 Hz, H-300 and H-500),
6.87 (1H, s, H-3), 6.98 (2H, d, J = 7.9 Hz, H-200 and H-600), 7.59–
7.69 (3H, m, Ar), 7.94 (2H, d, J = 7.5 Hz, H-20 and H-60), 8.12 (1H,
s, H-6), 11.92 (1H, s, NH); 13C NMR: d 14.1 (q), 20.5 (q), 20.8 (t),
27.2 (t), 59.9 (t), 64.9 (t), 99.5 (d), 115.9 (d), 117.0 (s), 123.7 (s),
125.9 (d), 126.3 (s), 127.5 (s), 127.9 (d), 128.8 (d), 128.9 (s),
129.0 (d), 130.7 (s), 133.4 (d), 135.7 (s), 136.4 (s), 140.6 (s), 157.9
(CO), 160.1 (CO). Anal calcd for C28H26N2O5S: C, 66.91; H, 5.21;
N, 5.57. Found: C, 66.85; H, 5.54; N, 5.42.
4.2.3. Cellular phototoxicity
Human chronic myeloid leukemia cells (K-562) and human
T-cell leukemia cells (Jurkat) were grown in RPMI-1640 medium,
human breast adenocarcinoma cells (MCF-7) and human keratino-
cytes (NCTC-2544) were grown in DMEM medium (Dulbecco’s
modified Eagle’s medium) and intestinal adenocarcinoma cells
(LoVo) were grown in Ham’s F12 medium. All cellular media were
supplemented with 115 U/mL of penicillin G, 115 lg/mL strepto-
mycin and 10% fetal bovine serum (Invitrogen, Milano, Italy). Indi-
vidual wells of a 96-well tissue culture microtiter plate were
inoculated with 100
l
L of complete medium containing 8 ꢁ 103
4.1.12.13. Ethyl 1-(p-methoxy-benzyl)-7-(phenylsulfonyl)-1,4,5,
9-tetrahydro-8H-pyrrolo[3,2-h] quinolin-8-one-2-carboxylate
(9m). This product was obtained from the reaction of 7l with phen-
ylsulfonylacetonitrile after 22 h reflux. The crude precipitate was
purified by column chromatography (Sepacore Büchi) using
DCM/AcOEt 9:1 as eluent. Brown solid; yield 37%; mp: 98–
K-562, Jurkat cells or 5 ꢁ 103 MCF-7, LoVo and NCTC-2544 cells.
Plates were incubated at 37 °C in a humidified 5% CO2 incubator
for 18 h before starting the experiments. Drugs were dissolved in
DMSO and then diluted with Hank’s Balanced Salt Solution (HBSS,
pH 7.2) for phototoxicity test (concentration range: 20–0.31
DMSO never exceeded 1%. After removal of the medium, 100
lM).
lL
100 °C; IR:
t
3297 (NH), 1701 (CO), 1664 (CO) cmꢀ1
;
1H NMR: d
of the drug solution were added to each well and incubated at
37 °C for 30 min and then irradiated (2.5 or 3.75 J/cm2). After irra-
diation, drug solutions were replaced with medium and plates
were incubated for 72 h. Cell viability was assayed by MTT [(3-
(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide)]
test, as previously described.18
1.23 (3H, t, J = 7.1 Hz, CH3), 2.69 (2H, t, J = 7.2 Hz, CH2), 2.85 (2H,
t, J = 7.2 Hz, CH2), 3.40 (3H, s, CH3), 4.21 (2H, q, J = 7.1 Hz, CH2),
6.34 (2H, s, CH2), 6.74 (2H, d, J=7.2 Hz, H-300 and H-500), 6.86 (1H,
s, H-3), 6.90 (2H, d, J = 7.2 Hz, H-200 and H-600), 7.57–7.69 (3H, m,
Ar), 7.95 (2H, d, J = 6.8 Hz, H-20 and H-60), 8.14 (1H, s, H-6), 11.95
(1H, s, NH); 13C NMR: d 14.1 (q), 20.8 (t), 27.2 (t), 47.7 (t), 54.9
(q), 64.9 (t), 99.5 (d), 113.6 (d), 115.9 (d), 117.2 (s), 124.1 (s),
127.5 (d), 127.6 (s), 127.9 (d), 129.0 (d), 129.7 (s), 130.6 (s),
131.3 (s), 131.6 (s), 133.4 (d), 138.1 (s), 140.6 (s), 159.1 (CO),
160.2 (CO). Anal calcd for C28H26N2O6S: C, 64.85; H, 5.05; N, 5.40.
Found: C, 64.70; H, 5.42; N, 5.53.
4.2.4. Externalization of phosphatidylserine
For the assessment of phosphatidylserine surface exposure by
apoptotic cells, 100,000 Jurkat cells were irradiated with 2
solutions of test compounds. After 30 min, 1 and 2 h from the
incubation, samples were centrifuged and 100 L of Annexin
lM
l

4842
P. Barraja et al. / Bioorg. Med. Chem. 18 (2010) 4830–4843
V-FITC/PI solution were added, according to the kit instructions (An-
nexin V-FLUOS Staining kit, Roche, Penzberg Germany). Samples
were incubated for 15 min in the dark and then analyzed by BD FACS
Calibur (Becton Dickinson, New York, USA) flow cytometer which
measures the fluorescence of FITC (green, FL1) and of PI (red,
FL3).19 At least 10,000 events for each sample were acquired.
the compounds. After irradiation, two aliquots of sample were
incubated at 37 °C with Fpg (Formamido pyrimidine glycosilase)
and Endo III (Endonuclease III), respectively, as described by Epe
et al.36 Before starting the run, buffer (0.25% bromophenol blue,
0.25% xylene cyanol, 30% glycerol in water) was added to DNA.
Samples were loaded on 1% agarose gel and the run was carried
out in TAE buffer (40 mM Tris–acetate, 1 mM EDTA, pH 8.0) at
80 V for 2 h. After staining with ethidium bromide solution, gel
was washed with water and the DNA bands were detected under
UV irradiation with a UV transilluminator. Photographs were taken
by a digital photocamera Kodak DC256 and the quantification of
the bands was achieved by image analyzer software Quantity
One (Bio Rad, Milano, Italy). The fractions of supercoiled (Form I)
and open circular DNA (Form II) were calculated as described by
others.27
4.2.5. Cell cycle analysis
The flow cytometric analysis of DNA content, was executed irra-
diating 5 ꢁ 105 Jurkat cells, in exponentially growth, in the pres-
ence of 1 lM of pyrroloquinolinones. After the removal of drug
solutions, cells were incubated for 24 h in RPMI-1640 medium,
then fixed with ice-cooled ethanol (70%), treated overnight with
phosphate-buffer saline (PBS) containing RNAse (10 KU/mL) and fi-
nally stained with propidium iodide (PI, 10 lg/mL). Samples were
analyzed on a BD FACS Calibur flow cytometer. Results of cell cycle
analysis, were examined using WinMDI 2.9 (Windows Multiple
Document Interface for Flow Cytometry). Data were expressed as
fractions of cells in the different cycle phases.
4.2.11. Studies on isolated proteins
Solutions of BSA and RNAse A (2 mg/mL) in phosphate buffer
were irradiated in the presence of the test compounds for various
times in a quartz cuvette. The degree of protein oxidation was
spectrophotometrically monitored through the methods of Levine
et al.28 by derivatization with 2,4-dinitrophenylhydrazine.
4.2.6. Mitochondrial membrane potential
The mitochondrial membrane potential was assessed using the
lipophilic cation JC-1. Jurkat cells were collected by centrifugation
2 and 4 h after the irradiation, and resuspended in HBSS containing
4.2.12. Lipid peroxidation
the JC-1 at the concentration of 1
lg/mL. The cytofluorimetric anal-
Jurkat cells (500,000) were irradiated (2.5 J/cm2) in the pres-
ence of the most active compounds and then incubated for 24 h.
ysis (BD FACS Calibur) was performed collecting green (FL1) and
orange (FL2) fluorescence.21 At least 10,000 events for each sample
were acquired.
To verify lipidic peroxidation, after cell centrifugation, 900
surnatant were collected and put at 253 K after having added
90 L of 2,6-di-tert-butyl-p-cresol (BHT, 2% in absolute ethanol).
Cells were washed, resuspended in 500 L of water, and 400
of cells were lysed with 400 L of SDS (Sodium dodecyl sulfate,
1% in water). This suspension was divided into two aliquots:
50 L of BHT were added in 500 L; 300 L were used for protein
lL of
l
4.2.7. Reactive oxygen species assessment
l
lL
ROS production was assessed by flow cytometry using two dif-
ferent probes: hydroethidine (HE), which reveals superoxide anion,
and 20,70-dichlorodihydrofluorescin diacetate (DCFDA), which re-
veals the presence of hydrogen peroxide. After 6 h from irradiation,
cells were collected by centrifugation and resuspended in HBSS
(Hank’s balanced salt solution) containing the fluorescence probes
l
l
l
l
quantification with Peterson method.37 Lipid peroxidation was
measured using a thiobarbituric acid assay as described by Mor-
liere et al.30 A standard curve of 1,1,3,3 tetraethoxypropane was
used to quantify the amount of produced malondialdehyde. Data
were expressed in terms of nanomoles of TBARS normalized to
the total protein content in an aliquot of the cell extract.
HE, DCFDA at the concentration of 2.5 and 5 lM, respectively. Cells
were then incubated at 37 °C for 15 or 30 min. The fluorescence
was recorded with the flow cytometer (BD FACS Calibur) using
the 488 nm wavelength as excitation and the emission at 620 nm
for HE and at 530 nm for DCFDA.23 At least 10,000 events for each
sample were acquired.
Acknowledgment
This work was financially supported by Ministero dell’Istruzi-
one dell’Università e della Ricerca.
4.2.8. Lysosomal stability assessment
Jurkat cells were assessed for lysosomal stability using the acri-
dine orange (AO) uptake method.24 After 2 and 4 h from irradia-
tion, cells were collected by centrifugation and resuspended in
Supplementary data
RPMI-1640 containing AO at the concentration of 1 lM. The fluo-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.080.
rescence was directly recorded with the flow cytometer (BD FACS
Calibur) using the 488 nm wavelength as excitation and the emis-
sion in FL3 channel. At least 10,000 events for each sample were
acquired.
References and notes
1. Parrish, J. A.; Fitzpatrick, T. B.; Pathak, M. A.; Tanenbaum, L. N. Engl. J. Med.
1974, 291, 1207.
2. Dall’Acqua, F.; Viola, G.; Vedaldi, D. In CRC Handbook of Organic Photochemistry
and Photobiology; Hoorspool, W. M., Lenci, F., Eds.; CRC Press: London, 2004; p
1.
3. (a) Stern, R. S. N. Engl. J. Med. 2007, 357, 682; (b) Danno, K. J. Dermatol. Sci. 1999,
19, 85; (c) Falabella, R.; Barona, M. I. Cell Melan. Res. 2008, 22, 42.
4. Edelson, R.; Berger, C.; Gasparro, F.; Jegasothy, C. B.; Heald, P.; Wintroub, B.;
Vonderheid, E.; Knobler, R.; Wolff, K.; Plewig, G.; McKiernan, G.; Christiansen,
I.; Oster, M.; Honigsmann, H.; Wilford, H.; Koroska, E.; Rehle, T.; Stingl, G.;
Laroche, L. N. Engl. J. Med. 1987, 316, 297.
5. Dalla Via, L.; Marciani Magno, S. Curr. Med. Chem. 2001, 8, 1405.
6. Kitamura, N.; Kohtani, S.; Nakagaki, R. J. Photochem. Photobiol. C: Photochem.
Rev. 2005, 6, 168.
4.2.9. Linear dichroism
LD measurements were performed with a Jasco J500A circular
dichroism spectropolarimeter (Jasco, Cremella, Italy), converted
for LD and equipped with an IBM PC and Jasco J interface. For these
analyses, the sample orientation was obtained using a flow device
designed by Wada and Kozawa,35 which presents a cylindrical
rotating cuvette, a 0.14 cm optical path and a constant flow of
800 rpm. LD spectra were performed using different [Drug]/
[DNA] ratios, dissolved in phosphate buffer (10 mM, pH 7.2).
7. (a) Martin, J. A.; Laube, S.; Edwards, C.; Gambles, B.; Anstey, A. V. Photodermatol.
Photoimmunol. Photomed. 2001, 23, 68; (b) Stern, R. S.; Thibodeau, L. A.;
Kleinerman, R. A.; Parrish, J. A.; Fitzpatrick, T. B. N. Engl. J. Med. 1979, 300, 809;
(c) Momtaz, K. T.; Fitzpatrick, T. B. Dermatol. Clin. 1998, 16, 227.
4.2.10. pBR322 DNA stand breaks
Each pBR322 DNA sample (100 ng) dissolved in phosphate buf-
fer (10 mM, pH 7.2) was irradiated with UV-A in the presence of

P. Barraja et al. / Bioorg. Med. Chem. 18 (2010) 4830–4843
4843
8. Dall’Acqua, F.; Vedaldi, D.; Guiotto, A.; Rodighiero, P.; Carlassare, F.; Baccichetti,
F.; Bordin, F. J. Med. Chem. 1981, 24, 806.
19. Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutelingsperger, C. J. Immunol.
Methods 1995, 184, 39.
9. (a) Jakobs, A. E.; Piette, J. J. Photochem. Photobiol., B 1994, 22, 9; (b) Jakobs, A. E.;
Piette, J. J. Med. Chem. 1995, 38, 869; (c) Rodighiero, P.; Chilin, A.; Pastorini, G.;
Guiotto, A. J. Heterocycl. Chem. 1987, 24, 1041; (d) Bordin, F.; Marzano, C.;
Carlassare, F.; Guiotto, A.; Caffieri, S.; Baccichetti, F. J. Photochem. Photobiol., B
1996, 34, 159; (e) Marzano, C.; Baccichetti, F.; Carlassare, F.; Chilin, A.; Lora, S.;
Bordin, F. J. Photochem. Photobiol. 2000, 71, 263.
10. (a) Barraja, P.; Diana, P.; Lauria, A.; Montalbano, A.; Almerico, A. M.; Dattolo, G.;
Cirrincione, G.; Viola, G.; Dall’Acqua, F. Bioorg. Med. Chem. Lett. 2003, 13, 2809;
(b) Barraja, P.; Diana, P.; Montalbano, A.; Dattolo, G.; Cirrincione, G.; Viola, G.;
Vedaldi, D.; Dall’Acqua, F. Bioorg. Med. Chem. 2006, 14, 8712; (c) Barraja, P.;
Sciabica, L.; Diana, P.; Lauria, A.; Montalbano, A.; Almerico, A. M.; Dattolo, G.;
Cirrincione, G.; Disarò, S.; Basso, G.; Viola, G.; Dall’Acqua, F. Bioorg. Med. Chem.
Lett. 2005, 15, 2291; (d) Barraja, P.; Diana, P.; Montalbano, A.; Carbone, A.;
Cirrincione, G.; Viola, G.; Salvador, A.; Vedaldi, D.; Dall’Acqua, F. Bioorg. Med.
Chem. 2008, 16, 9668.
20. Darzynkiewicz, Z.; Juan, G. In Curr. Protoc. Cytom.; Robinson, J. P., Ed.; John
Wiley & Sons, Inc.: New York, 2001. 7.5.1.
21. Salvioli, S.; Ardizzoni, A.; Franceschi, C.; Cossarizza, A. FEBS. Lett. 1997, 411,
77.
22. Zamzami, N.; Marchetti, P.; Castedo, M.; Decaudin, D.; Macho, A.; Hirsch, T.;
Susin, S. A.; Petit, P. X.; Mignotte, B.; Kroemer, G. J. Exp. Med. 1995, 182, 367.
23. Rothe, G.; Valet, G. J. Leukoc. Biol. 1990, 47, 440.
24. Zhao, M.; Eaton, J. W.; Brunk, U. T. FEBS Lett. 2000, 485, 104.
25. Nordén, B.; Kurucsev, T. J. Mol. Recognit. 1994, 7, 141.
26. Armitage, B. Chem. Rev. 1998, 98, 1171.
27. Ciulla, T. A.; Van Camp, J. R.; Rosenfeld, E.; Kochevar, I. J. Photochem. Photobiol.
1989, 49, 293.
28. Levine, L. R.; Garland, D.; Oliver, C. N.; Amici, A.; Climent, I.; Lenz, A. G.; Ahn, B.
G.; Shaltiel, V.; Stadtaman, E. R. Methods Enzymol. 1990, 186, 464.
29. See for example: Hsieh, Y.; Wu, C.; Chang, C.; Yu, J. J. Cell. Physiol. 2003, 194,
363.
11. See for example: Rodighiero, P.; Guiotto, A.; Chilin, A.; Bordin, F.; Bacicchetti,
F.; Carlassare, F.; Vedaldi, D.; Caffieri, S.; Pozzan, A.; Dall’Acqua, F. J. Med. Chem.
1996, 39, 1293.
30. Morlière, P.; Moysan, A.; Santus, R.; Hüppe, G.; Mazière, J.; Dubertret, L.
Biochem. Biophys. Acta 1991, 1084, 261.
12. Massa, S.; Stefancich, G.; Artico, M.; Corelli, F.; Silvestri, R. Il Farmaco, Ed. Sc.
1987, 42, 567.
13. (a) Kakushima, M.; Hamel, P.; Frenette, R.; Rokach, J. J. Org. Chem. 1983, 48,
3214; (b) Tani, M.; Ariyasu, T.; Ohtsuka, M.; Koga, T.; Ogawa, Y.; Yokoyama, Y.;
Murakami, Y. Chem. Pharm. Bull. 1996, 44, 55.
31. Barraja, P.; Spanò, V.; Diana, P.; Carbone, A.; Cirrincione, G.; Vedaldi, D.;
Salvador, A.; Viola, G.; Dall’Acqua, F. Bioorg. Med. Chem. Lett. 2009, 19, 1711.
32. Viola, G.; Fortunato, E.; Cecconet, L.; Del Giudice, L.; Dall’Acqua, F.; Basso, G.
Toxicol. Appl. Pharmacol. 2008, 227, 84.
ˇ
33. Bartošová, J.; Kuzelová, K.; Pluskalová, M.; Marinov, I.; Halada, P.; Gašová, Z. J.
14. Plattner, J. J.; Parks, J. A. J. Heterocycl. Chem. 1983, 20, 1059.
15. Stanovnik, B.; Svete, J. Chem. Rev. 2004, 104, 2433.
16. Ghose, A. K.; Crippen, G. M. J. Comb. Chem. 1986, 7, 565.
17. Caffieri, S. Photochem. Photobiol. Sci. 2002, 1, 149.
Photochem. Photobiol., B: Biol. 2006, 85, 39.
34. Canton, M.; Caffieri, S.; Dall’Acqua, F.; Di Lisa, F. FEBS Lett. 2002, 522, 168.
35. Wada, A.; Kozawa, S. J. Polym. Sci., Part A 1964, 2, 853.
36. Epe, B.; Pflaum, M.; Boiteux, S. Mutat. Res. 1993, 299, 135.
37. Peterson, G. L. Anal. Biochem. 1977, 83, 346.
18. Mosmann, T. J. Immunol. Methods 1983, 65, 55.