Pyrrolo[2,3-h]quinolinones: A new ring system with potent photoantiproliferative activity

Article abstract of DOI:10.1016/j.bmc.2006.07.061

A new class of compounds, the pyrrolo[2,3-h]quinolin-2-ones, nitrogen isosters of the angular furocoumarin Angelicin, was synthesized with the aim of obtaining new photochemotherapeutic agents with increased antiproliferative activity and lower undesired toxic effects than the lead compound. Two synthetic pathways were approached to allow the isolation both of the dihydroderivatives 10-17 and of the aromatic ring system 23. Compounds 10-17 showed a remarkable phototoxicity and a great UVA dose dependence reaching IC₅₀ values at submicromolar level. Intracellular localization of these compounds has been evaluated by means of fluorescence microscopy using tetramethylrhodamine methyl ester and acridine orange, which are specific fluorescent probes for mitochondria and lysosomes, respectively. A weak co-staining was observed with mitochondrial stain, whereas a specific localization in lysosomes was observed. Studies directed to elucidate the mode of action of this series of compounds revealed that they do not intercalate with DNA and do not induce photodamage to the macromolecule. On the contrary, they induce significative photodamage to lipids and proteins.

Full text of DOI:10.1016/j.bmc.2006.07.061

Bioorganic & Medicinal Chemistry 14 (2006) 8712–8728
Pyrrolo[2,3-h]quinolinones: A new ring system with
potent photoantiproliferative activity
Paola Barraja,a Patrizia Diana,a Alessandra Montalbano,a Gaetano Dattolo,a
b,
b
Girolamo Cirrincione,a,* Giampietro Viola, Daniela Vedaldi and
*
Francesco Dall’Acquab
a
`
Dipartimento Farmacochimico, Tossicologico e Biologico Universita degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy
b
`
Dipartimento di Scienze Farmaceutiche Universita degli Studi di Padova, Via Marzolo 5, 35131 Padova, Italy
Received 10 May 2006; revised 25 July 2006; accepted 28 July 2006
Available online 20 September 2006
Abstract—A new class of compounds, the pyrrolo[2,3-h]quinolin-2-ones, nitrogen isosters of the angular furocoumarin Angelicin,
was synthesized with the aim of obtaining new photochemotherapeutic agents with increased antiproliferative activity and lower
undesired toxic effects than the lead compound. Two synthetic pathways were approached to allow the isolation both of the dihyd-
roderivatives 10–17 and of the aromatic ring system 23. Compounds 10–17 showed a remarkable phototoxicity and a great UVA
dose dependence reaching IC50 values at submicromolar level. Intracellular localization of these compounds has been evaluated by
means of fluorescence microscopy using tetramethylrhodamine methyl ester and acridine orange, which are specific fluorescent
probes for mitochondria and lysosomes, respectively. A weak co-staining was observed with mitochondrial stain, whereas a specific
localization in lysosomes was observed. Studies directed to elucidate the mode of action of this series of compounds revealed that
they do not intercalate with DNA and do not induce photodamage to the macromolecule. On the contrary, they induce significative
photodamage to lipids and proteins.
ꢀ 2006 Elsevier Ltd. All rights reserved.
O
1. Introduction
Y
Psoralen 1 (Fig. 1) belongs to a class of naturally or syn-
thetic active substances which exhibit interesting photo-
biological properties.1 They have been used in the
PUVA therapy (Psoralen plus ultraviolet A radiation)
for the treatment of skin diseases such as psoriasis and
vitiligo or cutaneous T-cell lymphoma, in which oral
or topical application of psoralens is combined with
the exposure to UVA radiation. Since the introduction
of extracorporeal photochemotherapy2 (ECP, photo-
phoresis) represents an evolution of the PUVA therapy
for the treatment of cutaneous T-cell lymphoma
(CTCL), it has also been applied for T-cell-mediated
autoimmune disease (progressive systemic sclerosis, lu-
pus erythematosus, pemphigo vulgaris and AIDS).3–7
O
O
O
X
1
2 X=Y=O
3 X=O Y=NH
4 X=O Y=S
Figure 1. Structures of Psoralen 1, Angelicin 2 and heteroanalogues 3
and 4.
It has also successfully been used to reverse allograft
rejection after organ transplantation.8–11
During the last 40 years, much effort has been spent in
photobiological and photochemical studies, in order to
clarify the mechanism(s) of the desired phototherapeuti-
cal effects and undesired side effects of psoralens. It
turned out that these heterocyclic carbonyl compounds
can undergo photocycloadditions to biomolecules such
as DNA, RNA, proteins and fatty acids. In particular
the mechanism of photocycloaddition to DNA is now
Keywords: Photochemotherapy; Angelicin; Pyrroloquinolinones; Ant-
itumour activity.
*
Corresponding authors. Tel.: +39 0916161606; fax: +39 0916169999.
(G.C.), tel.: +39 04982755336; fax: +39 0498275366. (G.V.); e-mail
addresses: gcirrinc@unipa.it; giampietro.viola.1@unipd.it
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.061

P. Barraja et al. / Bioorg. Med. Chem. 14 (2006) 8712–8728
8713
O
well understood. In the dark, psoralen molecules may
form complexes with DNA via intercalation of the furo-
coumarin moiety between base pairs of the nucleic acid.
After photoexcitation, they may form photocycload-
ducts to pyrimidine bases (most effectively to thymidine)
involving either the C@C double bond of the furan ring
(2,3-position) or that of the pyrone ring (5,6-position) or
both. In this latter case ‘‘inter-strand cross links’’ (ISC)
are formed. Moreover photoexcited psoralens may also
take part in electron transfer reactions to molecular oxy-
gen leading to the formation of superoxide anion (Oꢀꢁ)
or hydroxyl radicals (OHꢀ).1 Additionally, they may also
produce singlet molecular oxygen O2 via excitation ener-
gy transfer.1
OH
OHC
i
Me
Me
N
R
(70-94%)
N
R
5a-c
6a-c
a R= Ph, b R= Me, c R= Bn
ii
(80-90%)
PhO2S
HC
CN
O
O
Et2NHC
H
C
iii
Me
(50%)
Me
N
R
N
Ph
8
Unfortunately the antiproliferative effect, of this class of
linear furocoumarins, is associated with undesirable side
effects. In particular the induction of ISC was regarded
as the main cause of short- and long-term side effects,
such as skin erythemas, genotoxicity and mutagenici-
ty,12 and for this reason new compounds, capable of
forming only monoadducts with DNA, have been syn-
thesized. Angelicin 2, for example, is an angular furo-
coumarin which, for its molecular geometry, cannot
produce ISC resulting less toxic than its linear conge-
ner.13 In this context, several angelicin isosters have
been prepared and studied through the years such as
furoquinolinones 3 and thioangelicin derivatives 4
(Fig. 1).14
7a-c
iii
iv
(30-80%)
O
O
2
C-NH2
O
R1
H
3
PhO2S
N1
4
9
8
H
Me
Me
5
N7
R
N
6
Ph
10-17
9
10 R= Ph, R1=SO2Ph
11 R=Ph, R1=COPh
12 R=Ph, R1=CN
Considering our interest in the chemistry of the pyrrole
and the indole nucleus, in the search of new derivatives
with photobiological activity, we focused our attention
on the synthesis of the new ring system pyrrolo[2,3-
h]quinolin-2-one, an angelicin isoster in which both oxy-
gen atoms are replaced with nitrogens. This ring system
incorporates the quinolin-2-one moiety, which confers
many different biological properties to the nucleus to
which it is condensed. In particular, some compounds
show potent antitumour14a or cardiac stimulant
activity.15
13 R=Ph, R1=COOEt
14 R=Me, R1=SO2Ph
15 R=Me, R1=COPh
16 R=Bn, R1=SO2Ph
17 R=Bn, R1=COPh
Scheme 1. Synthesis of compounds 10–17. Reagents and conditions:
(i) t-BuOK, HCOOEt, benzene, rt; (ii) HNEt2, benzene, rt; (iii)
CNCH2R1 (R1 = SO2Ph, COPh, CN, COOEt, CONH2), ethanol,
reflux; (iv) NEt2, ethanol, reflux.
to the 4,5-indole position, and can be obtained by con-
densation of 2-acetonyl-1,4-cyclohexanedione with the
proper amine in acetic acid as previously described.17
The ketones 5a-c were formylated to give the intermedi-
ates 6a–c in yields from good to excellent. The formyl
derivatives 6 were subjected to reaction with diethyl-
amine to achieve in excellent yields the enamino ketones
7a–c, key intermediates for the synthesis of the desired
ring system. Considering that it was reported, that 1,3-
dinucleophiles with a C–C–N sequence react with a vari-
ety of open-chain and cyclic enamino ketones to achieve
the ring closure to a 2-pyridone nucleus,18 we investigat-
ed the reactivity of our intermediates 7a–c with cyanom-
ethylene active compounds such as ethylcyanoacetate,
benzoylacetonitrile, cyanoacetamide, malononitrile and
phenylsulfonylacetonitrile. Such reactions were carried
out in ethanol, and prolonged refluxing time (24–48 h)
proved to be necessary to accomplish the cyclization
to give compounds 11, 12, 14–17. Only in the case of
reaction of 7a with phenylsulfonylacetonitrile, an inter-
mediate of type 8 was formed, thus indicating that the
reaction occurs between the nucleophilic methylene
In this paper, we wish to present the two synthetic
approaches and a detailed investigation on the photocy-
totoxicity exerted by these compounds. We have also
extended our studies on the photochemical damages in-
duced by these new derivatives on biological molecules,
such as proteins, lipids and DNA, to better characterize
the cellular targets involved in their phototoxic reac-
tions. Herein, these photobiological features will be pre-
sented and discussed.
2. Results and discussion
2.1. Chemistry
We have previously reported, in a short communication,
the synthetic pathway used for the synthesis of some of
the compounds assessed in this study (Scheme 1).16
The 1,5,6,7-tetrahydro-indol-4-ones 5a–c are versatile
precursors suitable for the annelation of heterocycles

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P. Barraja et al. / Bioorg. Med. Chem. 14 (2006) 8712–8728
O
group and the a-enamino carbon atom first, which is the
most electrophilic site of the enaminone. The tricyclic
derivative 10 was obtained by heating under reflux the
intermediate 8 with stoichiometric amount of diethyl-
amine in ethanol. For this compound, the general proce-
dure used for the other derivatives did not allow the
isolation of the tricyclic derivative which was instead ob-
tained in 30% yield by reacting the diethylamino methy-
lene 1-phenyl-indolone 7a with the anion of
ethylcyanoacetate.
O
Et2NHC
Et2NHC
i
Me
Me
N
R
N
R
7a-c
19a-c
i
ii
OH
OH
OHC
OHC
i
In general, it seems likely that, by prolonging refluxing
time, the hydrolysis of the cyano group to carboxamide
causes the reaction to proceed towards the direct closure
to the 2-pyridone ring, in moderate to high yields with-
out isolation of the intermediates 9. Such a mechanism
seems to be confirmed by the fact that the N-phenyl-
substituted enaminone 7a (R = Ph) reacts either with
malononitrile or with cyanoacetamide producing the
same 3-cyano-substituted pyrroloquinolinone derivative
12 thus, indicating that the carboxamido functionality is
the one involved in the ring closure by nucleophilic at-
tack of the nitrogen to the annular carbon.
Me
Me
(74-80%)
N
R
N
R
18a-c
6a-c
a R= Ph, b R= Me, c R= Bn
Scheme 2. Oxidation of compounds 7a–c. Reagents and conditions: (i)
DDQ, dichloromethane, rt; (ii) HNEt2, benzene, rt.
O
O
Me
N
H
H
N
Me
A different result was obtained when 7a–c were reacted
with the anion of the ethylcyanoacetate, obtained
in situ with sodium ethoxide. In these cases in fact, the
formation of the desired compounds was not observed
even after many hours of heating with the sole exception
of the reaction of 7a with ethylcyanoacetate, from which
the desired pyrroloquinolinone 13 was recovered from
the reaction mixture in poor yield (30%).
i, ii
H2N
NHNH2
20
21
iv
iii
O
O
N
8
H
N
In order to obtain flat molecules which could intercalate
into double stranded DNA, aromatization of derivatives
10–17 was attempted. However, due to the poor solubil-
ity of the title compounds in most of organic solvents,
reactions with many of the commonly used methods
for this purpose (i.e., DDQ in refluxing benzene, tolu-
ene, dioxane, tetrahydrofurane, dichloromethane; cys-
teine/bubbling oxygen in refluxing ethanol; 10% Pd on
charcoal/bubbling oxygen in refluxing benzene; 10%
Pd on charcoal in cumene at 150 ꢁC or 1-methylnaphta-
lene at 250 ꢁC)19a–19d did not give the expected result
even when a phase transfer catalyst was added to the
reaction mixture (TDA-1).19e It was reasonable to try
the oxidation of the diethylamino indoles 7a–c, more
soluble in most of organic solvents which could be fur-
ther cyclized into the desired ring system. In this case
however, due to the instability of the enaminoketones,
the only products isolated from the reaction mixture
were the aromatic derivatives 18a–c in traces. However,
good yields of these compounds were obtained starting
from the corresponding dihydroindole derivatives 6a–c
(74–80%). Any attempt to react 18a–c with diethylamine
to give compounds 19a–c in the same reaction condi-
tions used for the dihydro derivatives failed, even using
the secondary amine as solvent (Scheme 2).
Me
H
v
Me
Me
N
H
Me
6
C
N
N
H
Me
23
22
Scheme 3. Synthesis of compound 23. Reagents and conditions: (i)
NaNO2, hydrochloric acid, ꢁ5 ꢁC; SnCl2, hydrochloric acid, ꢁ12 ꢁC;
(ii) NaOH; (iii) acetone, ethanol, reflux; (iv) acetone, acetic acid, reflux,
Na2CO3 10%; (v) ZnCl2 280–290 ꢁC.
we prepared the 7-aminoquinolin-4-methyl-2-one 20,
which was used as building block for the synthesis of
the desired aromatic tricyclic system. Diazotization with
sodium nitrite of the amine 20 afforded the diazonium
salt, which was subsequently reduced with a solution
of stannous chloride in hydrochloric acid.21 From the
resulting mixture, hydrazine hydrochloride was filtered
off and carefully treated with a solution of sodium
hydroxide as to get the free base 21. This latter was sub-
sequently reacted with an excess of acetone in refluxing
ethanol to give the hydrazone 22 in excellent yields
(85%). When acetic acid was used as solvent, a mixture
of the hydrazone 22 (60%) and the desired cyclic deriv-
ative 23 (20%) was obtained. However, attempts to con-
vert the hydrazone into the pyrroloquinolinone 23 with
polyphosphoric acid did not lead to the expected tricy-
clic system but produced a tarry mixture from which it
Therefore, a different synthetic pathway was considered.
It consists in the annelation of the pyrrole ring on the
preformed aromatic quinoline (Scheme 3). In this light
according to a procedure described in the literature,20

P. Barraja et al. / Bioorg. Med. Chem. 14 (2006) 8712–8728
8715
was not possible to isolate compound 23. On the con-
trary when anhydrous zinc chloride was used to accom-
plish the cyclization of the pure hydrazone 23, the
reaction occurred in a short time avoiding undesirable
byproducts and making the work-up and purification
of the reaction mixture easier. Such Fisher cyclization
only furnished the angular derivative 23 (65%), even if
positions 6 and 8 were both available for the ring closure
on the quinoline moiety (Scheme 3).
human tumour such as HL-60 (human promyelocytic
leukaemia), HT-1080 (human fibrosarcoma) and LoVo
(human intestinal adenocarcinoma). Table 2 shows the
extent of cell survival expressed as IC50 which is the con-
centration, which induces 50% of inhibition of cell
growth, after irradiation at different UVA doses. Con-
trol experiments with UVA light or drugs alone were
carried out without significant cytotoxic effects (data
not shown). It can be noted the compounds exhibit dif-
ferent values of IC50 depending on the substitution pat-
tern, and a remarkable dose UVA-dependence.
2.2. Biology
2.2.1. Physico-chemical properties. The test compounds
represent isosters of the typical trycyclic system of angel-
icin with substituents in positions 3, 7 and 8. All com-
pounds bear an electron-withdrawing group in
position 3 so as to deactivate the photoreactive bond
in positions 3–4. The only exception is compound 23
which presents an additional methyl group in position
4. The double bond in positions 8–9 results to be activat-
ed by the methyl group in position 8 lying on the trycy-
clic plane. Both absorption and emission maxima
wavelengths of the compounds exhibit (Table 1) a
remarkable bathochromic shift with respect to angelicin
due to the conjugation with the substituents in positions
3 and 7. Position 3 is particularly sensitive to the nature
of the substituent as demonstrated by the values of the
molar extinction coefficients. In particular, the deriva-
tives bearing the sulfonyl group (10, 14, and 16) are en-
dowed with an higher molar extinction coefficient than
those bearing the carbonyl group (11, 13, 15, and 17).
This could be due to the higher electronic density and
to the increased conjugation but also to the structural
rigidity of the molecule. The relative fluorescence quan-
tum yields are measured at 298 K.22 As it can be ob-
served from Table 1, the 3-phenylsulfonyl derivatives
show values of fluorescence quantum yields, markedly
higher than the other compounds. In fact, the insertion
of such substituents into the quinoline nucleus markedly
affects the fluorescence both in terms of the energy and
of the yields. Moreover, the partition coefficients of
the test compounds were determined by a computational
method.23 The results obtained indicate that the pyrrol-
oquinolinones are highly hydrophobic in comparison to
angelicin (clogP = +2.08) taken as reference compound.
All the compounds inhibit the proliferation of tumour
cell lines at submicromolar/micromolar concentration.
In some cases the IC50 values are comparable, if not
lower than 8-methoxypsoralen (8-MOP) and angelicin
used as reference drugs. The most interesting derivatives
appear to be 10, 11, 17 and 15 only in the leukaemia cell
line. These IC50 values appear particularly interesting
considering the cell lines derived from solid tumours
(HT-1080 and LoVo).
From a structure–activity relationship point of view the
following considerations can be done: in all compounds
the nature of the substituent on the pyrrole nitrogen (po-
sition 7) plays a major role in the induction of phototox-
icity, in fact, with the exception of 12 (R = Ph, R0 = CN)
the antiproliferative activity generally decreases follow-
ing the rank order phenyl > benzyl > methyl.
Moreover, also the substituent in position 3 plays a role
in the modulation of the antiproliferative activity. In
fact, derivatives bearing the sulfonyl group become
more active than the compounds bearing other elec-
tron-withdrawing groups.
Surprisingly compound 23, which is devoid of the sub-
stituents in positions 3 and 7, appears to be inactive in
the three cell lines at all UVA doses used.
2.2.3. Effect of scavengers on pyrroloquinolinone-induced
phototoxicity. In order to evaluate the possible mecha-
nism of action of the antiproliferative activity of these
derivatives, a series of experiments were performed in
which, various scavengers were included in the cell cul-
tures during irradiation of the title compounds 10, 11,
and 17. The results of these studies are presented in
Figure 2. The scavengers used were sodium azide
2.2.2. Phototoxicity. The phototoxicity of title com-
pounds was investigated on three cultured cell lines of
Table 1. Physico-chemical properties of Pyrroloquinolinones
e (Mꢁ1 cmꢁ1
)
/
clogPb
a
f
Compound
kmax Absorb. (nm)
kmax Emiss. (nm)
10
11
421.3
441.5
451
460
440
463
458
484
464
484
445
413
34243
23201
29974
24350
31307
22139
33629
23310
10584
9350
0.34
+3.93
+4.38
+3.02
+2.83
+2.58
+3.29
+4.26
+4.64
+2.83
+2.08
2.6 · 10ꢁ3
1.8 · 10ꢁ4
1. 3 · 10ꢁ3
0.33
12
13
331.6–383.2
423.0
423.1
14
15
445.2
421.2
3.3 · 10ꢁ3
0.10
1.6 · 10ꢁ4
2.8 · 10ꢁ2
1.8 · 10ꢁ2
16
17
442.7
337.1–351.2
299
23
Ang.
a Relative fluorescence quantum yield determined in 0.1 N H2SO4 as described in Ref. 22. The estimated error is 15%.
b Calculated as described in Ref. 23.

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P. Barraja et al. / Bioorg. Med. Chem. 14 (2006) 8712–8728
Table 2. Photocytotoxicity of test compounds against three different human tumour cell lines
HL-60a
(IC50, lM)b
Dose UVAc 10
11
12
13
14
15
16
17
23
8-MOP
Ang.
(J cmꢁ2
2.6
3.2
)
1.2 0.2
8.5 0.3
3.9 0.5
5.4 1.1
4.7 0.5
0.45 0.04 0.51 0.08 1.3 0.7
7.7 3.0
5.5 2.1
1.3 0.5
>20
8.9 0.1
6.2 0.01
2.8 0.8 0.86 0.06
12.4 1.2 4.0 0.2
6.9 0.2 2.2 0.2
1.8 0.7 1.1 0.1
>20 1.4 0.1 1.5 0.2
>20 1.2 0.1 0.9 0.1
>20 0.7 0.1 0.6 0.1
0.82 0.02
13.2 2.1
6.5
HT-1080a
(IC50, lM)b
Dose UVAc 10
11
12
13
14
15
16
17
23
8-MOP
Ang.
(J cmꢁ2
2.6
3.2
)
3.6 0.5
2.3 0.8
0.4 0.04
6.6 1.6
4.6 0.9
1.7 0.2
>20
13.1 1.5
11.1 1.4
12.2 2.1 >20
7.9 0.9 >20
>20
>20
>20
15.1 1.2
1.7 0.2
14.7 0.9
9.0 1.1
1.9 0.02 >20 1.5 0.2
>20 7.8 0.7 15.7 1.9
>20 2.1 0.3
2.6 0.2
2.5 0.3
6.5
2.1 0.6 16.4 1.4 >20
LoVoa
(IC50, lM)b
Dose UVA 10
(J cmꢁ2 c
11
12
13
14
15
>20
16
17
23
8-MOP
Ang.
)
2.6
3.2
6.5
1.2 0.2
0.6 0.1
0.4 0.02
4.1 0.6
2.8 0.3
1.3 0.2
9.8 1.1
8.9 0.3
8.3 1.1
6.9 1.2
3.1 1.3
1.1 0.9
>20
>20
6.5 0.7
>20 1.1 0.4 1.6 0.2
>20 0.7 0.1 0.9 0.1
>20 0.4 0.1 0.8 0.1
17.5 4.7 >20
6.6 1.1 8.9 0.9
10.7 1.3 3.6 1.3
2.9 0.3 0.6 0.1
a Human cell lines: HL-60 promyelocytic leukaemia, HT-1080 fibrosarcoma cells, LoVo intestinal adenocarcinoma.
b Concentration of compound required to inhibit the cell growth by 50% after 72 h of exposure as determined by MTT assay.
c UVA Dose expressed in J cmꢁ2 as measured at 365 nm with a Cole-Parmer radiometer.
(NaN3) and 1,4-diazabicyclo[2,2,2]octane (DABCO),
two singlet oxygen scavengers; superoxide dismutase
(SOD) and catalase (CAT) which scavenge O2 and
intracellular localization of pyrroloquinolinones, we
used two fluorescent probes: TMRM, a lipophilic cation
commonly used for the assessment of the mitochondrial
potential28,29, and acridine orange (AO), which stains
lysosomes.25 These markers fluoresce in the visible re-
gion (about 550 nm), while the test compounds emit in
the blue region. Both fluorescences can be easily separat-
ed using suitable bandpass optical filters. After an incu-
bation in HT-1080 fibrosarcoma cells, the most
interesting compounds were found to incorporate and
associate with subcellular structures.
ꢀꢁ
H2O2, respectively; N-N0-dimethyl thiourea (DMTU)
and Mannitol (MAN), which scavenge hydroxyl radicals
(OHÆ); and 2,6-di-tert-butylhydroxyanisole (BHA) and
glutathione reduced form (GSH), two free radical scav-
engers. The concentrations of the compounds and UVA
doses were chosen on the basis of the results shown in
Table 2 (dose UVA = 3.2 J cm2). Scavengers alone did
not affect the cell viability during UVA exposure (data
not shown). A strong protective effect is generally ob-
served with GSH and BHA, and MAN for the three
compounds investigated and DMTU for 10 and 17, indi-
cating that the mechanism of action of pyrroloquinoli-
nones is principally mediated by free radicals and in
particular OH Å radicals. A weak protective effect is gen-
erally observed with NaN3 and DABCO, thus suggest-
ing that singlet oxygen could be only partially involved
in the cytotoxic activity of the test compounds.
It can be observed from Figure 3 that compound 10,
chosen as representative, diffuses in the cytoplasm and
only partially co-localizes with TMRM. On the con-
trary, a more evident accumulation in lysosomes can
be observed suggesting that lysosomes may be a major
site of photosensitization for this class of compounds.
2.2.5. Photodegradation/photostability. All derivatives
underwent a remarkable photodegradation, as shown
by the changes in absorption spectra upon UVA irradi-
ation in buffered aqueous solution (see Section 5). Dis-
tinct isosbestic points were observed, suggesting that
photoproducts are most likely formed. Interestingly,
the only exception is represented by compound 23
which, in comparison with the other derivatives, does
not show any variation of the absorption spectrum even
after a long time of irradiation (see Section 5).
2.2.4. Intracellular localization. The importance of the
cellular localization for the cytotoxic action of photo-
sensitizers has been demonstrated.24,25 The strength of
a photosensitizer is determined by the nature of the
excited states or by its ability to form reactive species
while, in a phototoxicological context, other factors
such as subcellular localization are important parame-
ters. Thus, it is critically important to evaluate the distri-
bution of photosensitizers inside cells. The subcellular
localization of a photosensitizer is due to its physico-
chemical properties such as hydrophobicity, charge,
pKa, etc. For example, lysosomes are preferential sites
of two fluoroquinolones such as lomefloxacin and cipro-
floxacin,26 whereas mitochondria are the primary targets
in photodynamic therapy.27 In order to investigate the
In order to obtain precise informations about the photo-
degradation kinetics, at appropriate time intervals the
irradiated solutions were analysed by HPLC. Kinetic
analysis of the photodegradation process reveals an
apparent first order kinetics for the test compounds.
The rate constants (k) were obtained from the slopes

P. Barraja et al. / Bioorg. Med. Chem. 14 (2006) 8712–8728
8717
photoproducts in high yields. The other two compounds
10 and 17 produce many photoproducts among which
those with mass value of ꢁ10, ꢁ2, and +32 in respect
to the mass of the starting compound can be observed
as for compound 11.
100
80
60
40
20
0
*
*
*
*
These values indicate that an oxidative pathway takes
place and the fact that the differences of mass values
are always the same indicates that the tricyclic system
is not involved in the photodegradation process. The po-
tential involvement of photoproducts generated upon
UVA irradiation has also been studied. HL-60 cells
and LoVo cells were incubated for 72 h in the presence
of a solution of pyrroloquinolinones which were previ-
ously irradiated for 30 min. The cell viability was as-
sayed as described before. The obtained results (data
not shown) have revealed no cytotoxic effects, suggest-
ing that the events that lead to cellular death are solely
due to a phototoxic damage and not to a production
of toxic photoproducts.
10
100
80
60
40
20
0
*
*
*
2.2.6. Lipid peroxidation. Having hydrophobic charac-
ter, as their partition coefficients indicate, (see Table 1)
the pyrroloquinolinones may be expected to be localized
mainly in plasma and/or in subcellular membranes, as
confirmed by fluorescence microscopy studies, making
these structures particularly sensitive to photodamage.30
The thiobarbituric assay (TBA test) was used in order to
determine whether lipid peroxidation occurs upon irra-
diation of HT-1080 cells incubated in the presence of
test compounds. The TBA test was performed on the
supernatants medium and in the cell homogenates as de-
scribed in the experimental section.31 Figure 7 shows the
results obtained for compounds 10 and 11 as a function
of light doses. Thiobarbituric reactive substances
(TBARS) are significantly produced when the cells are
exposed to the compounds and UVA both in the super-
natant and in the cell extract. A good correlation is ob-
served between the extent of lipid peroxidation
monitored by the TBA test and the degree of
phototoxicity.
11
120
100
80
60
40
20
0
*
*
*
*
17
2.2.7. Protein photodamage. The photosensitization
capacity of test compounds (10, 11) towards other com-
ponents of cellular membranes, such as proteins, was
estimated by measuring the photoinduced cross-linking
in erythrocyte ghost proteins.32 Light-induced cross-
linking of spectrin, a protein associated with the cyto-
plasmic side of the RBC membrane, in the presence of
these compounds was detected by the partial or total
disappearance of the two spectrin bands, (220,000 and
245,000 Da) on SDS–PAGE, while cross-linked aggre-
gates cannot run inside the gel and remain at the top.
Figure 2. Effect of different scavengers on the phototoxicity induced by
compounds 11 (upper panel) 10 (middle panel) and 17 (lower panel) in
HT-1080 fibrosarcoma. Cell viability was assayed by MTT test after
72 h from the irradiation in the presence of compounds 11, 10 and 17
and SOD (2000 U.I./ml), CAT (2000 U.I./ml), BHA (10ꢁ5 M), Man
(10 mM), GSH (1 mM) DMTU (1 mM) NaN3 (10 mM) and DABCO
(1 mM).
of the plots by linear regression analysis and the results
are presented in Table 3. It can be noted that pyrrolo-
quinolinones 10, 11, 12, 16, and 17 underwent a very ra-
pid degradation, their half life being in the range of 13–
63 min. The potential involvement of photoproducts
generated upon UVA irradiation has been studied. Fur-
thermore, a partial characterization of the photoprod-
ucts was performed by LC–MS. In Figures 4–6 are
reported the TIC (total ion current) of the most active
compounds (10, 11, and 17) and the mass values of
the principal peaks are reported in Table 4. As it can
be observed, irradiation of 11 produces three
Figure 8(A and B) shows the pictures of the SDS–PAGE
of ghosts irradiated in the presence of compounds 11
and 10. Under aerobic conditions the two compounds
cause an UVA dose-dependent disappearance of the
two spectrin subunit bands whereas in the presence of
nitrogen this effect is reduced indicating the involvement
of reactive oxygen species. To further investigate the
photosensitizing properties of the title compounds to-
wards proteins, solutions containing bovine serum albu-

8718
P. Barraja et al. / Bioorg. Med. Chem. 14 (2006) 8712–8728
Figure 3. Fluorescence microphotographs showing the intracellular localization of 10 in HT-1080 fibrosarcoma cells in the presence of TMRM
(panels b and c) and AO (panels e and f). The cells were treated as described in Material and Methods. a, fluorescence image of 10; b, fluorescence
image of TMRM; c, Overlay image of 10 localization generated by transferring the blue color of the compounds seen in panel a, onto the
corresponding fluorescence image of panel b. Pink is used as a false color to indicate the localization of 10. d, fluorescence image of the 10; e,
fluorescence image of AO; f, Overlay image of 10 localization generated by transferring the blue color of the compounds seen in panel d onto the
corresponding fluorescence image panel e. Clear green is used as a false color to indicate the localization of 10.
Table 3. Photodegradation kinetic parameters of the title compounds
40000
0 J cm-2
Compounda
k (minꢁ1) · 10ꢁ2
t1/2 (min)
30000
20000
10000
10
11
12
16
17
5.31
4.66
1.10
4.81
2.17
13.0
13.7
63.0
14.4
31.9
0
16000
1.3 J cm-2
12000
8000
4000
a The rate constant and the half-life of photodegradation were deter-
mined in phosphate buffer 10 mM, pH-7.2.
a
a
b
b
c
0
16000
12000
8000
4000
0
16000
0 J cm-2
7.8 J cm-2
12000
8000
4000
0
g
c
e
f
d
16000
12000
1.3 J cm-2
8000
4000
0
0
5
10
15
20
25
30
35
40
Time (min)
12000
8000
4000
2.6 J cm-2
b
Figure 5. LC-MS profile of upon UVA irradiation in phosphate buffer
under aerobic conditions. Compound 10.
a
c
0
20000
16000
12000
8000
4000
0
b
-2
7.8 J cm
17. No effects were observed when BSA alone was irra-
diated in the same conditions. When N2 was bubbled
through the solution prior and during the irradiation
in the presence of test compounds, a significant protec-
tion for the protein photodamage was observed. No sig-
nificant effects were observed when the protein was
irradiated in the presence of drugs and superoxide dis-
mutase (SOD) or in the deuterated phosphate buffer
indicating that neither superoxide anion nor singlet oxy-
gen is involved in the protein photodamage.
c
0
5
10
15
20
25
30
35
40
Time (min)
Figure 4. LC-MS profile of upon UVA irradiation in phosphate buffer
under aerobic conditions. Compound 11.
min (BSA), as a model33, and the drugs in phosphate
buffer were irradiated for different times. Tryptophane
(Trp) content was directly analysed by monitoring the
characteristic fluorescence of Trp residues.34 As depicted
in Figure 9, a rapid decrease of the emission fluorescence
was observed under aerobic conditions for 11, 10, and
Another protein model used in this study was RNAseA
which is a protein that does not possess Trp residues but
has tyrosine (Tyr) residues in its sequence.35,36 Its
emission band centred at about 350 nm significantly

P. Barraja et al. / Bioorg. Med. Chem. 14 (2006) 8712–8728
8719
Dark
UVA alone
11
10
90000
60000
30000
0 J cm-2
2.0
0
90000
1.5
1.3 J cm-2
60000
30000
1.0
0
60000
0.5
2.6 J cm-2
30000
a
b
0.0
0
0
7.8
15.6
20000
UVA dose (J cm-2)
7.8 J cm-2
a
c
g
b
10000
0
f
e
d
h
Dark
0
5
10
15
20
25
30
35
40
1.00
0.75
0.50
0.25
0.00
UVA alone
11
Time (min)
10
Figure 6. LC-MS profile of upon UVA irradiation in phosphate buffer
under aerobic conditions. Compound 17.
Table 4. MS spectral data of photodegradation products of test
compounds
Compound/photoproducts
Mass valuesa
0
7.8
15.6
10
a
b
c
417.14
UVA dose (J cm-2)
407.11 (ꢁ10)
495.17 (+78)
415.12 (ꢁ2)
449.13 (+32)
421.14 (+4)
451.13 (+34)
381.17
Figure 7. Lipid peroxidation induced in HT-1080 fibrosarcoma cells
24 h after the irradiation in the presence of 11 and 10 at the
concentration of 20 lM. The TBA test was performed on the
supernatants medium (upper panel) and in the cell homogenates
(lower panel) as described in the experimental section.
d
e
f
11
a
b
c
413.17 (+32)
371.14 (ꢁ10)
379.15 (ꢁ2)
395.18
fluorescence titrations carried out with the same deriva-
tives (data not shown) showed a poor fluorescence
quenching confirming that the title compounds are
loosely bound to DNA. These facts strongly suggest that
the new derivatives do not interact efficaciously, without
irradiation, with the macromolecule, as indeed demon-
strated with natural furocoumarins.1
17
a
b
c
385.18 (ꢁ10)
393.19 (ꢁ2)
409.15 (+14)
427.17 (+32)
427.17 (+32)
322.11 (ꢁ73)
427.17 (+32)
d
e
f
g
Further experiments were carried out in order to deter-
mine whether the new derivatives were able to photosen-
sitize DNA strand break activity. Their ability to
photodamage DNA was evaluated using supercoiled cir-
cular DNA because it is a very sensitive tool for damage
detection.39 In addition to frank strand break we have
also evaluated if purine and/or pyrimidine bases were in-
volved in the oxidative damage to DNA using base exci-
sion repair enzymes Formamido pyrimidin glycosilase
(Fpg) and Endonuclease III (Endo III), respectively.
a In parentheses is indicated the variation of mass in comparison to the
parent compound.
decreases after irradiation in the presence of compounds
11, 10, and 17. Again, this effect is markedly reduced in
anaerobic conditions. Deuterated phosphate buffer,
which increases singlet oxygen lifetime or SOD, did
not induce any variation of the degradation profile, as
described above for BSA.
Supercoiled circular DNA allows the detection of struc-
tural alterations such as strand breaks or damaged
bases. DNA strand break can be induced either directly
(frank strand breaks) or indirectly using DNA repair en-
zymes, in this case we determined the number of DNA
modification sensitive to the following repair enzymes
(i) Fpg protein which has been demonstrated to recog-
nize 8-hydroxy guanine, purines whose imidazole ring
is open (Fapy residues) and sites of base loss (apurinic
sites). (ii) Endonuclease III, which is known to recog-
2.2.8. DNA interactions and DNA-photodamage. In or-
der to understand the binding nature between the title
compounds and DNA, linear dichroism (LD) measure-
ments37,38 were performed on solutions with salmon tes-
tes DNA (st-DNA) and the title compounds at various
molar ratios. Inspection of the LD spectra of two deriv-
atives (10 and 23) chosen as representative (Fig. 10) in
the presence of st-DNA reveals that despite a strong
absorption in the 300–500 nm region, the two com-
pounds give no LD bands in this region. Furthermore,

8720
P. Barraja et al. / Bioorg. Med. Chem. 14 (2006) 8712–8728
Figure 8. Electrophoretic pattern of the photoinduced cross-link of spectrin in RBC ghosts irradiated at the indicates times (UVA dose =
0.24 J cmꢁ2 minꢁ1) in the presence of 11 (upper panels) and 10 (lower panels) at the concentration of 20 lM. Column A, aerobic condition; Column
B, N2 purged solution. Spectrin band is indicated by an arrow. St is a mixture of molecular weight standard, which value in KDa is depicted in the left
of the figure.
nize, in addition to apurinic sites, 5,6-dihydropyrimidine
derivatives. Damaged base release is followed by a b–d
reaction and a b-elimination step, respectively, resulting
in DNA breakage.40
reported, in fact, that the introduction of a nitrogen
atom on the furane ring decreases the photobiological
activity, as in the case of linear and angular pyrrolocou-
marines. Moreover, 5,6-dihydro furo and thienoquinoli-
nones bearing an electron-withdrawing substituent, such
as an ester functionality, are devoid of activity.19b
The obtained results (see Section 5) indicate that the new
compounds do not sensitize the formation of single
strand breaks at any of the UVA dose used. Further-
more, the treatment with the two base excision enzymes
reveals that they are not able to photooxidize DNA
bases. Altogether these results indicate that the new pyr-
roloquinolinones differently from psoralens do not effi-
caciously interact with DNA and other mechanisms
are operating for this new class of compounds.
Interestingly, the conjugation of the tricyclic ring with
phenylsulfonyl groups strongly increases their photoac-
tivity as demonstrated by the rapid photodegradation
and the remarkable induction of phototoxicity. Regard-
ing the mechanism of action, the lack of affinity of the
new compounds with DNA upon UVA irradiation
could be of great relevance in modulating the long-term
toxic effect such as skin cancer or mutagenesis exhibited
by psoralen. Indeed, it seems that lysosomes and/or
mitochondria could be target for pyrroloquinolinones
inducing photodamage to lipids and proteins. A first
indication about their mechanism of action comes from
the experiments of cytotoxicity carried out in the pres-
ence of different scavengers. Results demonstrate the
participation of free radicals and in particular the
involvement of the hydroxyl radicals. It will be necessary
to carry out further investigation on the photochemical
and photophysical aspect to better clarify their mecha-
nism of action.
3. Conclusions
The data reported herein indicate that pyrroloquinoli-
nones represent a new interesting class of potentially
useful compounds in photochemotherapy. They repre-
sent isosters of the tricyclic system of the angelicin with
the additional substituents in positions 3 and 7 that in-
crease the conjugation of the system. In fact, the tricyclic
compound 23 surprisingly is completely inactive. This
seems to be in contrast with previous studies. It was

P. Barraja et al. / Bioorg. Med. Chem. 14 (2006) 8712–8728
8721
A
B
100
90
80
70
60
50
40
100
90
80
70
60
50
0
0
0
5
10
15
0
5
10
15
UVA Dose (J cm-2)
UVA Dose (J cm-2)
C
D
100
90
80
70
60
50
100
90
80
70
60
50
40
0
5
10
15
5
10
15
UVA Dose (J cm-2)
UVA Dose (J cm-2)
E
100
90
80
70
60
50
40
F
100
90
80
70
60
50
0
5
10
15
5
10
15
UVA Dose (J cm-2)
UVA Dose (J cm-2)
Figure 9. Photosensitising effects on BSA (Panels A, C and E) and RNAseA (panels B, D and F) of compounds 11 (A,B), 10 (C,D), and 17 (E,F), at
the dose of 10 lM after irradiation, in different conditions: in aerated (d) and deaerated (m) solutions; plus SOD 2000 UI/ml(.); in deuterated
phosphate buffer (Ç); (j) BSA alone.
4. Experimental
4.1.1. General procedure for the 2-methyl-1-substituted-
1,5,6,7-tetrahydro-indole-4H-4-ones (5a–c). These com-
pounds were prepared by reacting the commercially
available 1,3-dihydroresorcin, according to the Stetter
and Lauterbach procedure,17 with chloroacetone and
potassium hydroxide in methanol at room temperature
for 48 h to afford the 1,3-acetonylcyclohexandione as a
mixture of the two tautomers which was used without
any further purification. Condensation with the suitable
amine in acetic acid at 60 ꢁC for 2 h yielded the 2-meth-
yl-1-substituted-1,5,6,7-tetrahydro-indole-4-ones 5a–c
(60–90%).
4.1. Chemistry
All melting points were taken on a Buchi–Tottoli cap-
illary apparatus and are uncorrected; IR spectra were
determined with a Jasco FT/IR 5300 spectrophotome-
ter; 1H and 13C NMR spectra were measured in
DMSO-d6 solutions, unless otherwise specified (TMS
as internal reference), at 200 and 50.3 MHz, respective-
ly, using a Bruker AC series 200 MHz spectrometer.
Column chromatography was performed with Merck
silica gel 230–400 Mesh ASTM or with a SEPACORE
chromatography apparatus BUCHI. Elemental analy-
ses (C, H, and N) were within 0.4% of the theoretical
values.
4.1.2. 2-Methyl-1-phenyl-1,5,6,7-tetrahydro-4H-indole-4-
one (5a). This product was obtained as white solid
(60%) by reaction of 1,3-acetonylcyclohexandione with

8722
P. Barraja et al. / Bioorg. Med. Chem. 14 (2006) 8712–8728
c
A1
A2
3
e
d
1.5
1.0
0.5
0.0
a
2
c
e
A
A
x10
b
a
1
0
b
c
b
0.000
-0.001
-0.002
-0.003
-0.004
-0.005
0.000
-0.002
-0.004
-0.006
a
b
a
e
B2
400
B1
500
c
250
300
350
400
450
250
300
350
Wavelength (nm)
Wavelength (nm)
Figure 10. Absorbance (A), and Linear dichroism (LD) spectra of mixtures of salmon testes DNA and compounds 10 (A1,B1) and 23 (A2,B2) at
different [Drug]/[DNA] ratio (a = 0.00, b = 0.02, c = 0.04, d = 0.08, e = 0.2).
phenylamine: mp 153–155 ꢁC; IR: 1648 (CO) cmꢁ1; H
NMR: r 1.94 (q, J = 6.2 Hz, 2H, CH2), 2.01 (s, 3H,
CH3), 2.33 (t, J = 6.2 Hz, 2H, CH2), 2.50 (t,
J = 6.2 Hz, 2H, CH2), 6.22 (s, 1H, H-3), 7.38 (d,
J = 6.9 Hz, 2H, H-20, H-60), 7.47–7.57 (m, 3H, H-30,
H-40, H-50,). 13C NMR: r 12.3 (CH3), 22.1 (CH2),
23.4 (CH2), 37.5 (CH2), 103.1 (CH), 119.5 (C), 127.4
(2· CH), 128.5 (CH), 129.5 (2· CH), 130.5 (C), 136.5
(C), 143.9 (C), 192.5 (CO). Anal. calcd for
C15H15NO: C, 79.97; H, 6.71; N, 6.22. Found: C,
80.01; H, 7.06; N, 6.36.
4.2. General procedure for the 5-carbaldehyde-4-hydroxy-
2-methyl-1-substituted-6,7-dihydro-1H-indoles (6a–c)
1
To a suspension of t-BuOK (8 g, 36 mmol) in anhydrous
benzene (45 mL), a solution of the suitable tetrahydroin-
dole (5a–c) (36 mmol) in the same solvent (70 mL) was
added with stirring under nitrogen atmosphere at room
temperature. After 1 h stirring, the solution was ice-
cooled and treated dropwise with a solution of ethyl for-
mate (2.6 mL, 36 mmol) in anhydrous benzene (30 mL)
under stirring. The solution was allowed to reach room
temperature and after 24 h, the solvent then was removed
in vacuo. To the crude residue diethyl ether was added and
extracted with water. The aqueous extracts were com-
bined, washed with diethyl ether and acidified at 0 ꢁC with
6 M hydrochloric acid. The solid which separated was fil-
tered off, air-dried recrystallized from cyclohexane to give:
4.1.3. 1,2-Dimethyl-1,5,6,7-tetrahydro-4H-indole-4-one
(5b). This product was obtained as a white solid (80%)
by reaction of 1,3-acetonylcyclohexandione with
1
methylamine: mp 100–102 ꢁC; IR: 1637 (CO) cmꢁ1; H
NMR: r 2.10 (q, J = 6.1 Hz, 2H, CH2), 2.24 (s, 3H,
CH3), 2.32 (t, J = 6.1 Hz, 2H, CH2), 2.77 (t,
J = 6.1 Hz, 2H, CH2), 3.48 (s, 3H, CH3), 6.11 (s, 1H,
H-3). 13C NMR: r 11.6 (CH3), 21.3 (CH2), 23.3
(CH2), 30.1 (CH3), 37.4 (CH2), 102.1 (CH), 118.7 (C),
130.2 (C), 143.5 (C), 192.0 (CO). Anal. calcd for
C10H13NO: C, 73.59; H, 8.03; N, 8.58. Found: C,
73.72; H, 8.14; N, 8.62.
4.2.1. 5-Carbaldehyde-4-hydroxy-2-methyl-1-phenyl-6,7-
dihydro-1H-indole (6a). This product was obtained as
white solid (80%) by formylation of tetrahydroindole
1
5a: mp 84–86 ꢁC; IR: 3020 (OH), 1637 (CO) cmꢁ1; H
NMR (CDCl3): r 2.00 (s, 3H, CH3), 2.40–2.50 (m, 4H,
2· CH2), 6.40 (s, 1H, H-3), 7.22 (d, J = 7.2 Hz, 2H, H-
20, H-60), 7.45–7.51 (m, 3H, H-30, H-40, H-50), 9.88 (s,
1H, CHO), 14.01 (bs, 1H, OH). 13C NMR (CDCl3): r
12.5 (CH3), 22.3 (CH2), 24.7 (CH2), 103.7 (CH), 108.1
(C), 119.8 (C), 127.4 (2 · CH), 128.7 (CH), 129.4
(2· CH), 132.2 (C), 136.8 (C), 143.1(C), 160.4 (C),
187.9 (CHO). Anal. calcd for C16H15NO2: C, 75.87; H,
5.97; N, 5.53. Found: C, 75.65; H, 5.80; N, 5.42.
4.1.4. 1-Benzyl-2-methyl-1,5,6,7-tetrahydro-4H-indole-4-
one (5c). This product was obtained as white solid (90%)
by reaction of 1,3-acetonylcyclohexandione with benzyl-
1
amine: mp 140–142 ꢁC; IR: 1638 (CO) cmꢁ1; H NMR:
r 1.94–1.98 (m, 2H, CH2), 2.10 (s, 3H, CH3), 2.29 (t,
J = 5.9 Hz, 2H, CH2), 2.67 (t, J = 5.9 Hz, 2H, CH2),
5.13 (s, 2H, CH2), 6.11 (s, 1H, H-3), 6.99 (dd, J = 7.5,
1.5 Hz, 2H, H-20, H-60), 7.22–7.39 (m, 3H, H-30, H-40,
H-50). 13C NMR: r 11.7 (CH3), 21.4 (CH2), 23.4
(CH2), 37.4 (CH2), 46.4 (CH2), 102.9 (CH), 119.1 (C),
125.9 (2· CH), 127.2 (CH), 128.7 (2· CH), 130.2 (C),
137.4 (C), 143.7 (C), 192.2 (CO). Anal. calcd for
C16H17NO: C, 80.30; H, 7.16; N, 5.85. Found: C,
80.60; H, 7.06; N, 5.80.
4.2.2. 5-Carbaldehyde-1,2-dimethyl-4-hydroxy-6,7-dihy-
dro-1H-indole (6b). This product was obtained as white
solid (70%) by formylation of tetrahydroindole 5b: mp
1
80–82 ꢁC; IR: 3024 (OH), 1632 (CO) cmꢁ1; H NMR
(CDCl3): r 2.19 (s, 3H, CH3), 2.57 (t, J = 6.3 Hz, 2H,
CH2), 2.69 (t, J = 6.3 Hz, 2H, CH2), 3.41 (s, 3H, CH3),
6.26 (s, 1H, H-3), 9.82 (s, 1H, CHO), 14.12 (bs, 1H,

P. Barraja et al. / Bioorg. Med. Chem. 14 (2006) 8712–8728
8723
OH). 13C NMR (CDCl3): r 12.1 (CH3), 21.7 (CH2), 24.6
(CH2), 30.3 (CH3), 103.1 (CH), 107.9 (C), 119.1 (C),
131.6 (C), 142.6 (C), 160.1 (C), 200.3 (CHO). Anal.
calcd for C11H13NO2: C, 69.09; H, 6.85; N, 7.32. Found:
C, 69.13; H, 6.70; N, 7.12.
101.4 (C), 103.1 (CH), 119.6 (C), 129.1 (C), 139.6 (C),
143.5 (CH), 182.4 (CO). Anal. calcd for C15H22N2O:
C, 73.13; H, 9.00; N, 11.37. Found: C, 73.00; H, 8.72;
N, 11.10.
4.3.3.
5-[(Diethylamino)methylene]-1-benzyl-2-methyl-
4.2.3. 1-Benzyl-5-carbaldehyde-4-hydroxy-2-methyl-6,7-
dihydro-1H-indole (6c). This product was obtained as
white solid (94%) by formylation of tetrahydroindole
1,5,6,7-tetrahydro-4H-indol-4-one (7c). This product
was obtained (85%) by reacting compound 6c with
1
diethylamine: mp 68–70 ꢁC; IR: 1633 (CO) cmꢁ1; H
1
5c: mp 93–95 ꢁC; IR: 3023 (OH), 1635 (CO) cmꢁ1; H
NMR: r 1.11 (t, J = 6.7 Hz, 6H, 2· CH2 CH3), 2.10 (s,
3H, CH3), 2.61 (t, J = 5.8 Hz, 2H, CH2), 2.79 (t,
J = 5.8 Hz, 2H, CH2), 3.25 (q, J = 6.7 Hz, 4H,
2· CH2CH3), 5.09 (s, 2H, CH2), 6.08 (s, 1H, H-3),
6.96 (d, J = 7.2 Hz, 2H, H-20, H-60), 7.21–7.36 (m, 3H,
H-30, H-40, H-50). 13C NMR: r 11.7 (CH3), 14.5
(2· CH3), 21.3 (CH2), 23.6 (CH2), 46.1 (CH2), 47.1
(2· CH2), 101.2 (C), 103.9 (CH), 120.1 (C), 125.9
(2· CH), 127.1 (CH), 128.7 (2· CH), 129.1 (C), 137.9
(C), 139.7 (C), 143.7 (CH), 182.5 (CO). Anal. calcd for
C21H26N2O: C, 78.22; H, 8.13; N, 8.69. Found: C,
78.00; H, 7.92; N, 8.58.
NMR (CDCl3):
r 2.14 (s, 3H, CH3), 2.53 (t,
J = 5.1 Hz, 2H, CH2), 2.62 (t, J = 5.1 Hz, 2H, CH2),
5.03 (s, 2H, CH2), 6.35 (s, 1H, H-3), 6.91 (d,
J = 7.2 Hz, 2H, H-20, H-60), 7.18–7.36 (m, 3H, H-30,
H-40, H-50), 9.94 (s, 1H, CHO), 14.04 (bs, 1H, OH).
13C NMR (CDCl3): r 11.9 (CH3), 21.7 (CH2), 24.5
(CH2), 47.0 (CH2), 103.7 (CH), 107.8 (C), 119.5 (C),
125.5 (2· CH), 127.6 (CH), 128.8 (2· CH), 131.6 (C),
136.4 (C), 142.7 (C), 160.2 (C), 187.6 (CHO). Anal.
calcd for C17H17NO2: C, 76.38; H, 6.41; N, 5.24. Found:
C, 76.18; H, 6.20; N, 5.12.
4.3. General procedure for the synthesis of 5-[(diethyl-
amino)methylene]-2-methyl-1-substituted-1,5,6,7-tetrahy-
dro-4H-indol-4-ones (7a–c)
4.4. General procedure for the synthesis of 3,7-disubsti-
tuted-8-methyl-1,5,6,7-tetrahydro-2H-pyrrolo[2,3-h]quin-
olin-2-ones (10–17)
A solution of the suitable hydroxy-methyl-1-substituted-
dihydroindole 6a–c (10 mmol) in benzene (40 mL) was
added dropwise at room temperature and under nitro-
gen atmosphere to a stirred solution diethylamine
(20 mmol) in benzene (30 mL). The mixture was stirred
at room temperature for 24 h and evaporated to dry-
ness. To the crude residue, Et2O was added and the
insoluble material was filtered off to afford the desired
compounds as white solids.
To a solution of the enaminones 7a–c (20 mmol) in
anhydrous ethanol (10 mL), the suitable cyanomethyl-
ene compound (22 mmol) was added and heated under
reflux for 24–48 h. The solid material which separated
from the solution was filtered off, washed with fresh eth-
anol and dried. Recrystallization from methanol gave
the desired compounds as yellow solids. In the case of
the reaction of 7a with phenylsulfonylacetonitrile, was
isolated the intermediate 8.
4.3.1.
5-[(Diethylamino)methylene]-2-methyl-1-phenyl-
4.4.1. 2-Methyl-1-phenyl-5-(20-phenylsulfonyl)-propane-
nitrile-1,5,6,7- tetrahydro-4H-indol-4-one 8. This product
was obtained as a yellow solid (50%): mp 234–235 ꢁC;
1,5,6,7-tetrahydro-4H-indol-4-one (7a). This product
was obtained (80%) by reacting compound 6a with
diethylamine: mp 146–148 ꢁC; IR: 1637 (CO) cmꢁ1; H
IR: 1637 (CO), 2216 (CN) cmꢁ1; H NMR: r 2.01 (s,
1
1
NMR: r 1.13 (t, J = 6.7 Hz, 6H, 2· CH2 CH3), 2.03 (s,
3H, CH3), 2.48 (t, J = 6.7 Hz, 2H, CH2), 2.78 (t,
J = 6.7 Hz, 2H, CH2), 3.27 (q, J = 6.7 Hz, 4H,
2· CH2CH3), 6.17 (s, 1H, H-3), 7.27 (s, 1H, CH), 7.36
(d, J = 6.2 Hz, 2H, H-20, H-60), 7.43–7.58 (m, 3H, H-
30, H-40, H-50). 13C NMR: r 12.4 (CH3), 14.5
(2· CH3), 21.9 (CH2), 23.5 (CH2), 47.1 (2· CH2), 101.1
(CH), 104.3 (CH), 120.5 (C), 127.3 (2· CH), 128.1
(CH), 129.3 (2· CH), 129.3 (C), 129.4 (C), 136.7 (C),
143.8 (C), 182.5 (CO). Anal. calcd for C20H24N2O: C,
77.89; H, 7.84; N, 9.08. Found: C, 77.50; H, 7.64; N,
8.86.
3H, CH3), 2.09–2.62 (m, 4H, 2· CH2), 5.29 (d,
J = 10.8 Hz, 1H, CH), 6.30 (s, 1H, H-3), 6.46 (d,
J = 10.8 Hz, 1H, CH), 7.38 (d, J = 8.1 Hz, 2H, Ar),
7.54–7.80 (m, 8H, Ar). 13C NMR: r 12.3 (CH3), 21.6
(CH2), 25.7 (CH2), 68.4 (CH), 103.9 (CH), 119.8 (C),
122.1 (CH), 127.4 (2· CH), 128.7 (CH), 129.0 (2· CH),
129.1 (2· CH), 129.6 (2· CH), 132.0 (C), 134.2 (CH),
136.0 (C), 137.5 (C), 142.5 (C), 143.8 (C), 164.0 (C),
180.8 (CO). Anal. calcd for C24H20N2SO3: C, 69.21;
H, 4.84; N, 6.73. Found: C, 69.00; H, 4.52; N, 6.68.
4.4.2. 8-Methyl-7-phenyl-3-phenylsulfonyl-1,5,6,7-tetra-
hydro-2H-pyrrolo[2,3-h]quinolin-2-one (10). This product
was obtained (80%) by reacting the intermediate 8 in
refluxing ethanol and stoichiometric amount of diethyl-
4.3.2. 5-[(Diethylamino)methylene]-1,2-dimethyl-1,5,6,7-
tetrahydro-4H-indol-4-one (7b). This product was ob-
tained (90%) by reacting compound 6b with diethyl-
amine: mp > 300 ꢁC; IR: 2921 (NH), 1635 (CO) cmꢁ1
;
amine: mp 107–108 ꢁC; IR: 1637 (CO) cmꢁ1
;
1H
1H NMR: r 2.02 (s, 3H, CH3), 2.56 (t, J = 7.6 Hz, 2H,
CH2), 2.80 (t, J = 7.6 Hz, 2H, CH2), 6.71 (s, 1H, H-9),
7.38 (d, J = 6.8 Hz, 2H, Ar), 7.50–7.69 (m, 6H, Ar),
7.96 (d, J = 6.8 Hz, 2H, Ar), 8.10 (s, 1H, H-4), 12.24
(s, 1H, NH). 13C NMR: 12.5 (CH3), 20.8 (CH2), 25.4
(CH2), 103.3 (CH), 107.0 (C), 111.8 (C), 118.1 (C),
127.2 (2· CH), 127.5 (2· CH), 128.4 (2· CH), 128.5
NMR: r 1.13 (t, J = 6.9 Hz, 6H, 2· CH2 CH3), 2.14 (s,
3H, CH3), 2.66 (t, J = 6.9 Hz, 2H, CH2), 2.80 (t,
J = 6.9 Hz, 2H, CH2), 3.26 (q, J = 6.9 Hz, 4H,
2· CH2), 3.36 (s, 3H, CH3), 6.00 (s, 1H, H-3), 7.20 (s,
1H, CH). 13C NMR: r: 11.8 (CH3), 14.5 (2· CH3),
21.1 (CH2), 23.5 (CH2), 29.7 (CH3), 47.1 (2· CH2),

8724
P. Barraja et al. / Bioorg. Med. Chem. 14 (2006) 8712–8728
(CH), 129.4 (2· CH), 130.8 (C), 132.5 (CH), 136.2 (C),
137.7 (C), 141.5(C), 142.0 (CH), 148.6 (C), 158.2 (CO).
Anal. calcd for C24H20N2O3S: C, 69.21; H, 4.84; N,
6.73. Found: C, 69.11; H, 4.69; N, 6.53.
NMR: r 2.07 (s, 3H, CH3), 2.40 (t, J = 6.6 Hz, 2H,
CH2), 2.70 (t, J = 6.6 Hz, 2H, CH2), 3.22 (s, 3H, CH3),
6.41 (s, 1H, H-9), 7.41–7.57 (m, 3H, H-30, H-50, H-40),
7.86 (dd, J = 8.1, 1.5 Hz, 2H, H-20 e H-60), 7.91 (s, 1H,
H-4), 11.84 (s, 1H, NH). 13C NMR: r 11.9 (CH3),
20.0 (CH2), 25.2 (CH2), 30.3 (CH3), 102.3 (CH), 106.4
(C), 117.5 (C), 127.5 (2· CH), 128.1 (C), 128.5
(2· CH), 128.7 (C), 131.1 (C), 132.6 (CH), 138.3 (C),
142.3 (CH), 148.3 (C), 157.5 (CO). Anal. calcd for
C19H18N2O3S: C, 64.39; H, 5.12; N, 7.90. Found: C,
64.00; H, 5.04; N, 7.58.
4.4.3. 8-Methyl-7-phenyl-3-benzoyl-1,5,6,7-tetrahydro-
2H-pyrrolo[2,3-h]quinolin-2-one (11). This product was
obtained (50%) by reacting the diethylamino methylene
1-phenyl-indolone
7a
with
benzoylacetonitrile:
mp > 300 ꢁC; IR: 2904 (b, NH), 1631 (b, CO) cmꢁ1
;
1H NMR: r 2.11 (s, 3H, CH3), 2.63 (t, J = 7.6 Hz, 2H,
CH2), 2.82 (t, J = 7.6 Hz, 2H, CH2), 6.81 (s, 1H, H-9),
7.40–7.64 (m, 8H, Ar), 7.74 (s, 1H, H-4), 7.76 (d,
J = 7.6 Hz, 2H, Ar), 12.1 (s, 1H, NH). 13C NMR: r
12.6 (CH3), 20.9 (CH2), 25.4 (CH2), 103.2 (CH), 107.6
(C), 111.8 (C), 119.8 (C), 127.3 (2· CH), 127.8
(2· CH), 128.5 (CH), 128.8 (2· CH), 129.4 (2· CH),
130.6 (C), 131.8 (CH), 136.3 (C), 136.9 (C), 138.7 (C),
144.1 (CH), 145.8 (C), 160.7 (CO), 193.8 (CO). Anal.
calcd for C25H20N2O2: C, 78.93; H, 5.30; N, 7.36.
Found: C, 78.80; H, 5.00; N, 6.98.
4.4.7. 3-Benzoyl-7,8-dimethyl-1,5,6,7-tetrahydro-2H-pyr-
rolo[2,3-h]quinolin-2-one (15). This product was obtained
(40%) by reacting the diethylamino methylene-1-methyl-
indolone 7b with benzoylacetonitrile: mp > 300 ꢁC; IR:
1
2915 (b, NH), 1624 (b, CO) cmꢁ1; H NMR: r 2.19 (s,
3H, CH3), 2.75–2.78 (m, 4H, 2· CH2), 3.44 (s, 3H,
CH3), 6.55 (s, 1H, H-9), 7.38–7.57 (m, 3H, H-30, H-50,
H-40), 7.67 (s, 1H, H-4), 7.68 (d, J = 6.2 Hz, 2H, H-20,
H-60), 11.87 (s, 1H, NH). 13C NMR: r 11.9 (CH3),
20.10 (CH2), 25.3 (CH2), 30.2 (CH3), 102.1 (CH),
107.1 (C), 110.6 (C), 118.8 (C), 127.7 (2· CH), 128.8
(2· CH), 130.6 (C), 131.6 (CH), 137.1 (C), 139.0 (C),
144.1 (CH), 146.6 (C), 160.7 (CO), 193.7 (CO). Anal.
calcd for C20H18N2O2: C, 75.45; H, 5.70; N, 8.80.
Found: C, 75.30; H, 5.30; N, 8.66.
4.4.4. 3-Cyano-8-methyl-7-phenyl-1,5,6,7-tetrahydro-2H-
pyrrolo[2,3-h]quinolin-2-one (12). This product was ob-
tained (30%) by reacting the diethylamino methylene
1-phenyl-indolone 7a with malononitrile or cyanoaceta-
mide: mp > 300 ꢁC; IR: 2948 (b, NH), 2210 (CN), 1627
(CO) cmꢁ1 1H NMR: r 2.06 (s, 3H, CH3), 2.50 (t,
;
J = 6.6 Hz, 2H, CH2), 2.64 (t, J = 6.6 Hz, 2H, CH2),
6.87 (s, 1H, H-9), 7.39 (d, J = 6.6 Hz, 2H, H-20, H-60),
7.41 (s, 1H, H-4), 7.50–7.61 (m, 3H, H-30, H-40, H-50),
11.6 (s, 1H, NH). 13C NMR: r 12.4 (CH3), 20.8
(CH2), 24.2 (CH2), 91.5 (C), 104.2 (CH), 111.4 (C),
113.5 (C), 118.0 (C), 127.4 (2· CH), 128.6 (CH), 129.5
(2· CH), 131.0 (C), 134.6 (CH), 136.1 (C), 138.5 (C),
150.3 (C), 161.2 (CO). Anal. calcd for C19H15N3O: C,
75.73; H, 5.02; N, 13.94. Found: C, 75.33; H, 4.88; N,
13.74.
4.4.8. 7-Benzyl-8-methyl-3-phenylsulfonyl-1,5,6,7-tetra-
hydro-2H-pyrrolo[2,3-h]quinolin-2-one (16). This prod-
uct was obtained (80%) by reacting the diethylamino
methylene 1-benzyl-indolone 7c with benzylsulfonyl-
acetonitrile: mp > 300 ꢁC; IR: 2942 (b, NH), 1635
(CO) cmꢁ1 1H NMR: r 2.10 (s, 3H, CH3), 2.73–
;
2.81 (m, 4H, 2· CH2), 5.17 (s, 2H, CH2), 6.62 (s,
1H, H-9), 7.98 (dd, J = 7.5, 1.5 Hz, 2H, Ar), 7.25–
7.38 (m, 3H, Ar), 7.51–7.64 (m, 3H, Ar), 7.96 (dd,
J = 6.9, 1.6 Hz, 2H, Ar), 8.02 (1H, s, H-4), 12.14
(1H, s, NH). 13C NMR: r 11.9 (CH3), 20.2 (CH2),
25.3 (CH2), 46.4 (CH2), 103.0 (CH), 106.6 (C), 110.7
(C), 118.0 (C), 125.9 (2· CH), 127.2 (CH), 127.5
(2· CH), 128.5 (2· CH), 128.7 (2· CH), 130.9 (C),
132.6 (CH), 137.3 (C), 138.2 (C), 141.4 (C), 142.4
(CH), 148.1 (C), 157.5 (CO). Anal. calcd for
C25H22N2O3S: C, 69.75; H, 5.15; N, 6.51. Found: C,
69.35; H, 4.96; N, 6.30.
4.4.5. 3-Ethoxycarbonyl-8-methyl-7-phenyl-1,5,6,7-tetra-
hydro-2H-pyrrolo[2,3-h]quinolin-2-one (13). This product
was obtained (30%) by reacting the diethylamino meth-
ylene 1-phenyl-indolone 7a with the anion of ethyl-
cyanoacetate generated in situ by the addition of
stoichiometric sodium ethylate and by heating 72 h un-
der reflux: mp > 300 ꢁC; IR: 2930 (b, NH), 1714 (CO),
1
1620 (CO) cmꢁ1; H NMR: r 1.22 (t, J = 7.0 Hz, 3H,
CH3), 2.01 (s, 3H, CH3), 2.47 (t, J = 7.4 Hz, 2H, CH2),
2.70 (t, J = 7.4 Hz, 2H, CH2), 4.14 (q, J = 7.0 Hz, 2H,
CH2), 6.71 (s, 1H, H-9), 7.33–7.54 (m, 5H, Ar), 7.89
(s, 1H, H-4), 11.91 (s, 1H, NH). 13C NMR: r 12.5
(CH3), 14.3 (CH3), 20.9 (CH2), 25.4 (CH2), 59.3
(CH2), 87.9 (C), 103.2 (CH), 116.3 (C), 127.3 (2· CH),
128.5 (CH), 129.4 (2· CH), 131.0 (C), 134.6 (C), 136.1
(C), 138.7 (CH), 138.5 (C), 143.5 (C), 157.1 (CO),
169.7 (CO). C23H20N2O3: C, 72.40; H, 5.79; N, 8.04.
Found: C, 72.00; H, 5.45; N, 7.92.
4.4.9.
3-Benzoyl-7-benzyl-1,5,6,7-tetrahydro-2H-pyr-
rolo[2,3-h]quinolin-2-one (17). This product was obtained
(50%) by reacting the diethylamino methylene 1-benzyl-
indolone 7c with benzoylacetonitrile: mp > 300 ꢁC; IR:
2892 (b, NH), 1635 (b, CO) cmꢁ1; H NMR: r 2.12 (s,
1
3H, CH3), 2.74–2.78 (m, 4H, 2· CH2), 5.17 (s, 2H,
CH2), 6.67 (s, 1H, H-9), 7.00 (d, J = 7.3, 2H, Ar),
7.23–7.57 (m, 6H, Ar), 7.68 (s, 1H, H-4), 6.70 (d,
J = 7.3 Hz, 2H, Ar), 12.0 (s, 1H, NH). 13C NMR: r
11.9 (CH3), 20.3 (CH2), 25.3 (CH2), 46.4 (CH2), 102.9
(CH), 111.3 (C), 119.2 (C), 125.9 (2· CH), 127.2 (CH),
127.8 (2· CH), 128.7 (2· CH), 128.7 (C), 128.8
(2· CH), 130.4 (C), 131.6 (CH), 137.0 (C), 137.4 (C),
138.9 (C), 144.0 (CH), 1146.2 (C), 160.7 (CO), 193.7
(CO). Anal. calcd for C26H22N2O2: C, 79.17; H, 5.62;
N, 7.10. Found: C, 78.87; H, 5.32; N, 6.90.
4.4.6. 7,8-Dimethyl-3-phenylsulfonyl-1,5,6,7-tetrahydro-
2H-pyrrolo[2,3-h]quinolin-2-one (14). This product was
obtained (80%) by reacting the diethylamino methylene
1-methyl-indolone 7b with phenylsulfonylacetonitrile:
1
mp > 300 ꢁC; IR 2925 (b, NH), 1624 (CO) cmꢁ1; H

P. Barraja et al. / Bioorg. Med. Chem. 14 (2006) 8712–8728
8725
4.5. Oxidation of 5-carbaldehyde-4-hydroxy-2-methyl-1-
substituted-6,7-dihydro-1H-indoles (6a–c)
able 1,3-phenylendiamine (17.3 g, 164 mmol) and
ethylacetoacetate (21.2 mL, 164 mmol) was heated at
130 ꢁC for 48 h. The white solid which separated upon
cooling was filtered off and recrystallized from methanol
to provide 20 (60%): mp 168–170 ꢁC; IR: 3407 (NH),
A warm solution of DDQ (5 mmol) in anhydrous
dichloromethane (30 mL) was added dropwise to a
warm and stirred solution of the suitable indole deriva-
tive 6a–c (5 mmol) in the same solvent (20–40 mL).
After the addition was completed, the reaction mixture
was stirred at room temperature for 3–4 h and then fil-
tered. The filtrate was washed with 1 M NaOH
(30 mL) and once with water (30 mL). The organic layer
was dried over sodium sulfate and evaporated under re-
duced pressure. The residue was recrystallized from eth-
anol to give the aromatic derivatives 18a–c.
3300 and 3184 (NH2), 1662 (CO) cmꢁ1 1H NMR
;
(CDCl3): r 2.29 (s, 1H, CH3), 5.73 (br s, 2H, NH2),
6.00 (s, 1H, H-3), 6.42 (d, J = 2.2 Hz, 1H, H-8), 6.50
(dd, J = 8.7, 2.2 Hz, 1H, H-6), 7.36 (d, J = 8.7 Hz, 1H,
H-5), 11.25 (s, 1H, NH). 13C NMR (CDCl3): r 18.4
(CH3), 96.8 (CH), 110.4 (C), 110.5 (CH), 114.6 (CH),
125.5 (CH), 140.7 (C), 148.0 (C), 151.0 (C), 162.5
(CO). Anal. calcd for C10H10N2O: C, 68.95; H, 5.79;
N, 16.08. Found: C, 68.70; H, 5.68; N, 15.96.
4.5.1. 5-Carbaldehyde-4-hydroxy-2-methyl-1-phenyl-1H-
indole (18a). This product was obtained as a white solid
(80%): mp 174–176 ꢁC; IR: 3150 (b, OH), 1633 (CO)
4.7. 7-Hydrazino-4-methylquinolin-2(1H)-one (21)
This compound was prepared according to the proce-
dure reported.20 A solution of the 7-aminoquinoline 20
(1.74 g, 10 mmol) in hydrochloric acid (10 mL) was diaz-
otized at ꢁ5 ꢁC with sodium nitrite (0.75 g, 11 mmol)
dissolved in water (3 mL). The mixture was stirred at
that temperature for 4 h and the diazonium salt was re-
duced by pouring it into a cold solution of stannous
chloride (5 g, 22 mmol) dissolved in hydrochloric acid
(5 mL). After stirring at ꢁ12 ꢁC for 1.5 h, the mixture
of the hydrazine hydrochloride and stannic chloride
was filtered off, dissolved in water and the solution basi-
fied with 1 N NaOH. The solid which separated was
filtered, dried to afford the free base (1.5 g, 80%) which
was used in the next step without any further purifica-
tion: mp 162–164 ꢁC; IR: 3450 (NH), 3264 and 3145
1
cmꢁ1; H NMR (CDCl3): r 2.00 (s, 3H, CH3), 6.60 (d,
J = 9.2 Hz, 1H, H-7), 6.71 (s, 1H, H-3), 7.35 (d,
J = 9.2 Hz, 1H, H-6), 7.40–7.47 (d, J = 7.2 Hz, 2H, H-
20, H-60), 7.55–7.64 (m, 3H, H-30, H-40, H-50), 8.50 (s,
1H, CHO), 10.05 (s, 1H, OH). 13C NMR (CDCl3): r
12.9 (CH3), 100.0 (CH), 103.1 (CH), 113.6 (C), 115.8
(C), 124.4 (CH), 127.6 (2· CH), 128.5 (CH), 129.7
(2· CH), 132.2 (C), 136.8 (C), 142.3(C), 151.8 (C),
193.9 (CHO). Anal. calcd for C16H13NO2: C, 76.48; H,
5.21; N, 5.57. Found: C, 76.40; H, 4.86; N, 5.42.
4.5.2. 5-Carbaldehyde-1,2-dimethyl-4-hydroxy-1H-indole
(18b). This product was obtained as white solid (75%):
1
mp 152–154 ꢁC; IR: 3230 (OH), 1629 (CO) cmꢁ1; H
NMR (CDCl3): r 2.37 (s, 3H, CH3), 3.61 (s, 3H,
CH3), 6.48 (s, 1H, H-3), 6.84 (d, J = 8.8 Hz, 1H, H-7),
7.16 (d, J = 8.8 Hz, 1H, H-6), 9.80 (s, 1H, CHO),
12.14 (bs, 1H, OH). 13C NMR (CDCl3): r 12.2 (CH3),
29.7 (CH3), 98.5 (CH), 102.9 (CH), 112.8 (C), 115.9
(C), 123.8 (CH), 137.2 (C), 142.0 (C), 155.9 (C), 195.3
(CHO). Anal. calcd for C11H11NO2: C, 69.83; H, 5.86;
N, 7.40. Found: C, 69.60; H, 5.76; N, 7.30.
(NH2), 1648 (CO) cmꢁ1 1H NMR: r 2.41 (s, 1H,
;
CH3), 4.14 (br s, 2H, NH2), 5.97 (s, 1H, H-3), 6.59 (d,
J = 2.0 Hz, 1H, H-8), 6.64 (dd, J = 7.6, 2.0 Hz, 1H, H-
6), 7.35 (s, 1H, NH), 7.37 (d, J = 7.6 Hz, 1H, H-5),
11.18 (s, 1H, NH). 13C NMR: r 18.4 (CH3), 94.2
(CH), 107.9 (CH), 110.7 (C), 114.8 (CH), 125.0 (CH),
140.7 (C), 147.8 (C), 154.1 (C), 162.3 (CO). Anal. calcd
for C10H11N3O: C, 63.48; H, 5.86; N, 22.21. Found: C,
63.28; H, 5.76; N, 22.10.
4.5.3. 1-Benzyl-5-carbaldehyde-4-hydroxy-2-methyl-1H-
indole (18c). This product was obtained as white solid
4.8. 4-Methyl-7-[2-(1-methylethylidene)hydrazine]quino-
lin-2(1H)-one (22)
(74%): mp 120–121 ꢁC; IR: 3288 (OH), 1635 (CO) cmꢁ1
;
1H NMR: r 2.33 (s, 3H, CH3), 5.43 (s, 2H, CH2), 6.60
(s, 1H, H-3,), 6.98 (d, J = 7.2 Hz, 2H, H-20, H-60), 7.08
(d, J = 8.0 Hz, 1H, H-7), 7.19–7.31 (m, 3H, H-30, H-40,
H-50), 7.36 (d, J = 8.0 Hz, 1H, H-6), 10.02 (s, 1H,
CHO), 11.71 (s, 1H, OH). 13C NMR: r 12.3 (CH3), 46.1
(CH2), 99.4 (CH), 103.3 (CH), 113.1 (C), 116.3 (C),
124.2 (CH), 126.1 (2· CH), 127.2 (CH), 128.6 (2· CH),
136.9 (C), 137.6 (C), 142.0 (C), 156.0 (C), 194.2 (CHO).
Anal. calcd for C17H15NO2: C, 76.96; H, 5.70; N, 5.28.
Found: C, 76.79; H, 5.56; N, 5.10.
To a solution of the hydrazine 21 (1.3 g, 6.9 mmol) in
ethanol (20 mL), acetone (0.8 mL, 7.5 mmol) was
added dropwise and the reaction mixture was heated
under reflux for 1 h. Upon cooling a white solid sep-
arated which was filtered off and dried in the dessic-
cator. Recrystallization from ethanol produced the
hydrazone 22 (85%): mp 240–241 ꢁC; IR: 3511
(NH), 3282 (NH), 1631 (CO) cmꢁ1 1H NMR: r
;
1.91 (s, 1H, CH3), 1.97 (s, 1H, CH3), 2.32 (s, 1H,
CH3), 6.04 (s, 1H, H-3), 6.91 (dd, J = 8.7, 2.4 Hz,
1H, H-6), 6.98 (d, J = 2.4 Hz, 1H, H-8), 7.46 (d,
J = 8.7 Hz, 1H, H-5), 9.09 (s, 1H, NH), 11.26 (s,
1H, NH). 13C NMR: r 16.8 (CH3), 18.4 (CH3),
25.0 (CH3), 96.0 (CH), 108.5 (CH), 112.0 (C), 115.7
(CH), 125.2 (CH), 140.4 (C), 145.5 (C), 147.7 (C),
148.2 (C), 162.2 (CO). Anal. calcd for C13H15N3O:
C, 68.10; H, 6.59; N, 18.33. Found: C, 67.84; H,
6.30; N, 18.00.
Compounds 18a–c were obtained in traces from the oxi-
dation of derivatives 7a–c under the same reaction con-
ditions used for the oxidation of compounds 6a–c.
4.6. 7-Amino-4-methylquinolin-2(1H)-one (20)
This compound was prepared according to the proce-
dure reported.19 A mixture of the commercially avail-

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P. Barraja et al. / Bioorg. Med. Chem. 14 (2006) 8712–8728
When the reaction was carried out in refluxing acetic
acid (12 mL), after the separation of the solid the acetic
filtrate was poured into crushed ice, and upon neutral-
ization with 10% sodium carbonate an insoluble materi-
al formed which was filtered off, recrystallized from
ethanol to afford a mixture of the hydrazone 22 (60%)
and of 23.
DMSO and diluted with Hanks’ Balanced Salt Solution
(HBSS, pH 7.2), was added to each well and incubated
at 37 ꢁC for 30 min and then irradiated. After irradia-
tion, the solution was replaced with the medium, and
the plates were incubated for 72 h. Cell viability was as-
sayed by the MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5
diphenyl tetrazolium bromide)] test, as described previ-
ously.41 Analogous experiments were performed in the
presence of various additives.
4.9. 4,8-Dimethyl-1,7-dihydro-2H-pyrrolo[2,3-h]quinolin-
2-one (23)
4.10.4. Fluorescence microscopy. HT-1080 fibrosarcoma
cells were grown in sterile microscope slides and treated
with the test compounds at the concentration of 20 lM
for 30 min, then washed with HBSS. Cellular fluores-
cence images were acquired with an Olympus IMT-2
inverted microscope, as previously described.42 For sub-
cellular co-localization studies tetramethyl rhodamine
methyl ester (TMRM) and acridine orange (AO) were
used as fluorescent probes which stain mitochondria
and lysosomes, respectively. Pyrroloquinolones’ excita-
tion was performed at 330 nm and emission was read
at 450 nm. Long pass emission filter settings were used
to separate the emission of the probes from that of the
test compounds. Data were acquired and analysed using
the Metamorph software (Universal imaging).
(20%): mp 198–200 ꢁC; IR: 3451 (NH), 3245 (NH), 1651
1
(CO) cmꢁ1; H NMR: r 1.94 (s, 1H, CH3), 2.41 (s, 1H,
CH3), 6.31 (s, 1H, H-9), 6.67 (s, 1H, H-3), 6.72 (d,
J = 9.3 Hz, 1H, H-6), 7.58 (d, J = 9.3 Hz, 1 H, H-5),
9.80 (s, 1H, NH), 12.1 (s, 1H, NH). 13C NMR: r 18.7
(CH3), 20.6 (CH3), 95.6 (CH), 110.5 (CH), 112.1 (CH),
111.1 (C), 113.6 (C), 125.9 (CH), 139.5 (C), 151.6 (C),
152.2 (C), 161.6 (C),169.1 (CO). Anal. calcd for
C13H12N2O: C, 73.57; H, 5.70; N, 13.20. Found: C,
73.27; H, 5.38; N, 13.00.
Compound 23 was also obtained upon reaction of the
hydrazone 22 (0.3 g, 1.3 mmol) with anhydrous zinc
chloride (1.5 g, 10 mmol). The reaction mixture was
heated to melting (280–290 ꢁC) for 1 h. After cooling,
the solid mass was suspended in water, filtered, dried
and purified by SEPACORE chromatography using a
4.10.5. Lipid peroxidation. For these experiments, HT-
1080 cells were seeded at the concentration of 50,000
cells/mL in 35-mm Petri dishes grown for two days to
reach confluency and then were irradiated in HBSS in
the presence of the test compounds. At different times
of irradiation supernatant was collected and frozen after
addition of 2,6-di-tert-butyl-p-cresol (BHT, 2% in etha-
nol). Cells were removed with the aid of a cell scraper
after addition of 500 ll water. Lipid peroxidation both
in the supernatants and cell extracts was measured using
¨
BUCHI apparatus (ethyl acetate/methanol, 95:5).
Recrystallization from ethanol afforded the cyclic deriv-
ative 23 (0.18 g, 65%).
4.10. Biology
4.10.1. Irradiation procedure. HPW 125 Philips lamps,
mainly emitting at 365 nm, were used for irradiation
experiments. The spectral irradiance of the source was
4.0 mW cmꢁ2 as measured, at the sample level, by a
Cole-Parmer Instrument Company radiometer (Niles,
IL), equipped with a 365-CX sensor.
a thiobarbituric acid assay as described previously.29
A
standard curve of 1,1,3,3-tetraethoxypropane was used
to quantitate the amount of malonaldehyde produced.
Data are expressed in terms of nanomoles of TBARS
normalized to the total protein content measured as de-
scribed,42 in an aliquot of the cell extract.
4.10.2. Spectrophotometric measurement. UV–vis
absorption spectra were recorded on a Perkin-Elmer
Lambda 15 spectrophotometer and emission spectra
were recorded on a Perkin-Elmer LS-50B luminescence
spectrophotometer.
4.10.6. Protein photo-cross-link. Erythrocytes’ plasmatic
membranes (ghosts) were prepared as described.43 The
test compounds were added to the membrane suspen-
sion (1.0 mg/mL protein concentration) and irradiated
for different times. The membrane samples were reduced
and denatured by addition of b-mercaptoethanol and
SDS at 90 ꢁC for 3 min, and bromophenol blue (BPB)
was added before polyacrylamide gel electrophoresis
analysis (5% running gel, 3% stacking gel). The quanti-
tation of the bands, stained with Coomassie brilliant
blue R-250 was achieved by image analyzer software
Quantity One (Bio-Rad, Milano, Italy).
4.10.3. Cellular phototoxicity. Human promyelocytic
leukaemia cells (HL-60) were grown in RPMI-1640
medium, (Sigma Co, MO, USA) human fibrosarcoma
cells (HT-1080) were grown in DMEM (Sigma Co,
MO, USA), and intestinal adenocarcinoma cells (LoVo)
were grown in Ham’s F12 medium (Sigma Co, MO,
USA) all supplemented with 115 U/mL of penicillin G
(Invitrogen, Milano, Italy), 115 lg/mL streptomycin
(Invitrogen, Milano, Italy) and 10% fetal calf serum
(Invitrogen, Milano, Italy). Individual wells of a 96-well
tissue culture microtitre plate (Falcon BD) were inocu-
lated with 100 ll of complete medium containing
8 · 103 HL-60 cells or 5 · 103 HT-1080 and LoVo cells.
The plates were incubated at 37 ꢁC in a humidified 5%
incubator for 18 h prior to the experiments. After medi-
um removal, 100 ll of the drug solution, dissolved in
4.10.7. Studies on isolated proteins. Solutions of Bovine
serum albumin (BSA) (0.5 mg/mL) in phosphate buffer
10 mM were irradiated in the presence of the test com-
pounds for various times in a quartz cuvette. At each
time the tryptophan (Trp) content was followed by mon-
itoring the characteristic Trp fluorescence as described
by Balasubramanian et al.34 Solutions of Ribonuclease

P. Barraja et al. / Bioorg. Med. Chem. 14 (2006) 8712–8728
8727
8. Rossetti, F.; Zulian, F.; Dall’Amico, R.; Messina, C.;
Montini, G.; Zacchello, F. Transplantation 1995, 59, 49–
51.
A (RNAseA), 0.5 mg/mL in phosphate buffer 10 mM,
were irradiated in the presence of the test compounds
for various times in a quartz cuvette. The fluorescence
was followed, as described above. Analogous experi-
ments were performed by bubbling nitrogen prior to
and during the irradiation and also in the presence of
superoxide dismutase. Further experiments were per-
formed in deuterated phosphate buffer.
9. Dall’Amico, R.; Rossetti, F.; Zulian, F.; Montini, G.;
Murer, L.; Andretta, B.; Messina, C.; Baraldi, E.;
Montesco, M. C.; Dini, G.; Locatelli, F.; Argiolu, F.;
Zacchello, G. Br. J. Hematol. 1997, 97, 848–854.
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11. Child, F. J.; Ratnavel, R.; Watkins, P.; Samson, D.;
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12. See for example: (a) Kirland, D. J.; Creed, K. L.;
Mannisto, P. Mutat. Res. 1983, 116, 73–82; (b) Venturini,
S.; Tamaro, M.; Monti-Bragadin, C.; Carlassare, F.
Mutat. Res. 1981, 88, 17–22.
4.10.8. Flow linear dichroism. Linear-dichroism (LD)
spectra were recorded in a ‘flow cell’ on a Jasco J500A
spectropolarimeter equipped with an IBM PC and a Jas-
co J interface. The determination and interpretation of
the data was performed as previously described.44
DNA concentration was 2.27 mM and the measure-
ments were performed at different [DNA]/[dye].
13. (a) Guiotto, A.; Rodighiero, P.; Mancini, P.; Pastorini, G.;
Bordin, F.; Bacicchetti, F.; Carlassare, F.; Vedaldi, D.;
Dall’Acqua, F.; Tamaro, M.; Recchia, G.; Cristofolini, M.
J. Med. Chem. 1984, 27, 959–966; (b) Bordin, F.;
Dall’Acqua, F.; Guiotto, A. Pharmacol. Ther. 1991, 52,
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Bacicchetti, F.; Carlassare, F.; Vedaldi, D.; Caffieri, S.;
Pozzan, A.; Dall’Acqua, F. J. Med. Chem. 1996, 39, 1293–
1302; (b) Jacobs, A. E.; Piette, J. J. Med. Chem. 1995, 38,
869–874.
4.10.9. HPLC separation and photoproducts character-
ization. LC-MS was performed using a Perkin-Elmer
Series 200 (Norwalk, CT, USA), equipped with Waters
‘X-Terra’ column (3.5 lm; 3.5· 150 mm i.d.) chromato-
graphic separations. The mobile phase consisted of: elu-
ent A, water/formic acid (99:1); eluent B, acetonitrile/
formic acid (99:1). Gradient: 5% B (0 min), 95% B
(30 min); flow rate 60 ll/min. ESI/MS measurements
were performed with an API-TOF. Mariner 5220
(Perseptive Biosystem Stratford, Texas, USA).
15. Alabaster, C. T.; Bell, A. S.; Campbell, S. F.; Ellis, P.;
Henderson, C. G.; Morris, D. S.; Roberts, D. A.;
Ruddock, K. S.; Samuels, G. M. R.; Stefaniak, M. J.
Med. Chem. 1989, 32, 575–583.
5. Supplementary material
16. Barraja, P.; Diana, P.; Lauria, A.; Montalbano, A.;
Almerico, A. M.; Dattolo, G.; Cirrincione, G.; Viola,
G.; Dall’Acqua, F. Bioorg. Chem. Lett. 2003, 13, 2809–
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17. Stetter, H.; Lauterbach, R. Liebigs Ann. Chem. 1962, 655,
20–27.
Agarose gel electrophoresis, UV–vis spectra of photo-
degradation and flow linear dichroism spectra of the
compounds. This material is available free of charge
via the Internet at www.sciencedirect.com.
18. Bondavalli, F.; Bruno, O.; Lo Presti, E.; Menozzi, G.;
Mosti, L. Synthesis 1999, 1169–1174.
Acknowledgments
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Eger, K.; Lanzner, K.; Rothenhausler, K. Liebigs Ann.
Chem. 1993, 465–470; (d) Remers, W. A.; Roth, R. H.;
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1989, 26, 917–922.
21. Ain Khan, M.; Brito Morley, M. L. J. Heterocycl. Chem.
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This work was financially supported by Ministero
`
dell’Istruzione dell’Universita e della Ricerca.
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