Pyrrolo[3,4-h]quinolinones a new class of photochemotherapeutic agents

Article abstract of DOI:10.1016/j.bmc.2011.02.023

Pyrrolo[3,4-h]quinolin-2-ones were synthesized as nitrogen isosters of the angular furocoumarin angelicin, with the aim of obtaining new photochemotherapeutic agents with increased antiproliferative activity and lower undesired toxic effects. A versatile synthetic pathway was approached to allow the isolation of derivatives of the new ring system with a good substitution pattern on the pyrrole moiety. Photobiological screenings of the new compounds revealed a potent phototoxic effect and a great UVA dose dependence, reaching IC₅₀ values at submicromolar level. The induced cellular photocytotoxicity was related to apoptosis with the involvement of mitochondria and lysosomes, alteration of cell cycle profile and membrane lipid peroxidation.

Full text of DOI:10.1016/j.bmc.2011.02.023

Bioorganic & Medicinal Chemistry 19 (2011) 2326–2341
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Pyrrolo[3,4-h]quinolinones a new class of photochemotherapeutic agents
Paola Barraja a, Patrizia Diana a, Alessandra Montalbano a, Anna Carbone a, Giampietro Viola b,
Giuseppe Basso b, Alessia Salvador c, Daniela Vedaldi c, Francesco Dall’Acqua c, Girolamo Cirrincione a,
⇑
a Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, Sezione di Chimica Farmaceutica e Biologica, Università degli Studi di Palermo, Via Archirafi 32, Palermo 90123, Italy
b Dipartimento di Pediatria, Laboratorio di Oncoematologia, Università degli Studi di Padova, Via Giustiniani 2, Padova 35128, Italy
c Dipartimento di Scienze Farmaceutiche Università degli Studi di Padova, Via Marzolo 5, Padova 35131, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyrrolo[3,4-h]quinolin-2-ones were synthesized as nitrogen isosters of the angular furocoumarin angel-
icin, with the aim of obtaining new photochemotherapeutic agents with increased antiproliferative activ-
ity and lower undesired toxic effects. A versatile synthetic pathway was approached to allow the isolation
of derivatives of the new ring system with a good substitution pattern on the pyrrole moiety. Photobio-
logical screenings of the new compounds revealed a potent phototoxic effect and a great UVA dose
dependence, reaching IC50 values at submicromolar level. The induced cellular photocytotoxicity was
related to apoptosis with the involvement of mitochondria and lysosomes, alteration of cell cycle profile
and membrane lipid peroxidation.
Received 29 September 2010
Revised 9 February 2011
Accepted 13 February 2011
Available online 18 February 2011
Keywords:
Pyrrolo[3,4-h]quinolinones
Angelicin heteroanalogues
Photochemotherapy
Phototoxicity
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
O
O
X
X
NH
Furocumarins are a family of naturally occurring photosensitiz-
ing drugs that find application in the treatment of several skin dis-
eases, such as psoriasis, vitiligo and cutaneous T-cell lymphoma
when used in conjuction with long-wave (320–400 nm) ultraviolet
light (UV-A).1,2 The effectiveness of this treatment, called PUVA, is
connected with the specific damage which these compounds can
induce to DNA. The photoexcited furocumarins are able to react
with biomolecules especially with pyrimidine bases of DNA. In fact,
the tricyclic planar structure allows the initial intercalation be-
tween nucleic acid base pairs followed by light activation and for-
mation of mono- and bis-adducts (inter-strand cross-links, ISC),
which join the two complementary strands of the macromolecule;
these latter are considered the main cause of the photoinduced cell
killing. Nowadays, human skin diseases such as psoriasis, and vit-
iligo are currently treated with PUVA therapy and thanks to the
development of the extracorporeal photochemotherapy (ECP), also
called photopheresis, T-cell lymphoma (CTCL) is also efficiently
cured. In this process, peripheral blood is exposed, in an extracor-
poreal system, to photoactivated 8-methoxypsoralen (8-MOP)
mainly for the treatment of disorders caused by aberrant T-lym-
phocytes.3 Photopheresis was approved by FDA for the cure of
T-cell lymphoma, but it is also effective against various tumors,
autoimmune diseases and in the prevention of rejection in organ
transplantation.4,5
O
N
R
X
2
1
a X-X= CH2-CH2
b X-X= CH=CH
O
Angelicin: X=O
Furoquinolinone: X=NH
O
2
1
NH
3
4
NH
R
9
7
N
8
N R
5
6
3
4
Chart 1. Structures of angelicin and furoquinolinone 1, pyrrolo[2,3-h]quinolin-2-
ones 2, pyrrolo[3,2-h]quinolin-2-ones 3 and pyrrolo[3,4-h]quinolin-2-ones 4.
Derivatives of angelicin 1 (Chart 1, X = O) have been synthesized
with the purpose to reduce long term side effects such as genotox-
icity and risk of skin cancer caused by ISC. In fact, this angular
isomer of psoralen possesses the ability of causing only monofunc-
tional damage to DNA because of its geometry.
Over the latest two decades, several heteroanalogues of angeli-
cin have been proposed with the aim to dissociate undesirable side
effects from the therapeutic ones.6 A new family of compounds,
namely the furoquinolinones 1 (Chart 1, X = NH) nitrogen isosters
of angelicin, has been extensively studied.
⇑
Corresponding author. Tel.: +39 091 6161606; fax: +39 091 23860854.
E-mail address: gcirrinc@unipa.it (G. Cirrincione).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.023

P. Barraja et al. / Bioorg. Med. Chem. 19 (2011) 2326–2341
2327
In particular, the 1,4,6,8- and 4,6,8,9- tetramethyl derivatives
(FQ) an (HFQ), respectively showed strong antiproliferative activity
against tumor cell lines not only upon UVA irradiation, which was
considerable higher than the most used drug in clinical photomed-
icine, 8-MOP, but also in the dark.7 However, despite their inability
of inducing ISC, they showed skin covalent DNA-protein cross-link
(DPC).8 Up to date several FQ analogues bearing other substituents
than methyl groups and in different positions on the furoquinoli-
none skeleton have been synthesized to better understand the
mechanism of action of this class of compounds even under no
irradiation.8,9
Quinolines represent a class of largely investigated heterocycles
for their antitumor activity.10 Among these, pyrrolo[3,2-f]quinolin-
9-ones and their geometric [2,3-h] isomers showed good antiprolif-
erative effect.11 Pyrroloquinolinones with photochemotherapeutic
activity were poorly explored. As part of our search for pyrrolo
fused heterocycles, we have recently reported the synthesis and
biological activity of ring systems containing the quinolinone moi-
ety, with the aim to investigate their antiproliferative effect either
in the dark or under UVA irradiation. In particular, pyrrolo
[2,3-h]quinolines 212 and the pyrrolo[3,2-h]quinolines 313 were
studied as nitrogen isosters of angelicin (Chart 1).
Transformation of 6b–d,h,i into the corresponding enamino
ketones 7b–d,h,i was achieved by reaction with stoichiometric
amount of diethylamine. Instead, the presence of an ethoxycar-
bonyl functionality at position 1 of the pyrrole ring allowed the di-
rect introduction of the enamino functionality even starting from
the NH derivative 5e. In fact, despite the numerous publications15
on such a reaction, 5e–g were the only ketones capable to react with
TBDMAM, producing the corresponding enaminones 7e–g. Annela-
tion of the pyridine ring on the isoindole moiety was accomplished
using enaminones as key intermediates and phenylsulfonylaceto-
nitrile as 1,3 dinucleophile. Such reactions were carried out in eth-
anol and, when reaction of the N-methyl derivative 7b was
conducted at room temperature for 24 h, the intermediate 8 (62%)
was isolated as the main product from the reaction mixture, indi-
cating the high electrophilicity of the a-enamino carbon atom com-
pared with the annular carbonyl. Prolonged heating of the mixture
(24–72 h) was necessary to accomplish the desired cyclization to
give compounds 4b–i (Table 1, 55–72%). The tricyclic derivative
4b (40%) was also obtained by heating under reflux (72 h) the inter-
mediate 8 with stoichiometric amount of diethylamine. In general,
it seems likely that, by prolonging refluxing time, the hydrolysis of
the cyano group to carboxamide 9 causes the reaction to proceed
towards the direct closure to the 2-pyridone ring. In order to obtain
pyrroloquinolinones with different solubility properties, the three
ethoxycarbonyl derivatives 4e–g (Table 1) were subjected to hydro-
lysis in basic media to give the corresponding carboxylic acid 4j–l
(Table 1) in very good yields (80–90%). Moreover N-methylation
of the pyridone moiety of derivatives 4b–g was achieved with stoi-
chiometric amount of iodomethane in DMF in the presence of so-
dium hydride as the base leading to the 1-methyl pyrrol
oquinolinones 4m–r (34–68%, Table 1). As expected, from the reac-
tion of 4e along with the desired 1,9-dimethyl pyrroloquinolinone
4p (34%), also the 8,9-dimethyl pyrroloquinolinone 4f (12%) and
the 1,8,9-trimethyl pyrroloquinolinone 4q (15%) were isolated from
the mixture. For the unsubstituted derivatives 4b–d, together with
the 1-N-methylated pyrroloquinolinones 4m–o (55–68%), the
O-methylated species 4s,t (18–20%) were also isolated as minor
products. Instead 4u was isolated only in traces (3%).
Both classes of compounds revealed remarkable phototoxicity
against human tumor cell lines and a great dose UV-A dependence
reaching IC50 values at submicromolar level (0.4–16.4
lM and 0.5–
5.5 M, respectively). For several derivatives, such a biological
l
activity was higher than angelicin itself, used as reference drug.
Studies on the mechanism of action for both classes of derivatives,
revealed a different behavior than the lead compound angelicin as
DNA is not the main target with a possible involvement of mito-
chondria in the photoinduced apoptosis. Undoubtedly, the most
important feature of the pyrrolo[3,2-h]quinolines is that they exert
their phototoxicity without any DNA damage (no DNA strand
breaks and no DNA oxidative damages) which is the main origin
of the long term side effects of the PUVA therapy. On the basis of
these results, we planned the synthesis of a new series of pyrrolo-
quinolinones, the pyrrolo[3,4-h]quinolin-2-ones 4, in which the
pyridine moiety is annelated to the isoindole ring with the purpose
to further explore the effect of the new condensation of the pyrrole
nucleus to the quinoline moiety. Such a ring system can be also re-
garded as a pyridine moiety fused to the isoindole system. We
focused our attention on the synthesis of dihydro derivatives, since
in the two previous series of pyrroloquinolinones non-aromatic
compounds of type 2a and 3 showed remarkable photoantiprolif-
erative activity whereas the aromatic pyrrolo[2,3-h]quinolin-
2-ones of type 2b were inactive. The phototoxicity of the title
compounds was studied and a relationship between structure
and photobiological properties was hypothesized. To confirm
whether also this class of pyrrolo quinolinones has a mode of
action different from that of angelicin, experiments were per-
formed to understand the mode of cell death and the cellular
targets involved.
2.2. Biology
2.2.1. Evaluation of some physico-chemical properties
The absorption and emission spectra of pyrrolo[3,4-h]quinolin-
2-ones were collected in phosphate buffer 10 mM pH 7.2. All
compounds absorbed between the range of 250–420 nm and pre-
sented bands in UV-A region. Absorption maxima wavelengths
(Table 2) exhibited a remarkable bathocromic shift compared to
angelicin due to the conjugation with the substituents in 3 and
8 positions.
All compounds demonstrated higher molar extinction coeffi-
cients than angelicin thanks to the presence of a phenylsulfonyl
group in position 3 and of the substituent in 8. This is probably
due to the greater electronic density, to the increased extension
of the molecule conjugation but also to the increased structural
rigidity for the steric hindrance of the phenylsulfonyl group. The
partition coefficients of the test compounds were calculated using
the computational method as described by Ghose and Crippen.17
The values obtained with this technique are close to the experi-
mental measured ones.18 The majority (11 out of 19) of pyrrol-
o[3,4-h]quinolin-2-ones were highly hydrophobic, reaching
c log P values of +6.44 in the case of 4r. The substituent in position
7 seems to play an important role in determining the c log P value.
Thus, derivatives bearing the ethoxycarbonyl functionality are
more hydrophobic; instead, unsubstituted or carboxylic acid deriv-
atives generally resulted more hydrophilic.
2. Results and discussion
2.1. Chemistry
We have already reported the synthesis of tetrahydroisoindole-
4-ones 5a–i as versatile precursors suitable for the annelation of
heterocycles to the 4,5 isoindole positions.14
Starting from derivatives 1,3-unsubstituted pyrroles 5a–d or
from the 3-methyl ketones 5h,i the 5-formyl derivatives 6b–d,h,i
were prepared using ethyl formate as formylating agent in the pres-
ence of t-BuOK, with the sole exception of 5a which was recovered
unchanged from the corresponding reaction mixture (Scheme 1).

2328
P. Barraja et al. / Bioorg. Med. Chem. 19 (2011) 2326–2341
O
O
O
PhO2S
iv
CN
R2
N R
R1
R2
N R
R1
R2
N R
R1
O
R23NHC
HOHC
ii
i
N Me
7b-i
8
6b-d,h,i
5a-i
5e-g
iii
7b-d,h,i R3=Et
7e-g R3=Me
a R=R1=R2=H; b R=Me, R1=R2=H
c R=Bn, R1=R2=H; d R=Ph, R1=R2=H
e R=H, R1=CO2Et, R2= Me; f R=Me, R1=CO2Et, R2= Me
g R=Bn, R1=CO2Et, R2= Me; h R=R2=Me R1=H
i R=Bn, R1=H, R2=Me
v
vi
O
O
O
OMe
2
1
H
PhO2S
Me
3
NH2
O
PhO2S
+
N
PhO2S
PhO2S
N
R2
8
N R
N
R2
N R
R1
R2
NR
R1
9
4
viii
b-g
N R
5
7 R1
6
4b-i
4j-l R1=CO2H
vii
4s-u
4m-r
e-g
9
Scheme 1. Synthesis of compounds 4b–u. Reagents and conditions: (i) t-BuOK, HCOOEt, benzene, rt, 52–92%; (ii) HNEt2, benzene, rt, 24 h, 50–95%; (iii) TBDMAM, toluene,
reflux, 57–87%; (iv) PhSO2CH2CN, ethanol, rt, 24 h, 62%; (v) HNEt2, ethanol, reflux, 72 h, 40%; (vi) PhSO2CH2CN, ethanol, reflux, 24–72 h, 55–72%; (vii) KOH, EtOH, reflux, 24 h,
80–90%; (viii) NaH, DMF, 0 °C to rt, 1 h then iodomethane at 0 °C to rt 2 h, 8–68%.
Table 1
Table 2
Pyrrolo[3,4-h]quinolin-2-ones 4b–u
Physico-chemical properties of pyrrolo[3,4-h]quinolin-2-ones
R1
R2
Substrate Melting point (°C) Yield (%)
kMAXabsa ea (Mꢀ1 cmꢀ1) kMAXabsb
kMAXfluob
/
F
c log Pd
c
Compound
R
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
Me
Bn
Ph
H
H
H
H
H
H
H
7b
7c
7d
345–346
293–294
217–220
324–325
239–240
194–195
244–245
252–254
243–244
230–231
185–186
230–231
164–165
257–258
248–249
165–166
138–139
202–203
186–187
257–258
65
55
60
70
64
65
68
72
80
85
90
55
62
68
34
65
68
18
20
3
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
409
410
414
405
353
401
413
410
406
404
405
408
409
413
352
350
370
355
354
299
18849.5
34338.6
27901.0
23928.0
18641.2
17332.8
24463.2
26883.3
26281.3
11084.5
19301.0
38804.6
36753.2
42799.2
29983.3
33957.8
37263.2
29148.4
15740.5
9350.0
396
408
394
393
395
380
399
401
396
399
399
395
396
401
357
364
396
351
353
302
438
462
437
431
429
429
448
486
442
460
456
435
430
441
429
422
428
460
454
460
0.018
0.059
0.197
0.790
0.870
0.734
0.023
0.064
0.006
0.004
0.004
0.040
0.175
0.662
0.387
0.361
0.615
0.014
0.058
0.016
+2.01
+3.88
+3.88
+3.98
+4.58
+5.84
+2.47
+4.34
ꢀ0.28
+0.51
+2.00
+2.36
+3.87
+4.23
+4.17
+4.93
+6.44
+2.01
+3.52
+2.00
CO2Et Me 7e
7f
CO2Et Me 7g
H
H
Me CO2Et Me
Bn
Me
Bn
H
Me 7h
Me 7i
Me 4e
Me 4f
Me 4g
H
H
H
CO2H
Me CO2H
Bn
Me
Bn
Ph
H
CO2H
H
H
4b
4c
4d
H
CO2Et Me 4e
4f
CO2Et Me 4g
H
H
H
Me CO2Et Me
Bn
Me
Bn
Ph
4s
4t
4u
H
H
H
4b
4c
4d
4s
4t
Ang
a
b
c
In DMSO.
In phosphate buffer 10 mM pH 7.2.
Relative fluorescence quantum yield determined in 0.1 N H2SO4 as described in
2.2.2. Cellular phototoxicity
Ref. 16.
After having assessed that pyrrolo[3,4-h]quinolin-2ones they
did not present any antiproliferative activity in the absence of irra-
diation, their phototoxicity was evaluated on a panel of cultured
human cell lines. The phototoxicity tests were carried out against
different human tumour cell lines: HL-60 (promyelocytic leukae-
mia), Jurkat (T-cell leukaemia), MCF-7 (breast adenocarcinoma)
and LoVo (colon carcinoma). Analogous experiments were per-
formed in an immortalized cell line of human keratinocytes,
NCTC-2544. Table 3 showed the IC50 in human cell lines after
72 h from the irradiation with two different UV-A doses (2.5 and
3.75 J/cm2).
Among pyrrolo[3,4-h]quinolin-2-ones, compounds 4h, 4j–m, 4o
were found to be non phototoxic at the employed concentrations,
whereas compounds 4b and 4c demonstrated some activity only
against leukaemia cell lines at micromolar level at the UVA dose
of 3.75 J/cm2. Derivatives 4d, 4e and 4p are active at micromolar
d
Calculated as reported in Ref. 17.
13.2
l
M, respectively). Pyrroloquinolines 4f, 4i, 4m, 4r and 4t show
M)
generally good activity in the micromolar range (1.3–14.4
l
against all the cell lines used for this study with exception of 4i
for the LoVo cell line.
The best results are indeed obtained for 4g, 4q and 4s which
showed high phototoxicity comparable and in some cases better
than angelicin (Ang) used as the reference drug, also against solid
tumors (0.2–2.5
molar range.
A relationship between activity and structure can be hypothe-
sized thanks to the fair number of evaluated compounds, starting
from the structure of the most phototoxic compounds. In the com-
pounds bearing the carbonyl group in position 2, the importance of
the presence of a substituent in position 8 and the ethoxycarbonyl
group in position 7 was observed. In fact, compounds that had no
lM) reaching the submicromolar and low micro-
concentration (5.8–12.5
l
M) against leukaemia cell lines at 3.75 J/
M and
cm2, and excepted for 4d, against LoVo tumor lines (13.6
l

P. Barraja et al. / Bioorg. Med. Chem. 19 (2011) 2326–2341
2329
Table 3
Phototoxicity of pyrrolo[3,4-h]quinolin-2-ones in different human cell lines
aIC50
MCF-7
(lM)
bHL-60
Jurkat
LoVo
NCTC-2544
3.75
c2.5
3.75
2.5
3.75
2.5
3.75
2.5
3.75
2.5
4b
4c
4d
4e
4f
4g
4h
4i
>20
>20
5.6 0.6
14.5 1.5
8.0 1.0
9.1 0.8
1.8 0.2
0.8 0.02
>20
>20
>20
>20
18.0 2.0
2.0 0.2
2.9 0.3
>20
13.0 1.5
>20
>20
>20
>20
>20
>20
>20
10.0 1.0
3.3 0.4
>20
>20
>20
>20
>20
>20
>20
5.1 0.6
1.4 0.1
>20
7.7 0.8
>20
>20
>20
>20
17.5 1.5
7.9 0.8
4.0 0.4
>20
>20
>20
>20
>20
>20
13.6 0.5
4.0 0.4
2.3 0.2
>20
>20
>20
>20
>20
>20
>20
10.1 1.0
4.0 0.5
>20
>20
>20
>20
>20
>20
15.0 1.4
6.2 0.6
2.5 0.3
>20
12.2 1.0
13.7 1.0
3.6 0.5
1.2 0.2
>20
7.4 0.7
10.0 1.0
1.4 0.2
1.9 0.2
>20
4.0 0.4
>20
2.5 0.3
>20
3.0 0.5
>20
14.4 0.8
>20
4j
4k
4l
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
4m
4n
4o
4p
4q
4r
4s
4t
Ang
>20
4.4 0.4
>20
>20
2.8 0.3
>20
5.8 0.6
0.9 0.02
2.6 0.3
0.9 0.1
7.3 0.7
0.9 0.2
>20
3.6 0.4
>20
>20
2.5 0.3
>20
12.5 1.3
0.8 0.1
1.3 0.1
0.2 0.01
6.3 0.6
0.9 0.1
>20
6.6 0.7
>20
>20
1.7 0.1
>20
2.5 0.3
10.0 1.2
4.4 0.5
>20
3.7 0.4
>20
>20
3.9 0.4
>20
>20
2.8 0.3
>20
13.2 1.3
0.4 0.03
8.7 0.8
0.9 0.09
8.7 0.6
1.1 0.4
>20
8.2 0.9
>20
>20
2.4 0.2
>20
4.9 0.5
15.3 1.4
4.2 0.5
>20
5.8 0.6
>20
>20
>20
>20
>20
>20
1.2 0.1
5.4 0.6
1.6 0.1
8.3 0.8
1.2 0.1
1.2 0.1
2.9 0.3
1.0 0.08
8.5 0.5
1.0 0.2
1.3 0.1
14.4 1.5
2.0 0.1
7.5 0.8
1.5 0.2
0.7 0.05
12.1 1.2
1.9 0.2
9.9 0.1
4.0 0.4
1.3 0.2
14.3 1.5
1.1 0.1
9.6 0.5
0.9 0.1
a Concentration of compound required to inhibit the cell growth by 50% after 72 h of exposure as determined by MTT assay. Values are means SEM of three experiences.
b
Human cells: HL-60, promyelocytic leukaemia; Jurkat, T-cell leukaemia; MCF-7, adenocarcinoma; LoVo, colon carcinoma; NCTC-2544, immortalized keratinocytes.
c
UV-A doses are expressed in J/cm2.
substituent or a carboxylic acid functionality in position 7 (4j, 4k
and 4l) exhibited moderate (4d and 4i) or no phototoxicity (4h,
4j, 4k and 4l). Among the compounds with the ethoxycarbonyl
group in position 7, the presence of a substituent of any nature
(methyl or benzyl groups) in position 8 was fundamental for activ-
ity: in fact, 4e and 4p demonstrated a reduced phototoxicity. De-
spite the small number of compounds with the methoxyl group
in position 2, we found a general increase in the phototoxicity. In
fact, 4s was one of the most active molecule and it is worthy to
note that the correspondent compound (4b) with the carbonyl
group in 2 was almost inactive.
the presence of some scavengers. These tests were performed with
the most active compounds 4g, 4q and 4s using MCF-7 cells. We
employed the radical scavengers reduced glutathione (GSH) and
2,6-di-tert-butylhydroxyanisole (BHA); the hydroxyl radical scav-
engers dimethylthiourea (DMTU) and mannitol (MAN), and the
singlet oxygen scavengers 1,4-diazabicyclo[2,2,2]octane (DABCO),
sodium azide (NaN3) as described before.13 The results of these
studies were presented in Figure 2. For 4g and 4s, the protective
effect of scavengers was very slight and non-specific; whereas a
major increase in cellular survival was detected with 4q. In partic-
ular, all scavengers seemed to demonstrate a protective effect in 4q
experiments but 80% of cell viability was assessed when the irradi-
ation was carried out in the presence of MAN and DABCO, indicat-
ing that hydroxyl radical and singlet oxygen may be involved in 4q
phototoxicity. However, from these data, an univocal mechanism
of photosensitization was not distinguished.
2.2.3. Photodegradation and evaluation of photoproducts
toxicity
All derivatives underwent a remarkable photodegradation, as
shown by the changes in absorption spectra upon UVA irradiation
in DMSO. In Figure 1, the absorption spectra of 4i, 4q and 4s are de-
picted as representatives, recorded after exposure to increasing
doses of UV-A light. Distinct isosbestic points were observed, sug-
gesting that photoproducts are most likely formed. A similar
behavior was observed for furocoumarins19 and other tricyclic
derivatives such as pyrrolo[2,3-h]quinolinones12 and pyrrolo
[3,2-h]quinolinones.13
Caffieri et al. demonstrated the ability of furocoumarins to gen-
erate cytotoxic products (POPs), upon photoirradiation that could
play a relevant role in PUVA therapy.20 Thus, the possible contribu-
tion of photoproducts in antiproliferative action was analysed.
After having irradiated (6.5 J/cm2) solutions of the most active
compounds, Jurkat cells were incubated with that mixture for
72 h. The results (data not shown) revealed no cytotoxic effects,
suggesting that the events that lead to cellular death are solely
due to a phototoxic damage and not to a production of toxic
photoproducts.
2.2.5. Externalization of phosphatidylserine
The mode of cell death photoinduced by pyrrolo[3,4-h]quinolin-
2-ones was investigated by cytofluorimetric analysis using propidi-
um iodide (PI) and Annexin V–FITC conjugates, which stain DNA and
PS residues, respectively. Thus, this cytofluorimetic measure dis-
criminated between intact (Aꢀ/PIꢀ), early apoptotic (A+/PIꢀ), late
apoptotic (A+/PI+) or necrotic cells (Aꢀ/PI+).21 The biparametric anal-
ysis was conducted in Jurkat cells after 24 h from the irradiation in
the presence of various concentrations of 4g and 4q. As control, the
same tests were performed in non irradiated Jurkat or in irradiated
cells.
A significant increase of early apoptotic cells was determined
when the irradiation was conducted in the presence of the test
compounds (Fig. 3).
A percentage of cells was positive to both probes and this was
compatible with a later stage of apoptosis. A small part of treated
cells (up to 5%) resulted necrotic but this amount was comparable
to the irradiated control. From these data, a main apoptotic path-
way can be associated to the cellular death photoinduced by
pyrrolo[3,4-h]quinolin-2-ones.
2.2.4. Effect of scavengers on pyrroloquinolinone-induced
phototoxicity
With the purpose to evaluate the possible mechanism of action
of the test compounds, we performed analogous experiments in

2330
P. Barraja et al. / Bioorg. Med. Chem. 19 (2011) 2326–2341
0.6
0.4
0.2
0.0
100
0 J/cm2
4g
4i
1.3 J/cm2
2.6 J/cm2
5.2 J/cm2
7.8 J/cm2
10.4 J/cm2
14.3 J/cm2
80
60
40
20
250
300
350
400
450
500
0
100
Wavelength (nm)
0.8
0.6
0.4
0.2
4q
0 J/cm2
1.3 J/cm2
3.8 J/cm2
6.4 J/cm2
10.2 J/cm2
14.1 J/cm2
80
60
40
20
4q
0
0.0
0.6
250
300
350
400
450
500
550
100
Wavelength (nm)
0 J/cm
4s
4s
2
80
60
40
20
0
1.3 J/cm
2
2.5 J/cm
2
5.1 J/cm
0.4
2
8.9 J/cm
2
12.7 J/cm
et al
2
15.2 J/cm
0.2
20
2
0.0
250
300
350
400
450
500
550
Wavelength (nm)
Figure 1. Photodegradation of pyrrolo[3,4-h]quinolin-2-ones. Absorption spectra of
compound 4i (upper panel), 4q (middle panel) and 4s (lower panel) at the
concentration of 20 lM in DMSO were recorded after exposure to increasing UV-A
Figure 2. Effect of different scavengers on the phototoxicity induced by 4g, 4q and
4s in MCF-7 cells. Cell viability was assayed by MTT test 72 h from the irradiation
(2.5 J/cm2) in the presence of the compounds and the scavengers. Data were
expressed as means SEM for at least three independent experiments.
doses. Red squares in the graphs indicate the presence of isosbetics points.
2.2.6. Cell cycle analysis
Nevertheless, the most important modification was the concen-
tration-dependent onset of a subG1 peak, which represented apop-
To investigate the effects of test compounds upon UVA irradia-
tion on the cell cycle, Jurkat cells were treated with the most active
compounds at different concentrations and at the light dose of
2.5 J cmꢀ2. After 24 h from the irradiation, the cells were fixed
and labelled with propidium iodide and the different phases of
the cell cycle were analyzed by flow cytometry. From the data in
Table 4, an arrest of the cell cycle in S phase was observed along
with a reduction of G1 cells. These data suggest that in response
to phototoxic stress induced by the drugs, the progress of the cell
cycle can be arrested at certain checkpoints that serve to maintain
genomic integrity.
totic cells. The subG1 was
a consequence of the ordered
degradation of DNA which occurred during apoptosis. This phe-
nomenon is considered a later event than PS externalization, thus
these data were well in agreement with the ones of the previous
experiment.
2.2.7. Intracellular localization of pyrrolo[3,4-h]quinolin-2-ones
It has recently been demonstrated the importance of the cellu-
lar localization for the cytotoxic action of photosensitizers.12,22,23
The strength of a photosensitiser is determined by the nature of

P. Barraja et al. / Bioorg. Med. Chem. 19 (2011) 2326–2341
2331
4q
CellMask
Overlay
4q
4q
4q
Rho 123
Overlay
ER tracker
Overlay
60
50
40
30
20
10
0
EARLY APOPTOSIS
LATE APOPTOSIS
NECROSIS
LysoTracker
Overlay
Figure 4. Intracellular localization of pyrrolo[3,4-h]quinolin-2-ones in NCTC-2544
cells. Fluorescence microphotographs showing the intracellular localization of 4q in
NCTC-2544 cells in the presence of Cellmask Orange, Rho 123, ER Tracker Green and
Lysotracker RED. The cells were treated as described in Experimental Section and
cellular fluorescence images were acquired with a video-confocal microscope, using
a Nir Apo 60X/1.0W water immersion objective. Overlay images of 4q localization
are generated by transferring the blue color of the compound, onto the corre-
sponding fluorescence image of the probe.
NIC
IC
10
5
10
5
μΜ
μΜ
μΜ
μΜ
4q
4g
Figure 3. Upper panel. Representative biparametric histograms obtained 24 h after
the irradiation (3.75 J cmꢀ2) of Jurkat cells in the presence of 4q at the indicated
concentrations. The cell were labelled with Annexin-V-FITC and Propidium iodide
(PI) as described in Section 4 and analyzed by flow cytometry. Lower panel.
Percentage of early apoptotic cells (A+/PIꢀ), late apoptotic cells (A+/PI+) and necrotic
cells (Aꢀ/PI+) after 24 h from the irradiation (2.5 J/cm2) in the presence of 4g and 4q
the excited states or by its ability to form reactive species whereas,
in a phototoxicological context, other factors such as subcellular
localization, are essential parameters. Thus, it is critically impor-
tant to evaluate the distribution of photosensitisers inside cells.
In order to investigate the intracellular localization of pyrroloquin-
olinones, we used different fluorescent probes: Rhodamine 123
(Rho 123), a lipophilic cation commonly used for the assessment
of the mitochondrial potential, Lyso tracker RED, a fluorescent
dye that specifically stains lysosomes,24 cell mask orange, a plasma
membrane staining and ER-tracker, which is an highly selective
marker for the endoplasmic reticulum in living cells. These mark-
ers fluoresce in the visible region (about 550–630 nm) whereas
the test compounds emit in the blue region. Both fluorescences
can be easily separated using suitable bandpass optical filters. After
an incubation in NCTC-2544 cells, compound 4q, chosen as repre-
sentative, was found to incorporate and associate with subcellular
structures. It can be observed from Figure 4 that 4q diffuses largely
in the cytoplasm and co-localizes with Rho 123, Lyso tracker and
ER tracker, indicating that this compound is broadly distributed
in the cytoplasm and in many subcellular organelles without a par-
ticular specificity.
at the indicated concentrations (lM). NIC = non irradiated control; IC = irradiated
control. Data were expressed as mean SEM of three independent experiments.
Table 4
Percentage of Jurkat cells in each cell cycle phase 24 h after irradiation
G1c
Sc
G2/Mc
SubG1d
NICa
ICb
63.0
64.4
27.6
25.3
9.4
10.3
0.1
0.5
4g
5
10
l
l
M
M
53.8
44.8
41.8
54.4
4.4
0.8
3.8
12.5
4q
5
10
l
l
M
M
48.9
39.1
46.9
55.6
4.2
5.3
10.5
43
4s
5
10
l
l
M
M
49.6
40.1
45.4
50.4
5.0
9.5
4.0
15.9
a
b
c
Not irradiated control.
Irradiated control.
The percentage of each phase of the cell cycle was calculated on living cells.
Percentage of the cell population with hypodiploid DNA content peak (apop-
2.2.8. Mitochondrial and lysosomial integrity assay
Mitochondrial dysfunction was examined to better investigate
cell death mode through the assessment of the mitochondrial
d
totic cells).
membrane potential (Dwmt). The determination of Dwmt was

2332
P. Barraja et al. / Bioorg. Med. Chem. 19 (2011) 2326–2341
carried out through a flow cytometric analysis using the probe
JC-1. This cytofluorimetric analysis was performed after 6, 12 and
24 h from the irradiation of Jurkat cells in the presence of 4g and
2.2.9. DNA interaction
As DNA is considered one of the most important target for psor-
alen, the affinity of pyrrolo[3,4-h]quinolin-2-ones for this macro-
molecule was checked. Linear dichroism is an efficient tool to
evaluate the binding mode between an organic compound and nu-
cleic acids.28 Linear dichroism (LD) measurements were performed
on solutions with salmon testes DNA (st-DNA) and the title com-
pounds at various molar ratios. The sign and the extend of LD sig-
nal is related to the orientation of the compound with respect to
DNA helix axis.
Inspection of the spectra of DNA in the presence of the most ac-
tive derivatives (4g, 4q and 4s) shows (Fig. 6) that there is an in-
crease in the signal intensity for the DNA band (230–300 nm)
indicating that the DNA becomes more oriented within the hydro-
dynamic field because of stiffening of the helix upon interaction of
these compounds. However, even for the highest concentrations of
test compounds, the spectra do not show any LD bands in the chro-
mophore absorption region (300–450 nm), suggesting a poor inter-
action with this macromolecule.
Moreover, the interaction of pyrrolo[3,4-h]quinolin-2-ones with
DNA was also monitored by fluorimetric titrations, where possible
changes in emission spectra of aqueous solution of compounds
were searched after the addition of st-DNA. Results are presented
in Figure 7.
Fluorescence titrations carried out with compounds 4g, 4q and
4s showed a poor fluorescence quenching even when DNA concen-
tration was 10 times higher than the one of the compounds. Alto-
gether these data indicate that the compounds are not intercalated
into DNA or are loosely bound to it, in contrast with natural fur-
ocoumarins which bound DNA by intercalation.1
4q. With normal cells (high
Dwmt), JC-1 displays a orange fluores-
cence (590 nm). This is caused by spontaneous and local formation
of aggregates that are associated with a large shift in the emission.
In contrast, when the mitochondrial membrane is depolarized
(low
Dw
mt), JC-1 forms monomers which emit at 530 nm.25
As depicted in Figure 5 (upper panel), a rapid mitochondrial
involvement in inducing apoptosis was assessed: in fact, after 6 h
from irradiation, the percentage of treated cells with low mito-
chondrial potential significantly increased compared to controls
at both concentration used. The disruption of
Dwmt is associated
with the appearance of Annexin-V positivity in the treated cells
when they are in an early apoptotic stage. In fact, the dissipation
of
Dwmt is characteristic of apoptosis and has been observed with
many photosensitisers including furocoumarins, fluoroquinolones,
phenothiazines, and antimalarial drugs.26a–d
In order to investigate the integrity of lysosomes after irradia-
tion with the test compounds, a flow cytometric analysis was
performed using the fluorescent dye acridine orange (AO).27 As de-
picted in Figure 5 (lower panel), a concentration and time-depen-
dent increase of the number of damaged lysosomes is observed
after irradiation in the presence of 4g and 4q), indicating that lyso-
somes, together with mitochondria, were deeply involved in cell
death.
60
6 h
12 h
50
24 h
2.2.10. DNA photodamage
40
DNA photodamage experiments were carried out to determine
whether the new derivatives were able to photosensitize DNA
strand break activity even if they did not demonstrate a high affin-
ity for this biomolecule. pBR322 plasmid DNA was used as a model
system to evaluate DNA breaking activity: frank strand breaks, the
oxidative damage of purine and/or pyrimidine bases. The irradia-
tion of supercoiled plasmid pBR322 was carried out in the presence
of the test compounds at the molar ratio [DNA]/[Drug] = 1/3. After
the irradiation, pBR322 aliquots were also incubated with two base
excision repair enzymes formamido pyrimidin glycosilase (Fpg)
and Endonuclease III (Endo III), respectively to evaluate, in addition
to the formation of frank strand breaks, also oxidative damage to
DNA bases.29 The results (see Fig. 8 upper panel) pointed out that
the new compounds did not provoke any formation of open circu-
lar or linear bands, indicating they did not sensitize frank strand
breaks. On the contrary, the treatment with the two base excision
enzymes revealed that they were able to photooxidize DNA bases
in a great amount.
All compounds were able to photoinduce base oxidation but 4q
resulted the most active especially after treatment of irradiated
DNA with Endo III, suggesting pyrimidinic bases as its preferential
targets. To better understand the mechanism of base photooxida-
tion, the irradiation of pBR322 was carried out in the presence of
nitrogen flux: a dramatic reduction of base oxidative damages
was observed (Fig. 9), indicating that the process of photoinduced
DNA damage is strongly oxygen dependent. Thus, the formation of
reactive oxygen species was essential for DNA photodamage by
pyrrolo[3,4-h]quinolin-2-ones.
30
20
10
0
μΜ
NIC
IC
10
5 μΜ 10 μΜ 5 μΜ
70
60
50
40
30
20
10
0
4g
4q
6 h
12 h
24 h
μΜ
NIC
IC
10
5
10μΜ
5
μΜ
μΜ
4q
4g
To further understand the mechanism of action, irradiation of
pBR322 was carried out in the presence of 4q and some scavengers.
We used MAN, a hydroxyl radical scavenger, DABCO, a singlet oxy-
gen scavenger, and GSH, a free radical scavenger (Fig. 10). A little
reduction in the formation of OC form was observed when irradia-
tion was conducted with GSH and MAN. Whereas, in the presence
Figure 5. Upper panel. Percentage of Jurkat with collapsed
the JC-1 monomeric form) after 6, 12 and 24 h from the irradiation (2.5 J/cm2) with
4g and 4q at the indicated concentrations ( M). Data were expressed as mean
SEM of three independent experiments. Lower Panel. Percentage of Jurkat cells with
damaged lysosomes after 6, 12 and 24 h from irradiation (2.5 J/cm2) in the presence
of 4g and 4q at the indicated concentrations. NIC = non irradiated control;
IC = irradiated control. Data are represented as mean S.E.M. of three experiments.
DwM (fluorescence of
l
-

P. Barraja et al. / Bioorg. Med. Chem. 19 (2011) 2326–2341
2333
d
a
1000
4g
0.5
0.3
4g
d
x10
800
a
b
c
600
0.1
b
400
-0.001
200
0.00
a
x10
300
350
400
450
0
-0.011
400
420
440
460
480
500
520
540
d
Wavelenght (nm)
250
300
350
400
450
Wavelength (nm)
0.6
0.4
0.2
1000
4q
d
a
4q
d
c
a
x10
800
600
400
200
0
b
b
0.0
0.000
x10
-0.00
a
300
350
400
450
380 400 420 440 460 480
500 520 540
d
Wavelenght (nm)
-0.015
250
300
350
400
450
Wavelength (nm)
300
d
4s
4s
250
200
150
100
50
0.5
0.3
a
b
d
x10
a
c
b
0.1
0.000
x10
-0.00
0
a
380 400 420 440 460 480
500 520 540
300
350
400
450
-0.011
Wavelenght (nm)
d
250
300
350
400
450
Figure 7. Fluorimetric titrations of stDNA (a = 0
4q, and 4s at a ligand concentration of 10 M.
lM; b = 100 lM) to compounds 4g,
l
Wavelength (nm)
Figure 6. Absorbance (A) and linear dichroism (LD) spectra of mixtures of st-DNA
and 4g and 4q (upper panels) and 4s (lower panel) at different [drug]/[DNA] ratios
(a = 0; b = 0.01; c = 0.02; d = 0.03) in phosphate buffer 10 mM, pH 7.2.
breaks) was observed. DNA damage was significantly enhanced
in D2O indicating the involvement of 1O2 since the lifetime of 1O2
is enhanced by a factor of ca. 14 in D2O.
2.2.11. Lipid peroxidation
of DABCO, the oxidations to DNA photoinduced by 4q were not de-
tected, suggesting an involvement of singlet oxygen in photosensi-
tization reaction. As DABCO is not very specific toward singlet
oxygen, these data were confirmed by irradiation in presence of
D2O buffer. Interestingly, when the aqueous buffer was replaced
by D2O, a dramatic increase of the formation of ssb (single strand
As most pyrrolo[3,4-h]quinolin-2-ones are hydrophobic (see
c log P in Table 2), they may be expected to be localized mainly
in plasmatic and/or in subcellular membranes, making these struc-
tures particularly sensitive to photodamage. Lipid peroxidation
was analyzed by measuring the level of malonyldialdehyde

2334
P. Barraja et al. / Bioorg. Med. Chem. 19 (2011) 2326–2341
pBR322 [DNA]:[4g]=1:3 [DNA]:[4q]=1:3 [DNA]:[4s]=1:3
[DNA]:[4g]=1:3 [DNA]:[4q]=1:3 [DNA]:[4s]=1:3 pBR322
a
b
c
a
b
c
a
b
c
a
b
c
a
b
c
a
b
c
a
b
c
a
b
c
a = no enz; b = + Endo III; c = + Fp
a = no enz; b = + Endo III; c = + Fp
30
no enz
60
no enz
+ Endo III
+ Fpg
+ Endo III
+ Fpg
25
20
15
10
5
40
20
0
0
pBR322
4g
4q
4s
pBR322
4g
4q
4s
Figure 9. DNA base modifications photoinduced by pyrrolo[3,4-h]quinolin-2-ones
in the presence of nitrogen flux. Upper panel. Supercoiled circular pBR322 was
irradiated (3.75 J/cm2) in the presence of [DNA]/[Drug] = 1/3 and then treated with
base excision enzyme: a = no enzyme; b = +Endo III; c = +Fpg. Lower panel.
Percentage of OC form after irradiation (3.75 J/cm2) in the presence of 4g, 4q and
4s at the ratio [DNA]/[Drug] = 1/3.
Figure 8. DNA base modifications photoinduced by pyrrolo[3,4-h]quinolin-2-ones.
Upper panel. Supercoiled circular pBR322 was irradiated (3.75 J/cm2) in the
presence of [DNA]/[Drug]=1/3 and then treated with base excision enzyme: a = no
enzyme; b = +Endo III; c = +Fpg. Lower panel. Percentage of II form after irradiation
(3.75 J/cm2) in the presence of 4g, 4q and 4s at the ratio [DNA]/[Drug] = 1/3.
(MDA) bound to thiobarbituric acid (TBA) in treated and untreated
Jurkat cells.30
carrying a methoxyl group in position 2 demonstrated some activ-
ity. Apoptosis represents the main mode of cell death photoin-
duced by these derivatives as demonstrated by the appearance of
a subG1 peak, an index of apoptotic DNA degradation and con-
firmed through Annexin-V-FITC/PI test.
As a consequence of their high hydrophobic character, the title
compounds easily entered the cell and diffused in the cytosol accu-
mulating into subcellular organelles. After irradiation, apart the
extensive lipid peroxidation exerted by the new derivatives, the
photodamage of mitochondria and lysosomes was also shown sug-
gesting the involvement of these organelles in the photoinduced
cell death.
Figure 11 shows the results obtained for compounds 4g, 4q and
4s as a function of light doses. Thiobarbituric acid reactive sub-
stances (TBARS) were significantly produced in a concentration-
dependent manner when the cells were exposed to the compounds
and UV-A. Thus, the induced oxidative damage to membrane lipids
is well correlated with the extent of cell death suggesting that an
extensive lipid peroxidation could play a major role in the photok-
illing mechanism of the test compounds.
3. Conclusions
Interactions with DNA have been investigated as this macro-
molecule is supposed to be the main cause of the antiproliferative
activity of furocoumarins. Pyrrolo[3,4-h]quinolin-2-ones did not
demonstrate a significative affinity towards DNA as they did not
share the planar nucleus of angelicin and psoralens: in fact, they
are not fully unsaturated derivatives. Of note, the lack of affinity
for the macromolecule is a characteristic shared by other isomers
of this series of compounds.13b Moreover, through a series of
plasmidic DNA photocleavage experiments, the formation of frank
strand break was not assessed, although a large number of DNA
bases oxidation was observed because of the production of singlet
oxygen. In this case, a photoreaction of II type could explain DNA
base oxidations: thus, DNA photoreaction of these new derivatives
would be different from that of psoralens, which are known to
mainly produce DNA-photoadducts (photoreaction of III type).
The high antiproliferative activity of the most active
compounds was similar to that of angelicin while the lack of
Pyrrolo[3,4-h]quinolin-2-ones were synthesized as heteroana-
logues of angular furocoumarins, in which oxygen atoms are re-
placed by nitrogens. The data reported herein indicate that
pyrrolo[3,4-h]quinolin-2-ones represent a new interesting class
of potential useful compounds in photochemotherapy with proba-
ble minor side effects than angelicins.
After assessing the lack of cytotoxicity by the title compounds
in the ground state, we found that several pyrrolo[3,4-h]quino-
lin-2-ones (4d, 4e, 4f, 4i, 4m, 4p, 4r and 4t) exhibited UVA dose
dependent phototoxicity at low micromolar concentrations against
many tumor cell lines. Three compounds 4g, 4q, and 4s resulted
the most active reaching submicromolar IC50 values in all cell lines.
Starting from these three molecules, a correlation between struc-
ture and activity was evidenced. Compounds carrying carbonyl in
position 2, ethoxycarbonyl group in position 7 and a substituent
in position 8, exhibited phototoxicity. Moreover, molecules

P. Barraja et al. / Bioorg. Med. Chem. 19 (2011) 2326–2341
2335
10
pBR322 No scavenger + DABCO + GSH
+ MAN
2
2.5 J/cm
2
3.75 J/cm
8
6
4
a
b
c
a
b
c a
b
c a
b
c
a
b
c
2
0
a = no enz; b = + Endo III; c = + Fpg
NIC
IC
2
10
4g
2
10
4q
2
10 (μΜ)
70
60
50
40
30
20
10
0
4s
no enz
+ Endo III
+ Fpg
Figure 11. Lipid peroxidation in Jurkat cells after 24 h from the irradiation (2.5 and
3.75 J/cm2) in the presence of 4g, 4q and 4s at the indicated concentrations (
M).
NIC = non irradiated control; IC = irradiated control. Data are represented as mean
l
SEM of three experiments.
Bruker Avance II series 200 MHz spectrometer. Column chroma-
tography was performed with Merck silica gel 230–400 Mesh
ASTM or with a SEPACORE BÜCHI chromatography apparatus. Ele-
mental analyses (C, H, N) were within 0.4% of the theoretical
values.
pBR322
4q
+DABCO + GSH + MAN
We have already reported the synthesis of ketones 5a–i.14
no enz
+ Endo III
+ Fpg
100
80
60
40
20
0
4.1.1. General procedure for the preparation of 5-
(hydroxymethylene)-2,5,6,7-tetrahydro-4H-isoindol-4-one (6b–
d,h,i)
To a suspension of t-BuOK (3.36 g, 30 mmol) in dry benzene
(30 mL), a solution of 5b–d,h,i (12 mmol) in dry benzene (40 mL)
was added dropwise at 0 °C. After 2 h stirring at room temperature
the reaction was cooled at 0 °C and a solution of ethyl formate
(1.45 mL, 18 mmol) in benzene (20 mL) was added and the mixture
was kept stirring at room temperature for 24 h, then the solvent
was removed under vacuum. The residue was dissolved in water
and the solution was washed with diethyl ether. The aqueous solu-
tion was then acidified with 6 M HCl and extracted with dichloro-
methane. The organic extracts were dried (Na2SO4) and evaporated
under reduced pressure. The residue was purified by chromatogra-
phy using dichloromethane/ethyl acetate (95:5) as eluent.
pBR322
Aqueous buffer
4q
pBR322
4q
D O buffer
2
Figure 10. Upper panel. DNA base modifications photoinduced by 4q in the
presence of some scavengers: DABCO, GSH and MAN. Supercoiled circular pBR322
was irradiated (3.75 J/cm2) in the presence of [DNA]/[4q] = 1/3 and then treated
with base excision enzyme: a = no enzyme; b = +Endo III; c = +Fpg. Middle panel.
Percentage of II form after irradiation (3.75 J/cm2) in the presence of 4q at the ratio
[DNA]/[Drug] = 1/3 and scavengers. Lower panel. Percentage of II form after
irradiation (3.75 J/cm2) in the presence of 4q at the ratio [DNA]/[Drug] = 1/3 in
aqueous buffered solution and deuterated buffer.
4.1.1.1.
5-(Hydroxymethylene)-2-methyl-2,5,6,7-tetrahydro-
4H-isoindol-4-one (6b). Brown oil; yield 60%; IR 3394 (OH),
1653 (CO) cmꢀ1
;
1H NMR: d 2.42–2.53 (4H, m, 2 ꢁ CH2), 3.64
(1H, s, CH3), 6.56 (1H, s, H-1); 7.31 (1H, s, H-3), 7.61 (1H, s, CH),
10.37 (1H, br s, OH); 13C NMR: d 20.6 (t), 24.8 (t), 36.1 (q), 109.3
(s), 118.7 (d), 123.9 (d), 126.1 (s), 164.9 (d), 189.5 (s), 201.6 (CO).
Anal. Calcd for C10H11NO2: C, 67.78; H, 6.26; N, 7.90. Found: C,
68.08; H, 6.41; N, 7.63.
affinity of the title compounds for DNA may be of great relevance
in modulating the long term toxic effect such as skin cancer or
mutagenesis exhibited by psoralen.
4.1.1.2. 2-Benzyl-5-(hydroxymethylene)-2,5,6,7-tetrahydro-4H-
isoindol-4-one (6c). Brown solid; yield 86%; mp 82–83 °C; IR
4. Experimental
4.1. Chemistry
2933 (OH), 1633 (CO) cmꢀ1 1H NMR (CDCl3): d 2.47 (2H, t,
;
J = 6.0 Hz, CH2), 2.63 (2H, t, J = 6.0 Hz, CH2), 5.00 (2H, s, CH2), 6.41
(1H, s, H-1), 7.14–7.34 (6H, m, Ar and H-3), 7.48 (1H, s, CH),
14.38 (1H, br s, OH); 13C NMR (CDCl3): d 21.0 (t), 25.7 (t), 53.9
(t), 109.2 (s), 117.3 (d), 122.5 (d), 125.7 (s), 127.4 (2 ꢁ d), 128.1
(d), 128.8 (2 ꢁ d), 136.3 (s), 165.4 (d), 186.7 (s), 200.5 (CO). Anal.
Calcd for C16H15NO2: C, 75.87; H, 5.97; N, 5.53. Found: C, 75.50;
H, 5.73; N, 5.77.
All melting points were taken on a Buchi-Tottoli capillary appa-
ratus and are uncorrected; IR spectra were determined with a Jasco
FT/IR 5300 spectrophotometer; 1H and 13C NMR spectra were mea-
sured in DMSO-d6 solutions, unless otherwise specified (TMS as
internal reference), at 200 and 50.3 MHz, respectively, using a

2336
P. Barraja et al. / Bioorg. Med. Chem. 19 (2011) 2326–2341
4.1.1.3. 5-(Hydroxymethylene)-2-phenyl-2,5,6,7-tetrahydro-4H-
isoindol-4-one (6d). Pale yellow oil; yield 85%; IR 2933 (OH),
2 ꢁ CH2), 7.22 (1H, s, H-1), 7.34–7.76 (7H, m, Ar, H-3 and CH);
13C NMR: d 14.6 (2 ꢁ q), 21.1 (t), 24.7 (t), 47.3 (2 ꢁ t), 102.6 (s),
112.0 (s), 114.4 (d), 118.6 (d), 119.5 (2 ꢁ d), 125.0 (s), 125.8 (d),
129.6 (2 ꢁ d), 139.5 (s), 145.6 (d), 182.6 (CO). Anal. Calcd for
1635 (CO) cmꢀ1 1H NMR (CDCl3): d 2.50 (2H, t, J = 5.9 Hz, CH2),
;
2.68 (2H, t, J = 5.9 Hz, CH2), 6.80 (1H, s, H-1), 7.15–7.44 (6H, m,
Ar and H-3), 7.58 (1H, s, CH), 14.42 (1H, br s, OH); 13C NMR (CDCl3):
d 20.8 (t), 25.4 (t), 109.2 (s), 115.8 (d), 120.0 (d), 120.5 (2 ꢁ d),
126.3 (s), 126.7 (d), 129.5 (2 ꢁ d), 139.5 (s), 166.8 (d), 185.8 (s),
200.5 (CO). Anal. Calcd for C15H13NO2: C, 75.30; H, 5.48; N, 5.85.
Found: C, 75.59; H, 5.64; N, 5.56.
C19H22N2O: C, 77.52; H, 7.53; N, 9.52. Found: C, 77.84; H, 7.75;
N, 9.25.
4.1.2.4. 5-[(Diethylamino)methylene]-2,3-dimethyl-2,5,6,7-tet-
rahydro-4H-isoindol-4-one (7h). Brown oil; yield 50%; IR 1633
(CO) cmꢀ1
;
1H NMR: d 1.13 (6H, t, J = 6.9 Hz, 2 ꢁ CH3), 2.41 (3H,
4.1.1.4. 5-(Hydroxymethylene)-2,3-dimethyl-2,5,6,7-tetrahydro-
s, CH3), 2.45–2.65 (4H, m, 2 ꢁ CH2), 3.26 (4H, q, J = 6.9 Hz,
2 ꢁ CH2), 3.44 (3H, s, CH3), 6.36 (1H, s, H-1), 7.29 (1H, s, CH); 13C
NMR: d 10.4 (q), 14.6 (2 ꢁ q), 21.3 (t), 25.1 (t), 32.7 (q), 47.1
(2 ꢁ t), 103.6 (s), 115.2 (d), 118.1 (s), 122.4 (s), 131.4 (s), 144.4
(d), 183.9 (CO). Anal. Calcd for C15H22N2O: C, 73.13; H, 9.00; N,
11.37. Found: C, 72.80; H, 9.32; N, 11.61.
4H-isoindol-4-one (6h). Pale yellow oil; yield 52%; IR 2933 (OH),
1635 (CO) cmꢀ1 1H NMR (CDCl3): d 2.42 (2H, t, J = 6.6 Hz, CH2),
;
2.51 (3H, s, CH3), 2.58 (2H, t, J = 6.6 Hz, CH2), 3.49 (3H, s, CH3),
6.27 (1H, s, H-1), 7.39 (1H, s, CH), 14.60 (1H, br s, OH); 13C NMR
(CDCl3): d 10.9 (q), 21.1 (t), 26.1 (t), 33.1 (q), 109.6 (s), 116.5 (d),
123.8 (s), 163.8 (d), 175.8 (s), 188.1 (s), 201.0 (CO). Anal. Calcd
for C11H13NO2: C, 69.09; H, 6.85; N, 7.32. Found: C, 69.32; H,
6.57; N, 6.99.
4.1.2.5. 2-Benzyl-5-[(diethylamino)methylene]-3-methyl-2,5,6,
7-tetrahydro-4H-isoindol-4-one (7i). Brown oil; yield 60%; IR
1633 (CO) cmꢀ1
;
1H NMR: d 1.14 (6H, t, J = 7.1 Hz, 2 ꢁ CH3), 2.37
4.1.1.5. 2-Benzyl-5-(hydroxymethylene)-3-methyl-2,5,6,7-tetra-
hydro-4H-isoindol-4-one (6i). Yellow oil; yield 58%; mp 64–
65 °C; IR 2933 (OH), 1635 (CO) cmꢀ1; 1H NMR (CDCl3): d 2.43–2.51
(5H, m, CH2 and CH3), 2.62 (2H, t, J = 6.1 Hz, CH2), 5.00 (2H, s, CH2),
6.34 (1H, s, H-1), 7.04 (2H, d, J = 6.8, H-20 and H-60), 7.26–7.39 (3H,
m, H-30, H-40 and H-50), 7.44 (1H, s, CH), 14.63 (1H, br s, OH); 13C
NMR (CDCl3): d 11.2 (q), 21.3 (t), 26.2 (t), 50.1 (t), 109.8 (s), 116.3
(d), 117.5 (s), 124.3 (s), 126.6 (2 ꢁ d), 127.8 (d), 128.9 (2 ꢁ d),
134.8 (s), 136.7 (s), 164.3 (d), 188.4 (CO). Anal. Calcd for
(3H, s, CH3), 2.52 (2H, t, J = 5.2 Hz, CH2), 2.68 (2H, t, J = 5.2 Hz,
CH2), 3.27 (4H, q, J = 7.1 Hz, 2 ꢁ CH2), 5.05 (2H, s, CH2), 6.50 (1H,
s, H-1), 7.09 (2H, d, J = 7.1 Hz, H-20 and H-60), 7.22–7.37 (4H, m,
H-30, H-40, H-50 and CH); 13C NMR: d 10.6 (q), 14.6 (2 ꢁ q), 21.3
(t), 25.1 (t), 47.2 (2 ꢁ t), 48.9 (t), 103.7 (s), 115.1 (d), 118.6 (s),
122.9 (s), 126.7 (2 ꢁ d), 127.2 (d), 128.6 (2 ꢁ d), 131.2 (s), 138.1
(s), 144.6 (d), 184.0 (CO). Anal. Calcd for C21H26N2O: C, 78.22; H,
8.13; N, 8.69. Found: C, 77.99; H, 7.82; N, 8.93.
C
4.89.
17H17NO2: C, 76.38; H, 6.41; N, 5.24. Found: C, 76.09; H, 6.88; N,
4.1.3. General procedure for the preparation of ethyl 5-[(dimeth
ylamino)methylene]-3-methyl-4-oxo-4,5,6,7-tetrahydro-2H-iso
indole-1-carboxylate (7e–g)
4.1.2. General procedure for the preparation of 5-[(diethylami
no)methylene]-2,5,6,7-tetrahydro-4H-isoindol-4-one (7b–d,h,i)
To a cooled solution of diethylamine (2.6 mL, 25 mmol) in dry
benzene (30 mL), a solution of 6b–d,h,i (25 mmol) in dry benzene
(30 mL) was added. After 24 h stirring at room temperature the
solvent was removed and the residue was washed with diethyl
ether and filtered off.
To a solution of 5e–g (4.5 mmol) in dry toluene (10 mL), tert-
butoxybis(dimethylamino) methane (2.79 mL, 13.5 mmol) was
added. The reaction was heated under reflux for a proper time.
Upon cooling at room temperature, a solid separated from the
reaction mixture, which was collected and dried.
4.1.3.1 Ethyl 5-[(dimethylamino)methylene]-3-methyl-4-oxo-
4,5,6,7-tetrahydro-2H-isoindole-1-carboxylate (7e)
pound was obtained after 5 h. Pale green solid; yield 87%; mp
193–194 °C; IR 3278 (NH), 1668 (CO), 1637 (CO) cmꢀ1 1H NMR:
d 1.28 (3H, t, J = 7.2 Hz, CH3), 2.43 (3H, s, CH3), 2.81–2.92 (4H, m,
2 ꢁ CH2), 3.03 (6H, s, 2 ꢁ CH3), 4.22 (2H, q, J = 7.2 Hz, CH2), 7. 27
(1H, s, CH), 11.75 (1H, br s, NH); 13C NMR: 12.6 (q), 14.4 (q), 22.1
(t), 24.3 (t), 43.1 (2 ꢁ q), 59.3 (t), 103.4 (s), 114.2 (s), 119.8 (s),
132.8 (s), 136.4 (s), 147.2 (d), 160.7 (CO), 183.6 (CO). Anal. Calcd
for C15H20N2O3: C, 65.20; H, 7.30; N, 10.14. Found: C, 65.44; H,
6.99; N, 10.36.
. This com-
4.1.2.1. 5-[(Diethylamino)methylene]-2-methyl-2,5,6,7-tetrahy-
dro-4H-isoindol-4-one (7b). Brown oil; yield 85%; IR 1635 (CO)
;
cmꢀ1
;
1H NMR: d 1.14 (6H, t, J = 7.3 Hz, 2 ꢁ CH3), 2.53 (2H, t,
J = 6.4 Hz, CH2), 2.69 (2H, t, J = 6.4 Hz, CH2), 3.28 (4H, q, J = 7.3 Hz,
2 ꢁ CH2), 3.59 (3H, s, CH3), 6.45 (1H, s, H-1), 7.06 (1H, s, H-3),
7.33 (1H, s, CH); 13C NMR: d 14.6 (2 ꢁ q), 21.2 (t), 25.1 (t), 35.9
(q), 47.2 (2 ꢁ t), 102.8 (s), 117.1 (d), 122.1 (d), 122.8 (s), 124.2
(s), 144.8 (d), 182.7 (CO). Anal. Calcd for C14H20N2O: C, 72.38; H,
8.68; N, 12.06. Found: C, 72.15; H, 8.81; N, 12.40.
4.1.2.2. 2-Benzyl-5-[(diethylamino)methylene]-2,5,6,7-tetrahy-
4.1.3.2. Ethyl 5-[(dimethylamino)methylene]-2,3-dimethyl-4-
dro-4H-isoindol-4-one (7c). Pale brown solid; yield 95% mp 80–
oxo-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylate
compound was obtained after 3 h. Brown solid; yield 72%; mp
78–79 °C; IR 1682 (CO), 1637 (CO) cmꢀ1 1H NMR: d 1.28 (3H, t,
J = 7.1 Hz, CH3), 2.52 (3H, s, CH3), 2.74–2.85 (4H, m, 2 ꢁ CH2),
3.03 (6H, s, 2 ꢁ CH3), 3.73 (3H, s, CH3), 4.21 (2H, q, J = 7.1 Hz,
CH2), 7.29 (1H, s, CH); 13C NMR: d 10.9 (q), 14.3 (q), 22.8 (t), 24.1
(t), 32.0 (q), 43.1 (2 ꢁ q), 59.3 (t), 103.2 (s), 115.8 (s), 118.7 (s),
133.6 (s), 138.8 (s), 147.4 (d), 161.1 (CO), 183.1 (CO). Anal. Calcd
for C16H22N2O3: C, 66.18; H, 7.64; N, 9.65. Found: C, 65.82; H,
7.33; N, 9.98.
(7f). This
81 °C; IR 1637 (CO) cmꢀ1 1H NMR: d 1.21 (6H, t, J = 7.1 Hz,
;
2 ꢁ CH3), 2.64 (2H, t, J = 7.1 Hz, CH2), 2.80 (2H, t, J = 7.1 Hz, CH2),
3.32 (4H, q, J = 7.1 Hz, 2 ꢁ CH2), 5.00 (2H, s, CH2), 6.34 (1H, s, H-
1), 7.14–7.35 (6H, m, Ar and H-3), 7.59 (1H, s, CH); 13C NMR: d
14.6 (2 ꢁ q), 21.5 (t), 25.4 (t), 47.2 (2 ꢁ t), 53.6 (t), 103.6 (s),
115.8 (d), 122.0 (d), 123.4 (s), 125.5 (s), 127.3 (2 ꢁ d), 127.7 (d),
128.6 (2 ꢁ d), 137.0 (s), 146.2 (d), 184.7 (CO). Anal. Calcd for
;
C20H24N2O: C, 77.89; H, 7.84; N, 9.08. Found: C, 77.51; H, 8.04; N,
9.33.
4.1.2.3. 5-[(Diethylamino)methylene]-2-phenyl-2,5,6,7-tetrahy-
4.1.3.3. Ethyl 2-benzyl-5-[(dimethylamino)methylene]-3-meth
yl-4-oxo-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylate
(7g). This compound was obtained after 6 h. Green solid; yield
dro-4H-isoindol-4-one (7d). Pale brown solid; yield 85%; mp
114–115 °C; IR 1637 (CO) cmꢀ1 1H NMR: d 1.23 (6H, t, J = 6.8 Hz,
;
2 ꢁ CH3), 2.57–2.80 (4H, m, 2 ꢁ CH2), 3.39 (4H, q, J = 6.8 Hz,
57%; mp 133–134 °C; IR 1685 (CO), 1639 (CO) cmꢀ1 1H NMR: d
;

P. Barraja et al. / Bioorg. Med. Chem. 19 (2011) 2326–2341
2337
1.21 (3H, t, J = 6.9 Hz, CH3), 2.46 (3H, s, CH3), 2.75–2.87 (4H, m,
2 ꢁ CH2), 3.04 (6H, s, 2 ꢁ CH3), 4.14 (2H, q, J = 6.9 Hz, CH2), 5.61
(2H, s, CH2), 6.92 (2H, d, J = 6.9 Hz, H-20 and H-60), 7.18–7.32 (4H,
m, H-30, H-40, H-50 and CH), 13C NMR: d 10.8 (q), 14.1 (q), 22.9
(t), 24.0 (t), 43.1 (2 ꢁ q), 47.2 (t), 59.4 (t), 103.2 (s), 115.5 (s),
119.3 (s), 125.7 (2 ꢁ d), 126.9 (d), 128.5 (2 ꢁ d), 134.2 (s), 138.0
(s), 138.9 (s), 147.6 (d), 160.9 (CO), 183.1 (CO). Anal. Calcd for
8.00 (2H, d, J = 7.8 Hz, H-200 and H-600), 8.12 (1H, s, H-9), 8.18 (1H,
s, H-4), 12.34 (1H, s, NH); 13C NMR: d 19.4 (t), 26.2 (t), 110.1 (s),
115.1 (s), 116.5 (d), 118.4 (d), 119.5 (2 ꢁ d), 121.9 (s), 123.2 (s),
126.3 (d), 127.8 (2 ꢁ d), 128.7 (2 ꢁ d), 129.9 (2 ꢁ d), 132.9 (d),
139.1 (s), 140.9 (s), 144.0 (d), 145.4 (s), 157.2 (CO). Anal. Calcd
for C23H18N2O3S: C, 68.64; H, 4.51; N, 6.96. Found: C, 69.00; H,
4.32; N, 6.68.
C22H26N2O3: C, 72.11; H, 7.15; N, 7.64. Found: C, 72.42; H, 6.92;
N, 7.53.
4.1.4.4. Ethyl 9-methyl-3-(phenylsulfonyl)-1,5,6,8-tetrahydro-
2H-pyrrolo[3,4-h]quinoline-2-one-7-carboxylate (4e). This prod-
uct was obtained from the reaction of 7e after 24 h. Yellow solid;
4.1.4. General procedure for the preparation of 8-substituted-3-
(phenylsulfonyl)-1,5,6,8-tetrahydro-2H-pyrrolo[3,4-h]quinolin-
2-ones (4b–i)
yield 70%; mp: 324–325 °C; IR:
m
3299 (broad, 2 ꢁ NH), 1702 (CO),
1643 (CO) cmꢀ1 1H NMR: d 1.29 (3H, t, J = 7.0 Hz, CH3), 2.51 (3H,
;
To a suspension of 7b–i (4 mmol) in anhydrous ethanol (30 mL),
phenylsulfonylacetonitrile (1.09 g, 6 mmol) in anhydrous ethanol
(50 mL) was added dropwise under nitrogen atmosphere, and the
reaction mixture was heated under reflux for 24–72 h. Upon cool-
ing, a precipitate formed which was filtered and purified by recrys-
tallization or by column chromatography (Sepacore Büchi) using
DCM/AcOEt 9:1 as eluent.
s, CH3), 2.72 (2H, t, J = 6.4 Hz, CH2), 2.88 (2H, t, J = 6.4 Hz, CH2),
4.24 (2H, q, J = 7.0 Hz, CH2), 7.54–7.66 (3H, m, H-30, H-40, H-50),
7.97 (2H, d, J = 7.2 Hz, H-20 and H-60), 8.14 (1H, s, H-4), 11.23 (1H,
s, NH), 11.97 (1H, s, NH); 13C NMR: d 12.5 (q), 14.3 (q), 20.3 (t),
26.4 (t), 59.6 (t), 113.4 (s), 116.0 (s), 119.5 (s), 127.8 (2 ꢁ d), 128.2
(s), 128.7 (2 ꢁ d), 130.2 (s), 133.0 (d), 134.2 (s), 140.9 (s), 141.7
(s), 143.2 (d), 157.4 (CO), 160.4 (CO). Anal. Calcd for C21H20N2O5S:
C, 61.15; H, 4.89; N, 6.79. Found: C, 60.54; H, 5.10; N, 6.58.
4.1.4.1. 8-Methyl-3-(phenylsulfonyl)-1,5,6,8-tetrahydro-2H-pyr-
rolo[3,4-h]quinolin-2-one (4b). This product was obtained from
the reaction of 7b after 72 h (yield 65%), or by heating for the same
time the intermediate 8 with stoichiometric amount of diethyl-
4.1.4.5. Ethyl 8,9-dimethyl-3-(phenylsulfonyl)-1,5,6,8-tetrahy-
dro-2H-pyrrolo[3,4-h]quinoline-2-one-7-carboxylate (4f). This
product was obtained from the reaction of 7f after 24 h reflux. Yel-
amine (yield 40%). Yellow solid; mp: 345–346 °C; IR:
m 3106
(NH), 1633 (CO) cmꢀ1 1H NMR: d 2.59–2.66 (2H, t, J = 6.0 Hz,
;
low solid; yield 64%; mp: 239–240 °C; IR:
m 2981 (NH), 1699 (CO),
1646 (CO) cmꢀ1; 1H NMR: d 1.30 (3H, t, J = 6.7 Hz, CH3), 2.53 (3H, s,
CH3), 2.73 (2H, t, J = 7.1 Hz, CH2), 2.88 (2H, t, J = 7.1 Hz, CH2), 3.77
(3H, s, CH3), 4.23 (2H, q, J = 6.7 Hz, CH2), 7.54–7.69 (3H, m, H-30,
H-40, H-50), 7.94 (2H, d, J = 7.8 Hz, H-20 and H-60), 8.11 (1H, s, H-
4), 11.97 (1H, s, NH); 13C NMR: d 11.7 (q), 14.1 (q), 21.1 (t), 26.5
(t), 32.4 (q), 59.5 (t), 113.5 (s), 117.4 (s), 118.6 (s), 127.7 (2 ꢁ d),
127.8 (s), 128.6 (2 ꢁ d), 130.9 (s), 132.9 (d), 136.40 (s), 140.4 (s),
140.9 (s), 142.6 (d), 157.7 (CO), 160.7 (CO). Anal. Calcd for
CH2), 2.66 (2H, t, J = 6.0 Hz, CH2), 3.62 (3H, s, CH3), 6.63 (1H, s, H-
7), 7.49–7.64 (4H, m, H-30, H-40, H-50, H-9), 7.96 (2H, d, J = 7.8 Hz,
H-20, H-60), 8.09 (1H, s, H-4), 12.23 (1H, s, NH); 13C NMR: d 19.5
(t), 26.4 (t), 36.4 (q), 109.2 (s), 112.7 (s), 119.3 (d), 120.4 (s),
121.5 (s), 123.7 (d), 127.7 (2 ꢁ d), 128.6 (2 ꢁ d), 132.9 (d), 141.1
(s), 143.7 (d), 146.4 (s), 157.4 (CO). Anal. Calcd for C18H16N2O3S:
C, 63.51; H, 4.74; N, 8.23. Found: C, 63.84; H, 4.60; N, 8.00. When
the same reaction from 7b was conducted at room temperature for
24 h the intermediate 3-(2-methyl-4-oxo-2,4,6,7-tetrahydro-5H-iso-
indol-5-ylidene)-2-(phenylsulfonyl) propanenitrile (8) was isolated.
C22H22N2O5S: C, 61.96; H, 5.20; N, 6.57. Found: C, 62.08; H, 5.36;
N, 6.30. This product was also isolated (12%) from the methylation
of 4e.
Yellow solid; yield 62%; mp: 221–222 °C; IR:
m 2198 (CN), 1695
(CO) cmꢀ1
;
1H NMR: d 2.59 (4H, m, 2 ꢁ CH2), 3.68 (3H, s, CH3),
4.1.4.6. Ethyl 8-benzyl-9-methyl-3-(phenylsulfonyl)-1,5,6,8-tet-
rahydro-2H-pyrrolo[3,4-h]quinoline-2-one-7-carboxylate
(4g). This product was obtained from the reaction of 7g after 24 h
5.24 (1H, d, J = 10.2 Hz, CH), 5.76 (1H, s, H-1), 5.88 (1H, d,
J = 10.2 Hz, CH), 6.63 (1H, s, H-3), 7.58–7.81 (5H, m, Ar-H-H); 13C
NMR: d 22.7 (t), 27.6 (t), 36.8 (q), 116.6 (s), 122.6 (d), 124.7 (s),
127.8 (d), 128.9 (2 ꢁ d), 129.0 (2 ꢁ d), 129.4 (d), 133.8 (d), 134.3
(d), 137.4 (s), 140.4 (s), 141.1 (s), 163.9 (CO). Anal. Calcd for
reflux. Yellow solid; yield 65%; mp: 194–195 °C; IR:
m 2900 (NH),
1695 (CO), 1643 (CO) cmꢀ1 1H NMR: d 1.22 (3H, t, J = 7.1 Hz,
;
CH3), 2.50 (3H, s, CH3), 2.81 (2H, t, J = 7.1 Hz, CH2), 2.95 (2H, t,
J = 7.1 Hz, CH2), 4.16 (2H, q, J = 7.1 Hz, CH2), 5.64 (2H, s, CH2),
6.92–6.95 (2H, m, H-200 and H-600), 7.21–7.34 (3H, m, H-300, H-400,
H-500), 7.54–7.67 (3H, m, H-30, H-40, H-50), 7.95–7.98 (2H, m, H-20
and H-60), 8.16 (1H, s, H-4), 11.53 (1H, s, NH); 13C NMR: d 11.6
(q), 14.1 (q), 21.2 (t), 26.5 (t), 47.5 (t), 59.7 (t), 117.2 (s), 125.6
(2 ꢁ d), 125.7 (s), 126.9 (d), 127.8 (2 ꢁ d), 128.5 (2 ꢁ d), 128.6 (s),
128.8 (2 ꢁ d), 131.7 (s), 133.1 (d), 136.7 (s), 137.8 (s), 140.8 (d),
140.9 (s), 141.0 (s), 144.8 (s), 157.7 (CO), 160.6 (CO). Anal. Calcd
for C28H26N2O5S: C, 66.92; H, 5.21; N, 5.57. Found: C, 67.18; H,
5.02; N, 5.26.
C
18H16N2O3S: C, 63.51; H, 4.74; N, 8.23. Found: C, 63.18; H, 4.86;
N, 8.44.
4.1.4.2. 8-Benzyl-3-(phenylsulfonyl)-1,5,6,8-tetrahydro-2H-pyr-
rolo[3,4-h]quinolin-2-one (4c). This product was obtained from
the reaction of 7c after 48 h. Yellow solid; yield 55%; mp: 293–
294 °C; IR:
m ;
2933 (NH), 1641 (CO), cmꢀ1 1H NMR: d 2.59–2.69
(4H, m, 2 ꢁ CH2), 5.10 (2H, s, CH2), 6.75 (1H, s, H-7), 7.22–7.36
(5H, m, Ar-H), 7.52–7.63 (4H, m, H-30, H-40, H-50, H-9), 7.96 (2H,
d, J = 7.8 Hz, H-20, H-60), 8.11 (1H, s, H-4), 12.29 (1H, s, NH); 13C
NMR: d 19.4 (t), 26.35 (t), 52.8 (t), 109.3 (s), 113.0 (s), 118.5 (d),
120.6 (s), 121.1 (d), 121.5 (s), 127.4 (d), 127.5 (2 ꢁ d), 127.7
(2 ꢁ d), 128.6 (2 ꢁ d), 128.7 (2 ꢁ d), 132.8 (d), 137.6 (s), 141.1 (s),
143.7 (d), 146.3 (s), 157.3 (CO). Anal. Calcd for C24H20N2O3S: C,
69.21; H, 4.84; N, 6.73. Found: C, 69.04; H, 4.60; N, 6.80.
4.1.4.7. 8,9-Dimethyl-3-(phenylsulfonyl)-1,5,6,8-tetrahydro-2H-
pyrrolo[3,4-h]quinolin-2-one (4h). This product was obtained
from the reaction of 7h after 24 h reflux. Brown solid; yield 68%;
mp: 244–245 °C; IR:
m ;
2915 (NH), 1635 (CO) cmꢀ1 1H NMR: d
2.33 (3H, s, CH3), 2.47 (2H, t, J = 6.0 Hz, CH2), 3.32 (2H, t,
J = 6.0 Hz, CH2), 3.42 (3H, s, CH3), 6.51 (1H, s, H-7), 7.46–7.53
(3H, m, H-30, H-40, H-50), 7.90 (2H, d, J = 7.0 Hz, H-20 and H-60),
8.02 (1H, s, H-4), 11.21 (1H, s, NH); 13C NMR: d 10.9 (q), 19.5 (t),
26.9 (t), 32.8 (q), 110.8 (s), 111.9 (s), 117.3 (d), 118.1 (s), 120.0
(s), 127.3 (2 ꢁ d), 128.2 (2 ꢁ d), 129.2 (s), 132.4 (d), 140.8 (s),
4.1.4.3. 8-Phenyl-3-(phenylsulfonyl)-1,5,6,8-tetrahydro-2H-pyr-
rolo[3,4-h]quinolin-2-one (4d). This product was obtained from
the reaction of 7d after 48 h. Yellow solid; yield 60%; mp: 217–
220 °C; IR:
m ;
2933 (NH), 1633 (CO) cmꢀ1 1H NMR: d 2.68–2.81
(4H, m, 2 ꢁ CH2), 7.31 (1H, s, H-7), 7.50–7.66 (6H, m, H-30, H-40,
H-50, H-300, H-400, H-500), 7.96 (2H, d, J = 7.8 Hz, H-20 and H-60),

2338
P. Barraja et al. / Bioorg. Med. Chem. 19 (2011) 2326–2341
141.7 (d), 148.8 (s), 157.0 (CO). Anal. Calcd for C19H18N2O3S: C,
64.39; H, 5.12; N, 7.90. Found: C, 64.14; H, 5.00; N, 8.18.
140.9 (s), 150.6 (s), 157.7 (CO), 162.2 (CO). Anal. Calcd for
26H22N2O5S: C, 65.81; H, 4.67; N, 5.90. Found: C, 65.55; H, 4.42;
C
N, 6.02.
4.1.4.8. 8-Benzyl-9-methyl-3-(phenylsulfonyl)-1,5,6,8-tetrahy-
dro-2H-pyrrolo[3,4-h]quinolin-2-one (4i). This product was ob-
tained from the reaction of 7i after 24 h reflux. Yellow solid; yield
4.1.6. General procedure for the preparation of 8-substituted 1-
methyl-3-(phenylsulfonyl)-1,5,6,8-tetrahydro-2H-pyrrolo[3,4-h]
quinolin-2-ones (4m–r) and 8-substituted 2-methoxy-3-(phenyl
sulfonyl)-1,5,6,8-tetrahydro-2H-pyrrolo[3,4-h]quinolines (4s–u)
To a solution of the proper 8-substituted 3-(phenylsulfonyl)-1,5,6,8-
tetrahydro-2H-pyrrolo[3,4-h]quinolin-2-one 4b–i (15 mmol) dissolved
in anhydrous DMF (20 mL), NaH (0.38 g, 16 mmoles) was added at 0 °C
and the reaction mixture was stirred for 1 h at rt, then iodomethane
(1 mL, 16 mmol) was added at 0 °C. The reaction mixture was stirred
for 2 h, then poured onto crushed ice and the precipitate was filtered
off. Column chromatography of the residue, using DCM as eluent, gave
the expected product.
72%; mp: 252–254 °C; IR:
m ;
2938 (NH), 1641 (CO) cmꢀ1 1H NMR
(CDCl3): d 2.55 (3H, s, CH3), 2.58–2.62 (4H, m, 2 ꢁ CH2), 5.06 (2H,
s, CH2), 6.49 (1H, s, H-7), 7.06–7.42 (8H, m, Ar-H), 7.89 (2H, d,
J = 7.3 Hz, H-20 and H-60), 8.16 (1H, s, H-4), 12.21 (1H, s, NH); 13C
NMR (CDCl3): d 12.4 (q), 20.9 (t), 28.3 (t), 51.0 (t), 112.1 (s),
112.7 (s), 117.7 (d), 121.3 (s), 122.5 (s), 127.2 (2 ꢁ d), 128.4 (d),
128.6 (2 ꢁ d), 128.8 (2 ꢁ d), 129.5 (2 ꢁ d), 130.1 (s), 133.0 (d),
137.0 (s), 141.3 (s), 144.4 (d), 148.0 (s), 159.3 (CO). Anal. Calcd
for C25H22N2O3S: C, 69.75; H, 5.15; N, 6.51. Found: C, 69.54; H,
5.30; N, 6.78.
4.1.5. General procedure for the preparation of the 9-methyl-3-
(phenylsulfonyl)-1,5,6,8-tetrahydro-2H-pyrrolo[3,4-h]quinoline
-2-one-7-carboxylic acids (4j–l)
4.1.6.1. 1,8-Dimethyl-3-(phenylsulfonyl)-1,5,6,8-tetrahydro-2H-
pyrrolo[3,4-h]quinolin-2-one (4m). This product was obtained
To a suspension of 4e–g (1 mmol) in ethanol (5 mL), KOH
(0.34 g, 6 mmol) dissolved in a small amount of water (2 mL)
was added. The mixture was heated under reflux for 24 h, then
poured onto crushed ice and extracted with diethyl ether. The
aqueous layer was acidified with 6 M hydrochloric acid and the
precipitate formed was filtered off and washed with small amount
of ethanol to afford the desired carboxylic acid derivatives.
from 4b. Yellow solid; yield 55%; mp: 230–231 °C; IR:
m 1643
(CO) cmꢀ1
;
1H NMR (CDCl3): d 2.65–2.74 (4H, m, 2 ꢁ CH2), 3.71
(3H, s, CH3), 3.73 (3H, s, CH3), 6.51 (1H, s, H-9), 7.04 (1H, s, H-3),
7.46–7.51 (3H, m, H-30, H-40, H-50), 8.10 (2H, d, J = 6.9 Hz, H-20,
H-60), 8.19 (1H, s, H-4); 13C NMR (CDCl3): d 20.2 (t), 29.3 (t), 33.7
(q), 36.7 (q), 112.1 (s), 114.2 (s), 118.3 (d), 120.9 (s), 123.3 (d),
124.2 (s), 128.4 (2 ꢁ d), 128.6 (2 ꢁ d), 132.7 (d), 140.7 (s), 141.8
(d), 148.4 (s), 157.9 (CO). Anal. Calcd for C19H18N2O3S: C, 64.39;
H, 5.12; N, 7.90. Found: C, 64.12; H, 5.40; N, 7.68. From the same
reaction mixture 2-methoxy-8-methyl-3-(phenylsulfonyl)-1,5,6,8-
tetrahydro-5H-pyrrolo[3,4-h]quinoline (4s) was isolated as white so-
4.1.5.1. 9-Methyl-3-(phenylsulfonyl)-1,5,6,8-tetrahydro-2H-pyr-
rolo[3,4-h]quinoline-2-one-7-carboxylic acid (4j). This product
was obtained from 4e. White solid; yield 80%; mp: 243–244 °C;
3401 (OH), 3299 (broad, 2 ꢁ NH), 1672 (CO), 1655 (CO) cmꢀ1
;
lid; yield 18%; mp: 202–203 °C; IR:
m ;
1572 (C@N) cmꢀ1 1H NMR
IR:
m
1H NMR: d 2.51 (3H, s, CH3), 2.71 (2H, t, J = 6.2 Hz, CH2), 2.89
(2H, t, J = 6.2 Hz, CH2), 7.53–7.65 (3H, m, H-30, H-40, H-50), 7.97
(2H, d, J = 7.5 Hz, H-20 and H-60), 8.14 (1H, s, H-4), 11.90 (1H, s,
NH), 12.02 (2H, br s, NH and OH); 13C NMR: d 13.0 (q), 20.7 (t),
26.9 (t), 113.4 (s), 113.7 (s), 117.4 (s), 119.9 (s), 128.2 (2 ꢁ d),
129.2 (2 ꢁ d), 130.5 (s), 133.6 (d), 134.3 (s), 141.1 (s), 142.6 (d),
149.2 (s), 157.9 (CO), 162.4 (CO). Anal. Calcd for C19H16N2O5S: C,
59.37; H, 4.20; N, 7.29. Found: C, 59.57; H, 4.10; N, 7.38.
(CDCl3): d 2.73 (2H, t, J = 7.5 Hz, CH2), 2.90 (2H, t, J = 7.5 Hz, CH2),
3.66 (3H, s, CH3), 3.91 (3H, s, CH3), 6.40 (1H, s, H-7), 7.13 (1H, s,
H-9), 7.45–7.51 (3H, m, H-30, H-40, H-50), 8.00 (2H, d, J = 6.8 Hz,
H-20, H-60), 8.10 (1H, s, H-4); 13C NMR (CDCl3): d 20.0 (t), 28.5 (t),
36.4 (q), 53.5 (q), 118.1 (d), 120.0 (d), 120.6 (s), 122.1 (s), 122.4
(s), 128.2 (2 ꢁ d), 128.4 (2 ꢁ d), 132.7 (d), 138.0 (d), 141.4 (s),
144.3 (s), 154.8 (s), 158.7 (s). Anal. Calcd for C19H18N2O3S: C,
64.39; H, 5.12; N, 7.90. Found: C, 64.56; H, 4.98; N, 8.08.
4.1.5.2. 8,9-Dimethyl-3-(phenylsulfonyl)-1,5,6,8-tetrahydro-2H-
pyrrolo[3,4-h]quinoline-2-one-7-carboxylic acid (4k). This
product was obtained from 4f. White solid; yield 85%; mp: 230–
4.1.6.2. 8-Benzyl-1-methyl-3-(phenylsulfonyl)-1,5,6,8-tetrahy-
dro-2H-pyrrolo[3,4-h]quinolin-2-one (4n). This product was
231 °C; IR:
m ;
3197 (OH), 2956 (NH), 1648 (CO), 1646 (CO) cmꢀ1
obtained from 4c. Yellow solid; yield 62%; mp: 164–165 °C; IR:
m
1643 (CO) cmꢀ1 1H NMR (CDCl3): d 2.67 (2H, t, J = 6.5 Hz, CH2),
;
1H NMR: d 2.52 (3H, s, CH3), 2.71 (2H, t, J = 7.0 Hz, CH2), 2.91
(2H, t, J = 7.0 Hz, CH2), 3.78 (3H, s, CH3), 7.54–7.71 (3H, m, H-30,
H-40, H-50), 7.96 (2H, d, J = 7.0 Hz, H-20, H-60), 8.11 (1H, s, H-4),
12.03 (2H, br s, NH and OH); 13C NMR: d 11.8 (q), 21.1 (t), 26.6
(t), 32.4 (q), 113.1 (s), 115.7 (s), 118.1 (s), 118.7 (s), 127.8 (2 ꢁ d),
128.7 (2 ꢁ d), 130.1 (d), 133.1 (s), 136.1 (s), 139.9 (s), 140.9 (d),
150.0 (s), 157.6 (CO), 162.3 (CO). Anal. Calcd for C20H18N2O5S: C,
60.29; H, 4.55; N, 7.03. Found: C, 60.58; H, 4.32; N, 7.20.
2.71 (2H, t, J = 6.5 Hz, CH2), 3.70 (3H, s, CH3), 5.09 (2H, s, CH2),
6.56 (1H, s, H-7), 7.15–7.54 (9H, m, Ar-H and H-3), 8.12 (2H, d,
J = 6.4 Hz, H-20, H-60), 8.20 (1H, s, H-4); 13C NMR (CDCl3): d 20.3
(t), 29.2 (t), 33.7 (q), 53.9 (t), 112.2 (s), 114.5 (s), 117.7 (d), 121.1
(s), 122.7 (d), 124.3 (s), 127.1 (2 ꢁ d), 128.2 (d), 128.4 (2 ꢁ d),
128.6 (2 ꢁ d), 128.9 (2 ꢁ d), 132.7 (d), 136.4 (s), 140.6 (s), 141.9
(d), 148.3 (s), 157.9 (CO). Anal. Calcd for C25H22N2O3S: C, 69.75;
H, 5.15; N, 6.51. Found: C, 70.02; H, 4.86; N, 6.84. From the same
reaction mixture 8-benzyl-2-methoxy-3-(phenylsulfonyl)-1,56,8-tet-
rahydro-2H-pyrrolo[3,4-h]quinoline (4t) was isolated as white solid;
4.1.5.3. 8-Methyl-9-benzyl-3-(phenylsulfonyl)-1,5,6,8-tetrahy-
dro-2H-pyrrolo[3,4-h]quinoline-2-one-7-carboxylic acid (4l).
This product was obtained from of 4g. White solid; yield 90%;
yield 20%; mp: 186–187 °C; IR:
m ;
1572 (C@N) cmꢀ1 1H NMR
(CDCl3): d 2.74 (2H, t, J = 6.9 Hz, CH2), 2.92 (2H, t, J = 6.9 Hz, CH2),
3.90 (3H, s, CH3), 5.05 (2H, s, CH2), 6.46 (1H, s, H-7), 7.15–7.55
(9H, m, Ar-H and H-9), 7.98 (2H, d, J = 6.9 Hz, H-20, H-60), 8.12
(1H, s, H-4); 13C NMR (CDCl3): d 20.1 (t), 28.5 (t), 53.5 (q), 53.7
(t), 117.6 (d), 117.9 (s), 119.5 (d), 120.9 (s), 122.2 (s), 122.6 (s),
127.1 (2 ꢁ d), 127.9 (d), 128.2 (2 ꢁ d), 128.4 (2 ꢁ d), 128.8
(2 ꢁ d), 132.7 (d), 137.3 (s), 138.2 (d), 141.4 (s), 148.6 (s), 158.7
(s). Anal. Calcd for C25H22N2O3S: C, 69.75; H, 5.15; N, 6.51. Found:
C, 69.48; H, 5.40; N, 6.42.
mp: 185–186 °C; IR:
m 3457 (OH), 2969 (NH), 1656 (CO), 1654
(CO) cmꢀ1 1H NMR: d 2.48 (3H, s, CH3), 2.78 (2H, t, J = 6.4 Hz,
;
CH2), 2.99 (2H, t, J = 6.4 Hz, CH2), 5.69 (2H, s, CH2), 6.96 (2H, d,
J = 6.5 Hz, H-200 and H-600), 7.21–7.34 (3H, m, H-300, H-400, H-500),
7.58–7.66 (3H, m, H-30, H-40, H-50), 7.96 (2H, d, J = 6.9 Hz, H-20,
H-60), 8.16 (1H, s, H-4), 12.03 (2H, br s, NH and OH); 13C NMR: d
11.7 (q), 21.3 (t), 26.6 (t), 47.3 (t), 113.9 (s), 116.4 (s), 117.8 (s),
118.8 (s), 125.8 (2 ꢁ d), 126.9 (d), 127.8 (2 ꢁ d), 128.5 (2 ꢁ d),
128.8 (2 ꢁ d), 131.6 (s), 133.1 (d), 136.2 (s), 138.0 (d), 140.7 (s),

P. Barraja et al. / Bioorg. Med. Chem. 19 (2011) 2326–2341
2339
4.1.6.3. 1-Methyl-8-phenyl-3-(phenylsulfonyl)-1,5,6,8-tetrahy-
dro-2H-pyrrolo[3,4-h]quinolin-2-one (4o). This product was
obtained from the reaction of 4d. Yellow solid; yield 68%; mp:
161.5 (CO). Anal. Calcd for C29H28N2O5S: C, 67.42; H, 5.46; N,
5.42. Found: C, 67.74; H, 5.23; N, 5.60.
257–258 °C; IR:
m
1645 (CO) cmꢀ1
;
1H NMR (CDCl3): d 2.67–2.77
4.2. Biology
(4H, m, 2 ꢁ CH2), 3.80 (3H, s, CH3), 6.97 (1H, s, H-7), 7.33–7.52
(9H, m, Ar-H and H-9), 8.12 (2H, d, J = 6.5 Hz, H-20, H-60), 8.24
(1H, s, H-4); 13C NMR (CDCl3): d 20.2 (t), 29.1 (t), 33.9 (q), 112.7
(s), 116.0 (s), 116.3 (d), 120.4 (d), 120.9 (2 ꢁ d), 122.1 (s), 125.4
(s), 127.0 (d), 128.4 (2 ꢁ d), 128.6 (2 ꢁ d), 129.8 (2 ꢁ d), 132.8
(d), 139.4 (s), 140.5 (s), 142.0 (d), 147.7 (s), 157.8 (CO). Anal. Calcd
for C24H20N2O3S: C, 69.21; H, 4.84; N, 6.73. Found: C, 69.02; H,
5.12; N, 6.54. From the same reaction mixture 8-phenyl-2-meth-
oxy-3-(phenylsulfonyl)-6,8-dihydro-5H-pyrrolo[3,4-h]quinoline (4u)
4.2.1. Chemicals
Plasmid pBR322 was bought from Fermentas (Burlington,
Canada). Base excision repair enzymes (BER), Formamydo-pyrimi-
dine glycosilase (Fpg) and Endonuclease III (Endo III), were a gen-
erous gift from Dr. S. Boiteux (CEA, Fontenay aux Roses, France). If
not otherwise indicated, all the chemicals were purchased from
Sigma-Aldrich (Milano, Italy).
was isolated as white solid; yield 8%; mp 284–286 °C; IR:
m
1572
4.2.2. Irradiation procedure
(C@N) cmꢀ1
;
1H NMR (CDCl3): d 2.80–3.01 (4H, m, 2 ꢁ CH2), 4.14
HPW 125 Philips lamps, mainly emitting at 365 nm, were used
for irradiation experiments. The spectral irradiance of the source
(3H, s, CH3), 6.95 (1H, s, H-7), 7.28–7.57 (9H, m, Ar-H, H-9), 8.06
(2H, d, J = 6.0 Hz, H-20, H-60), 8.12 (1H, s, H-4); 13C NMR: d 20.0
(t), 30.1 (t), 63.7 (q), 115.7 (d), 116.5 (s), 118.4 (s), 119.1 (d),
120.7 (2 ꢁ d), 123.2 (s), 126.6 (d), 128.0 (2 ꢁ d), 128.9 (2 ꢁ d),
129.7 (2 ꢁ d), 132.3 (s), 132.8 (d), 133.4 (d), 135.3 (s), 136.9 (s),
139.9 (s), 141.4 (s). Anal. Calcd for C24H20N2O3S: C, 69.21; H,
4.84; N, 6.73. Found: C, 69.42; H, 4.68; N, 6.86.
was 4.0 mW cmꢀ2 as measured, at the sample level, by
a
Cole-Parmer Instrument Company radiometer (Niles, IL), equipped
with a 365-CX sensor.
4.2.3. Spectrophotometric measurement
All spectrophotometric measures were performed using
a
Perkin–Elmer Lambda 15 spectrophotometer and emission spectra
were recorded with a Perkin–Elmer LS-50B fluorimeter.
4.1.6.4. Ethyl 1,9-dimethyl-3-(phenylsulfonyl)-1,5,6,8-tetrahy-
dro-2H-pyrrolo[3,4-h]quinoline-2-one-7-carboxylate (4p). This
product was obtained from the reaction of 4e. Yellow solid; yield
4.2.4. Cellular phototoxicity
34%; mp: 248–249 °C; IR:
m
3284 (NH), 1660 (CO), 1655 (CO) cmꢀ1
;
Human promyelocytic leukaemia cells (HL-60) and human lym-
phoblastoid cells (Jurkat) were grown in RPMI-1640 medium, hu-
man breast adenocarcinoma cells (MCF-7) and human kerat
inocytes (NCTC-2544) were grown in DMEM medium, intestinal
adenocarcinoma cells (LoVo) were grown in Ham’s F12 medium.
All cellular media were supplemented with 115 units/mL of penicil-
1H NMR: d 1.30 (3H, t, J = 6.8 Hz, CH3), 2.56 (3H, s, CH3), 2.88–
2.93 (4H, m, 2 ꢁ CH2), 3.85 (3H, s, CH3), 4.24 (2H, q, J = 6.8 Hz,
CH2), 7.57–7.73 (3H, m, H-30, H-40, H-50), 7.93 (2H, d, J = 6.8 Hz,
H-20, H-60), 8.11 (1H, s, H-4), 11.90 (1H, s, NH); 13C NMR: d 13.0
(q), 14.4 (q), 20.2 (t), 27.0 (t), 53.8 (q), 59.5 (t), 115.6 (s), 117.1
(s), 122.2 (s), 127.7 (2 ꢁ d), 129.1 (2 ꢁ d), 129.9 (s), 133.4 (d),
134.7 (s), 137.6 (d), 140.9 (s), 147.2 (s), 154.9 (s), 157.9 (CO),
160.5 (CO). Anal. Calcd for C22H22N2O5S: C, 61.96; H, 5.20; N,
6.57. Found: C, 61.82; H, 5.08; N, 6.84.
lin G, 115
lg/ml streptomycin and 10% foetal bovine serum (Invit-
rogen, Milano, Italy). Individual wells of a 96-well tissue culture
microtiter plate were inoculated with 100 ll of complete medium
containing 8 ꢁ 103 HL-60, 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 prior the experiments. Drugs were dis-
solved in DMSO and then diluted with Hank’s Balanced Salt Solu-
tion (HBSS, pH 7.2) for phototoxicity test. After medium removal,
4.1.6.5. Ethyl 1,8,9-trimethyl-3-(phenylsulfonyl)-2,5,6,8-tetra-
hydro-1H-pyrrolo[3,4-h]quinoline-2-one-7-carboxylate (4q).
This product was obtained from the reaction of 4f. Yellow solid;
yield 65%; mp: 165–166 °C; IR:
m
1684 (CO), 1682 (CO) cmꢀ1
;
1H
100 ll of the drug solution were added to each well and incubated
NMR: d 1.30 (3H, t, J = 7.2 Hz, CH3), 2.65 (3H, s, CH3), 2.88 (2H, t,
J = 6.5 Hz, CH2), 2.95 (2H, t, J = 6.5 Hz, CH2), 3.78 (3H, s, CH3),
3.86 (3H, s, CH3), 4.23 (2H, q, J = 7.2 Hz, CH2), 7.58–7.71 (3H, m,
H-30, H-40, H-50), 7.95 (2H, d, J = 6.8 Hz, H-20, H-60), 8.13 (1H, s, H-
4); 13C NMR: d 11.3 (q), 14.2 (q), 20.9 (t), 26.8 (t), 32.3 (q), 53.8
(q), 59.5 (t), 116.0 (s), 117.1 (s), 117.3 (s), 122.6 (s), 127.8 (2 ꢁ d),
129.1 (2 ꢁ d), 130.8 (s), 133.4 (d), 137.0 (s), 137.6 (d), 140.8 (s),
154.5 (s), 157.7 (CO), 160.9 (CO). Anal. Calcd for C23H24N2O5S: C,
62.71; H, 5.49; N, 6.36. Found: C, 62.90; H, 5.18; N, 6.63. This prod-
uct was also isolated (15%) from the methylation of 4e.
at 37 °C for 30 min and then irradiated (2.5 or 3.75 J/cm2). After
irradiation, 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.12 Analogous experiments were
performed in the presence of various additives.
4.2.5. Cellular localization studies
NCTC-2544 cells were allowed to attach in a sterile Petri dishes
and treated with 4q at the concentration of 10 lM for 2 h, then
washed with HBSS, and incubated for further 30 min in the presence
of Rhodamine 123 a fluorescence probe which stains mitochon-
dria,26b LysoTracker RED, as fluorescent probe to stain lysosomes,
CellMask™ Orange, a plasma membrane probes and ER Tracking™
a specific stain for the endoplasmic reticulum. All of these probes
were purchase d by Molecular Probes. Cellular fluorescence images
were acquired with an video-confocal microscope (NIKON), using a
Nir Apo 60X/1.0W water immersion objective (NIKON). Emission fil-
ter settings were used to separate the emission of the probes from
that of the test compound.
4.1.6.6. Ethyl-1,9-dimethyl-8-benzyl-3-(phenylsulfonyl)-1,5,6,8-
tetrahydro-2H-pyrrolo[3,4-h]quinoline-2-one-7-carboxylate
(4r). This product was obtained from the reaction of 4g. Yellow
solid; yield 68%; mp: 138–139 °C; IR:
m 1692 (CO), 1690 (CO)
cmꢀ1 1H NMR (CDCl3): d 1.30 (3H, t, J = 6.9 Hz, CH3), 2.64 (3H, s,
;
CH3), 2.93 (2H, t, J = 7.0 Hz, CH2), 3.11 (2H, t, J = 7.0 Hz, CH2), 3.90
(3H, s, CH3), 4.24 (2H, q, J = 6.9 Hz, CH2), 5.62 (2H, s, CH2), 6.95
(2H, d, J = 6.9 Hz, H-200 and H-600), 7.19–7.30 (3H, m, H-300, H-400,
H-500), 7.47–7.56 (3H, m, H-30, H-40, H-50), 8.01 (2H, d, J = 6.6 Hz,
H-20, H-60), 8.15 (3H, s, H-4), 12.3 (1H, s, NH); 13C NMR (CDCl3):
d 11.6 (q), 14.3 (q), 21.6 (t), 27.8 (t), 48.1 (t), 53.9 (q), 59.9 (t),
117.4 (s), 117.5 (s), 118.0 (s), 122.8 (s), 125.7 (2 ꢁ d), 127.0 (d),
128.3 (2 ꢁ d), 128.5 (2 ꢁ d), 128.6 (2 ꢁ d), 132.1 (s), 132.9 (d),
136.9 (s), 137.8 (d), 137.9 (s), 141.2 (s), 154.8 (s), 158.3 (CO),
4.2.6. Externalization of phosphatidylserine
Surface exposure of phosphatidylserine (PS) by apoptotic cells
was measured by flow cytometry with a Coulter Cytomics FC500
(Becton Dickinson) by adding Annexin V–FITC to cells according

2340
P. Barraja et al. / Bioorg. Med. Chem. 19 (2011) 2326–2341
to the manufacturer’s instructions (Annexin V Fluos Roche Diag-
nostic). Simultaneously cells were stained with PI. Excitation was
set at 488 nm and the emission filters were at 525 nm.19
4.2.12. Lipid peroxidation
500.000 Jurkat cells were irradiated (2.5 and 3.75 J/cm2) in the
presence of the most active compounds and then they were incu-
bated for 24 h. Lipid peroxidation was measured using a thiobarbi-
turic acid assay as described by Morliere et al.30 Briefly, after cell
4.2.7. Cell cycle analysis
For flow cytometric analysis of DNA content, 5 ꢁ 105 Jurkat cells
in exponentially growth were irradiated (2.5 J/cm2) in the presence
of various concentrations of compounds. After 24 h of incubation,
cells were fixed in ice-cold ethanol (70%), then treated with lysis
buffer containing RNAseA, and finally stained with propidium io-
centrifugation, 900
ꢀ20 °C after having added 90
(BHT, 2% in absolute ethanol). Cells were washed, resuspended in
500 l of water. Cells (400 l) were lysed with 400 l of SDS (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 quantifica-
ll of supernatant were collected and put at
ll
of 2,6-di-tert-butyl-p-cresol
l
l
l
l
dide (PI, 10
lg/ml). Samples were analyzed on a Coulter Cytomics
l
l
FC500 (Becton Dickinson) flow cytometer. For cell cycle analysis,
results were examined using MultiCycle for Windows (Phoenix
Flow Systems, San Diego, CA), as previously reported.14 Data were
expressed as fractions of cells in the different cycle phases.
tion with Peterson method.35 A standard curve of 1,1,3,3 tetraeth-
oxypropane 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.
4.2.8. Mitochondrial membrane potential
The mitochondrial membrane potential was measured with the
lipophilic cation 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazol-
carbocyanine (JC-1, Molecular Probes). After 6, 12 and 24 h from the
irradiation (2.5 J/cm2), Jurkat were collected by centrifugation and
resuspended in HBSS containing the JC-1 at the concentration of
Acknowledgment
This work was financially supported by Ministero dell’Istruzi-
one dell’Università e della Ricerca.
1
lg/ml. The cytofluorimetric analysis (Coulter Cytomics FC500)
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