Synthesis and biological evaluation of a new furo[2,3-h]quinolin-2(1H)-one

Article abstract of DOI:10.1021/jm010993r

A new furoquinolinone derivative, namely 4-hydroxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-one (HOFQ), was synthesized and its biological activity studied. By UVA activation, HOFQ induced strong antiproliferative effects in Ehrlich ascite cells, which lost their ability to transmit the tumor by transplantation. HOFQ exhibited poor genotoxicity and absence of skin phototoxicity. Actually, HOFQ sensitization forms DNA-protein cross-linkages but not interstrands cross-links. Therefore, HOFQ appears to be a new promising drug for PUVA photochemotherapy and photopheresis.

Full text of DOI:10.1021/jm010993r

1146
J . Med. Chem. 2002, 45, 1146-1149
Syn th esis a n d Biologica l Eva lu a tion of a New F u r o[2,3-h ]qu in olin -2(1H)-on e
Adriana Chilin,† Christine Marzano,† Adriano Guiotto,*,‡ Francarosa Baccichetti,‡ Francesco Carlassare,‡ and
Franco Bordin‡
Department of Pharmaceutical Sciences, University of Padua, Via Francesco Marzolo 5, I-35131 Padova, Italy, and
Centro di Studio sulla Chimica del Farmaco e dei Prodotti Biologicamente Attivi del CNR, associated with the National
Institute for the Chemistry of Biological Systems, Department of Pharmaceutical Sciences, University of Padua,
Via Francesco Marzolo 5, I-35131 Padova, Italy
Received J uly 19, 2001
A new furoquinolinone derivative, namely 4-hydroxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-
2(1H)-one (HOFQ), was synthesized and its biological activity studied. By UVA activation,
HOFQ induced strong antiproliferative effects in Ehrlich ascite cells, which lost their ability
to transmit the tumor by transplantation. HOFQ exhibited poor genotoxicity and absence of
skin phototoxicity. Actually, HOFQ sensitization forms DNA-protein cross-linkages but not
interstrands cross-links. Therefore, HOFQ appears to be a new promising drug for PUVA
photochemotherapy and photopheresis.
In tr od u ction
Furocoumarins, currently called psoralens, are active
sensitizers used in PUVA (Psoralen plus UVA) therapy
to cure several skin diseases1 and in photopheresis to
treat T-cell lymphoma and various autoimmune dis-
eases and to prevent rejection in organ transplants.2
PUVA therapy, performed by using linearly annu-
lated psoralens such as 8-methoxypsoralen (8-MOP)
F igu r e 1. Structure of 8-methoxypsoralen (8-MOP), 1,4,6,8-
(Figure 1), is highly effective, but some toxic effects are
to be expected, such as skin erythema,1 genotoxicity,3,4
and carcinogenicity,5 mostly attributed to the lesions
induced in DNA by psoralen sensitization. Indeed,
different kinds of DNA lesions can be recognized, i.e.,
covalent monoadducts (MA) with pyrimidine bases,6
diadducts with pyrimidine bases placed on the opposite
DNA strand, thus forming inter-strands cross-links
(ISC),6 and covalent DNA-protein cross-links (DPC).7
Since many authors considered ISC mainly respon-
sible for furocoumarin genotoxicity, several monofunc-
tional derivatives have been prepared and studied, such
as angularly annulated furocoumarins as well as an-
gular furoquinolinones.8 Among them, 1,4,6,8-tetram-
ethylfuro[2,3-h]quinolin-2(1H)-one (FQ) (Figure 1) showed
a very high antiproliferative activity, without inducing
ISC:9 this was attributed to its ability to form a
considerable amount of DPC, which can be considered
another kind of diadduct because the molecule is a part
of the covalent bridge linking together DNA and pro-
teins.10 Moreover, these lesions induced marked clas-
togenic effects in mammalian cells cultivated in vitro.11
We tried to modulate FQ activity reducing its toxic
effects without affecting its antiproliferative activity.
Considering the studies carried out on benzopsoralen
derivatives in which the insertion of a hydroxymethyl
group at 4 position improved the activity of such
tetramethylfuro[2,3-h]quinolin-2(1H)-one (FQ), and 4-hydroxy-
methyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-one (HOFQ).
derivatives without increasing their toxicity,12 we in-
troduced a slight modification into FQ molecular struc-
ture, replacing the 4-methyl group with a hydroxy-
methyl one. So, we describe the synthesis and the main
biological properties of a new furoquinolinone derivative,
4-hydroxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-
one (HOFQ) (Figure 1). As a reference compound, we
chose 8-MOP (Figure 1), as it is a well-known and widely
used linear furocoumarin.
Ch em istr y
The synthetic pathway consists of building the tricy-
clic nucleus already carrying the 4-hydroxymethyl group
protected as methyl ether and then cleaving the ether
function. As shown in Scheme 1, 2,4-diaminotoluene (1)
was reacted with methyl 4-methoxyacetoacetate to yield
7-amino-4-methoxymethyl-6-methylquinolin-2-one (2),
which was diazotized and hydrolyzed to give 7-hydroxy-
4-methoxymethyl-6-methylquinolin-2-one (3). Compound
3 was condensed with allyl bromide, giving the 7-O-allyl
ether 4, which was methylated by dimethyl sulfate to
give the corresponding N-methyl derivative 5. In this
reaction a small amount of 2-methoxyquinoline 6 was
also obtained. The 7-allyloxy-1,6-dimethyl-4-methoxy-
methylquinolin-2-one (5) was submitted to Claisen
rearrangement, yielding 8-allyl derivative 7. Compound
7 was acetylated and brominated at room temperature,
affording 8-dibromopropyl derivative 9, which was
submitted to cyclization in alkaline medium,13 to give
4-methoxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-
* Address correspondence to Prof. Adriano Guiotto, Dipartimento
di Scienze Farmaceutiche, Universita` degli Studi di Padova, Via
Marzolo, 5, 35131 Padova, Italy. Tel: 0039-49-8275351. Fax: 0039-
49-8275366. E-mail: adriano.guiotto@unipd.it.
† University of Padua.
‡ Centro di Studio sulla Chimica del Farmaco e dei Prodotti
Biologicamente Attivi del CNR.
10.1021/jm010993r CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/30/2002

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5 1147
Sch em e 1a
F igu r e 2. ISC formation in vitro in PM2-linearized DNA by
sensitization with HOFQ or 8-MOP (two drug molecules per
base pair) detected by electrophoresis. Lane 1: untreated
double-stranded DNA. Lane 2: untreated single-stranded
DNA. Lanes 3: DNA exposed to 3.3 kJ m-2 alone. Lanes 4, 5:
DNA exposed to 1.5 and 3 kJ m-2 in the presence of 8-MOP,
respectively. Lanes 6, 7: DNA exposed to 1.5 and 3 kJ m-2 in
the presence of HOFQ, respectively.
a
Reagents and conditions: (i) MeOCH2COCH2CO2Et, 150 °C;
(ii) H2SO4, NaNO2, 0 °C then H2SO4, 160 °C; (iii) CH2dCHCH2Br,
DMF, K2CO3, RT; (iv) Me2SO4, acetone, K2CO3, reflux; (v) N,N-
diethylaniline, reflux; (vi) Ac2O, AcONa, reflux; (vii) Br2, AcOH,
RT; (viii) KOH, abs. EtOH, reflux; (ix) HBr, AcOH, reflux; (x) Ac2O,
AcONa, reflux; (xi) KOH, MeOH, reflux.
F igu r e 3. DPC formation in CHO cells detected by alkaline
elution. Cells were exposed to UVA light (0.3 kJ m-2) in the
presence of HOFQ or 8-MOP (both at 2 µM concentration).
Symbols: ∆, controls only submitted to γ rays (30 Gy); O,
HOFQ + 30 Gy; b, 8-MOP + 30 Gy.
2(1H)-one (10). Compound 10 was reacted with hydro-
bromic acid, to give the 4-bromomethyl derivative 11,
then submitted to acetylation, yielding the correspond-
ing 4-acetoxymethyl derivative 12, and finally hydro-
lyzed in alkaline conditions to give the desired 4-hy-
droxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-
one (13). Direct alkaline hydrolysis of 11 to 13 was
carried out, but the reaction time was longer and the
yield lower than indirect hydrolysis through 12, owing
to the formation of many degradation products.
reported in Figure 3, 8-MOP generated an elution profile
just above that of untreated control cells: this means
that 8-MOP, tested under the same experimental condi-
tions of HOFQ, formed small numbers of DPC. On the
contrary, cells sensitized by HOFQ showed a high DNA
retention, due to the formation of a considerable amount
of DPC.
Mu ta gen esis in Esch er ich ia coli WP 2. The HOFQ
mutagenic activity was detected in two E. coli WP2
strains, TM6 and TM9 (Figure 4). In TM6 strain,
proficient in DNA repair, HOFQ and 8-MOP gave rise
to two superimposed curves; in TM9 strain, defective
in DNA excision repair, HOFQ appeared to be no
mutagenic, while 8-MOP induced large numbers of
revertants.
An tip r olifer a tive Activity in Ma m m a lia n Cells
in Vitr o. The antiproliferative effect of HOFQ was
tested in mammalian cells in vitro by studying the
inhibition of DNA synthesis in Ehrlich cells and of clonal
growth in CHO cells. Table 1 summarizes the data
obtained in various experiments showing the ID50
values, which represent the treatments producing a 50%
inhibition. HOFQ exhibited an antiproliferative activity
40-60-fold higher than 8-MOP in both tests. To obtain
further information on the mechanism of the HOFQ
Biologica l Section
Detection of DNA Da m a ge. (a ) F or m a tion of ISC
in Vitr o. The induction of ISC by UVA irradiation,
using PM2-linearized DNA, was tested. ISC formation
was evaluated by gel-electrophoresis separation of
single-stranded, not cross-linked DNA, from double-
stranded, cross-linked DNA. As shown in Figure 2,
HOFQ appeared to be completely incapable of inducing
ISC, even at increasing UVA dose, while 8-MOP, used
as reference, formed high levels of ISC, even at low UVA
dose. UVA irradiation alone up to 10 kJ m-2 did not
modify the electrophoretic migration of DNA (data not
shown).
(b) F or m a tion of DP C in Vivo. The DNA-damage-
inducing ability of HOFQ was also studied detecting the
formation of DPC in CHO cells by alkaline elution. As

1148 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5
Brief Articles
when the challenge was accomplished with 4 × 106
tumor cells.
(b) Sk in P h ototoxicity. Skin phototoxicity was
studied on albino guinea pig skin; HOFQ appeared to
be completely unable of inducing skin erythemas even
when tested at high concentration (30 µM cm-2) and
with large UVA doses (10 kJ m-2). On the contrary,
8-MOP induced skin phototoxicity even in mild experi-
mental conditions (4 µM cm-2, 5 kJ m-2).
Con clu sion s
The new furoquinolinone derivative HOFQ was tested
in various biological systems. Upon UVA activation,
HOFQ, like FQ,9,10 induced large amounts of DPC, as
assayed in vivo in CHO cells; moreover, HOFQ appeared
to be incapable of forming ISC, as detected in vitro in
PM2-linearized DNA. Unfortunately, at present a ra-
dioactive sample of HOFQ is not available; therefore we
can only suppose it shows a high capacity of photobin-
ding to DNA, like FQ.8 On the other hand, the induction
of a severe DNA damage was also demonstrated when
studying macromolecular synthesis in Ehrlich cells:
both DNA and RNA syntheses were strongly affected
by HOFQ sensitization, while protein synthesis re-
mained unchanged; these results are similar to those
previously obtained with FQ [unpublished results].
HOFQ retained the very high antiproliferative activ-
ity in mammalian cells typical of FQ, as detected
studying both DNA synthesis and clonal growth capac-
ity in Ehrlich and CHO cells, respectively.
F igu r e 4. Mutagenesis in E. coli WP2. Bacteria were exposed
to UVA light in the presence of HOFQ or 8-MOP (2 µM), and
surviving fraction and revertants per million survivors were
determined. The revertants were plotted against the surviving
fraction. Panel A: TM6 strain, proficient in DNA repair. Panel
B: TM9 strain, defective in DNA excision repair. Symbols: O,
HOFQ; b, 8-MOP.
Ta ble 1. Antiproliferative Effect in Mammalian Cells upon
UVA Irradiationa
compound
DNA synthesis
clonal growth
HOFQ
8-MOP
0.65 ( 0.02
36.6 ( 0.3
0.02 ( 0.006
0.84 ( 0.04
a
The data are expressed as ID50, which indicates the treatment
producing a 50% inhibition, calculated by probit analysis, that is,
the UVA light dose, expressed as kJ m-2, delivered in the presence
of a 2 µM drug concentration.
Ta ble 2. Protection against the Ehrlich Viable Cells
Transplanta
challenge
The HOFQ activity was further studied in Ehrlich
cells in vivo; when these cells are injected intraperito-
neously into recipient mice, a 100% mortality is ob-
served within 1 or 3 weeks, according to cell amount.
However, if Ehrlich cells were submitted to HOFQ
photosensitization, this property was completely can-
celed and all mice were able to survive a long period.
This means that all tumor cells lost their reproductive
capacity. Moreover, we observed that animals treated
with such photoinactivated cells developed a resistance
against a successive transplantation of untreated viable
tumor cells; this protection was only observed if tumor
challenge was performed at least three weeks after the
injection of inactivated cells; at shorter times, the
protection decreased, with a corresponding increase in
mortality in treated mice. This suggested that the
injection of the inactivated cells stimulated a host
immune response against tumor cells. These results can
be explained supposing that DNA damage induced by
HOFQ neutralizes cell reproductive ability and intro-
duces modifications which increase cell antigenic char-
acter, so stimulating a host immune response against
tumor challenge. This immune response mechanism is
the same postulated as that of photopheresis.2 On the
basis of these observations, the results achieved pho-
toinactivating Ehrlich cells in the presence of HOFQ
prompt the consideration of this furoquinolinone deriva-
tive as a potential useful drug for photopheresis therapy.
HOFQ genotoxicity was investigated on two WP2
Escherichia coli strains, TM6 and TM9, the former
proficient in DNA repair, the latter defective, uvrA-
being an essential gene of DNA excision repair.14 In the
defective strain TM9, HOFQ induced much lower
amounts of revertants than 8-MOP and seems to be a
weaker mutagen.
no. of viable
days after
the transplant
mean survival
time (days)
cells (×10-6
)
% mortality
0.3
0.3
0.3
1
8
15
21
21
21
70
35
0
30
100
21.3
25
>60
10.6
15.8
4
a
Before the challenge, mice (groups of 10 animals) were injected
ip with 5 × 106 Ehrlich cells exposed to UVA light (2.7 kJ m-2) in
the presence of HOFQ (2 µM).
activity by UVA irradiation, its ability to inhibit RNA
and protein syntheses was also checked in the experi-
mental conditions used for DNA. HOFQ sensitization
inhibits RNA synthesis, with an ID50 value (0.36 ( 0.02)
very close to that obtained for DNA synthesis. On the
contrary, protein synthesis remained completely un-
affected (ID50 > 10).
Exp er im en ts in Vivo. (a ) Eh r lich Tu m or Tr a n s-
p la n ta tion . The effect of HOFQ sensitization was
further studied in Ehrlich cells in vivo, at first testing
the tumor transmitting capacity into recipient mice. The
injection of untreated viable cells yielded a 100%
mortality, but when they were exposed to UVA light
(1.33 kJ m-2) in the presence of 2 µM HOFQ, the
mortality was abolished.
Then we studied the effect of the injection of such
inactivated cells on mice; therefore, groups of mice were
injected with the photoinactivated cells and then, at
different times, they were submitted to a transplanta-
tion of different amounts of viable tumor cells. As shown
in Table 2, the protection became total when the
challenge was performed 21 days after the injection of
photoinactivated cells. If 106 viable cells were injected,
a 30% mortality was observed, which reached 100%

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5 1149
4-Hyd r oxym eth yl-1,6,8-tr im eth ylfu r o[2,3-h ]qu in olin -
2(1H)-on e (13). To a solution of 12 (0.70 g, 2.3 mmol) in MeOH
(20 mL) was added a 5% methanolic KOH solution (20 mL),
and the mixture was refluxed for 1 h. The solution was diluted
with water (100 mL) and acidified with 2 N HCl. The collected
precipitate was crystallized from MeOH to give 13 (0.45 g,
76%).
A similar picture has been obtained studying skin
phototoxicity, a well-known side effect of 8-MOP; in fact,
while FQ showed a phototoxicity comparable to that of
8-MOP,9 HOFQ appeared to be incapable of inducing
erythemas on guinea pig skin.
Therefore, we can conclude that HOFQ represents a
new furoquinolinone derivative markedly more efficient
in promoting antiproliferative effects than 8-MOP and
very close to FQ.9,11 Pointing out some preliminary
interesting properties of HOFQ, including scarce geno-
toxicity and lack of skin phototoxicity, extensive studies
will be made in the future on the relationship between
the damage induced into DNA and the observed biologi-
cal effects.
Biologica l Assa ys. Detection of DNA damage, mutagen-
esis, antiproliferative activity in vitro and in vivo, and skin
phototoxicity have been recently described.10-12 For further
details, see Supporting Information.
Su p p or tin g In for m a tion Ava ila ble: Detailed procedures
for the syntheses of compounds 2 and 3; mp, NMR, and
microanalysis data for all compounds; and biological assays.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Exp er im en ta l Section
Ch em istr y. TLC, mp, 1H NMR spectra, and CHN were
obtained as noted.9 See Supporting Information for synthesis
of 2 and 3, mp, NMR, and microanalysis data.
7-Allyloxy-4-m eth oxym eth yl-6-m eth ylqu in olin -2-on e (4).
A mixture of 3 (7.4 g, 34.0 mmol), allyl bromide (6.1 g, 50.4
mmol), and K2CO3 (10.0 g) in DMF (150 mL) was stirred at
20 °C for 40 h. The mixture was diluted with water (350 mL)
and the solid collected, washed with water, and crystallized
from MeOH to give 4 (2.1 g, 24%).
Refer en ces
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7-Allyloxy-1,6-d im et h yl-4-m et h oxym et h ylq u in olin -2-
on e (5) a n d 7-Allyloxy-6-m eth yl-2-m eth oxy-4-m eth oxy-
m eth ylqu in olin e (6). A mixture of 4 (5.0 g, 19.3 mmol),
dimethyl sulfate (2.8 g, 22.5 mmol), and K2CO3 (10.0 g) in
acetone (250 mL) was refluxed for 15 h. After cooling, the solid
was filtered off and the filtrate evaporated under reduced
pressure. The residue was purified by column chromatography
to give 6 (0.44 g, 8%), followed by 5 (3.5 g, 66%).
8-Allyl-1,6-d im eth yl-7-h yd r oxy-4-m eth oxym eth ylqu in -
olin -2-on e (7). A solution of 5 (3.5 g, 12.8 mmol) in N,N-
diethylaniline (50 mL) was refluxed for 7 h. After cooling, the
precipitate was collected, washed with cyclohexane, and
crystallized from AcOEt to give 7 (2.5 g, 75%).
7-Acetoxy-8-a llyl-1,6-d im eth yl-4-m eth oxym eth ylqu in -
olin -2-on e (8). A mixture of 7 (5.4 g, 19.7 mmol) and
anhydrous AcONa (1.0 g) in acetic anhydride (30 mL) was
refluxed for 1 h. The mixture was cautiously diluted with water
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AcOEt/n-hexane to give 8 (4.2 g, 68%).
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7-Acetoxy-8-(2′,3′-dibr om opr opyl)-1,6-dim eth yl-4-m eth -
oxym eth ylqu in olin -2-on e (9). A solution of Br2 (1.4 g, 8.9
mmol) in acetic acid (10 mL) was added dropwise to a solution
of 8 (2.8 g, 8.9 mmol) in acetic acid (60 mL) at 20 °C, and after
the addition was completed the solution was stirred for 30 min.
The solvent was evaporated under reduce pressure, and the
residue was crystallized from MeOH to give 9 (3.9 g, 92%).
4-Meth oxym eth yl-1,6,8-tr im eth ylfu r o[2,3-h ]qu in olin -
2(1H)-on e (10). A solution of KOH (2.5 g) in absolute EtOH
(50 mL) was added to a solution of 9 (3.9 g, 8.2 mmol) in
absolute EtOH (100 mL), and the mixture was refluxed for 45
min. The cooled mixture was acidified with 2 N HCl and
diluted with water (500 mL). The solid was collected, washed
with water, and crystallized from AcOEt to give 10 (1.8 g, 83%).
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(5 mL), and acetic acid (50 mL) was refluxed for 10 h. The
mixture was poured into water (250 mL), and the collected
precipitate was crystallized from EtOAc to give 11 (0.97 g,
55%).
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4-Acet oxym et h yl-1,6,8-t r im et h ylfu r o[2,3-h ]q u in olin -
2(1H )-on e (12). Compound 12 (0.74 g, 86%) was prepared
from 11 (0.95 g, 2.9 mmol) as described for compound 8.
J M010993R