Synthesis and biological activity of some rigid analogues of flavone-8- acetic acid

Article abstract of DOI:10.1016/S0968-0896(99)00282-5

Some rigid analogues of flavone-8-acetic acid are described. Direct in vitro toxicity of the synthesised compounds was evaluated towards four tumoral cell lines and the ability of these compounds to stimulate mouse peritoneal macrophages in culture to bec

Full text of DOI:10.1016/S0968-0896(99)00282-5

Bioorganic & Medicinal Chemistry 8 (2000) 239±246
Synthesis and Biological Activity of Some Rigid Analogues of
Flavone-8-acetic Acid
Piero Valenti, a,* Alessandra Bisi, a Angela Rampa, a Federica Belluti, a Silvia Gobbi, a
Antonella Zampiron b and Maria Carrara b
aDepartment of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
bDepartment of Pharmacology, University of Padova, Largo Meneghetti 2, 35131 Padova, Italy
Received 18 June 1999; accepted 23 September 1999
AbstractÐSome rigid analogues of ¯avone-8-acetic acid are described. Direct in vitro toxicity of the synthesised compounds was
evaluated towards four tumoral cell lines and the ability of these compounds to stimulate mouse peritoneal macrophages in culture
to become tumoricidal (indirect toxicity) was also studied. All compounds were able to induce direct cytotoxicity only at very high
concentrations but showed a remarkable indirect activity. In particular compound 4d was able to signi®cantly increase macrophage
lytic properties and has been selected for further investigations. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
through the production of interferons,12 activation of
macrophage tumouricidal activity15 and the production
of nitric oxide.16 Therefore, the action of FAA involves
also the immune system, since it acts as a biological
response modi®er stimulating natural killer cells' activ-
ity13,14 and enhancing the lytic potential of macro-
phages.15 Moreover, there is evidence16 for FAA
induced increase in the expression of some cytokine
genes in mice, such as TNF gene, for which Mahadevan
et al.17 postulated a role in FAA induced tumour vas-
cular shutdown. A paper from Futami et al.18 demon-
strated that FAA can directly stimulate cytokine gene
expression in mouse but not in human leukocytes; this
di€erence in sensitivity to FAA between human and
mouse immune systems may account for the lack of
activity seen in human tumours.
Flavone-8-acetic acid (FAA, 1) is a ¯avonoid synthe-
sised by Atassi et al.1 with a unique pattern of pre-
clinical antitumour activity. A peculiar feature of FAA
is its low activity against fast-growing tumours, like
leukemia, and its striking, broad activity against slow-
growing solid tumours (colon, pancreatic and mammary
adenocarcinomas, Glasgow osteogenic sarcoma) that
are usually insensitive to most cytotoxic drugs.1±3 Its
toxicological pro®le is also di€erent from conventional
chemotherapeutic agents, since it does not cause myelo-
suppression.4 Anyway, its potency is low5 and high
doses and long exposure times are required for a direct
cytotoxic e€ect. Unfortunately, the activity of the drug
on murine tumours was not con®rmed by the sub-
sequent clinical trials, which led to disappointing results
showing no activity on human tumours.6,7
Extensive studies have been carried out to elucidate the
mechanism of action of FAA at molecular level.19±22
Despite these studies, it has not been fully elucidated yet
and synthesis and evaluation of novel analogues may
provide useful information, since the biological receptor
for FAA is presently unknown. Furthermore, the
structure±activity relationships for FAA have not been
thoroughly investigated. Recent papers23±25 reported
some novel modi®cations of the parent structure, but
these did not lead to major improvements in potency.
Remarkable activity and higer potency was shown by a
series of analogues of xanthenone-4-acetic acid (XAA,
2), where the xanthenone moiety can be regarded as a
`fused' ¯avone, expecially if substituted in positions 5
The activity of FAA seems to be due to indirect e€ects
more than to direct cytotoxicity. Factors involved are its
ability to induce haemorrhagic necrosis of subcutaneous
tumours,8 that may be attributed to vascular collapse,9±11
its ability to induce production of tumour necrosis factor-a
(TNF-a),12 increase of natural killer (NK) cells13,14
Keywords: ¯avone-8-acetic acid; antitumour compounds; human
colon adenocarcinoma; ovarian adenocarcinoma; macrophages lytic
properties.
*Corresponding author. Fax: +39-051-2099734; e-mail: pvalenti@
alma.unibo.it
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00282-5

240
P. Valenti et al. / Bioorg. Med. Chem. 8 (2000) 239±246
and/or 6: the 5,6-dimethylxanthenone-4-acetic acid (3)
proved to be the most potent compound synthesised so
far, and was selected for clinical evaluation.26±30
In continuing our studies on the structure±activity rela-
tionships of FAA,31,32 in this paper we report the
synthesis and in vitro study of some furochromene and
furoxanthene carboxylic acids of general formulas 4 and
5 in which the acetic chain of FAA and XAA is locked
in a rigid structure. This introduces a signi®cant
change in the a position to the carboxyl group, a change
shown not to greatly in¯uence activity in the xanthe-
none derivatives.30
Scheme 1. Reagents and conditions: (a) methylallylbromide, K2CO3
re¯ux; (b) 2,6-dimethylphenol 230±240 ꢀC, 8 h; (c) NBS; (d) CuCN; (e)
AcOH, H2SO4, H2O re¯ux, 2 h.
chromones (7a,b,d) were prepared via Baker±Venka-
taraman rearrangement of resacetophenone dibenzoates,
which were prepared from resacetophenone and benzoyl
chlorides, to the corresponding diketones followed by
cyclisation.33±35 The same procedure was used to pre-
pare the 7-hydroxy-2-thenoylchromone (7c). The sub-
stituted 7-hydroxychromones and 3-hydroxyxanthenone36
(8) were heated with methylallylbromide in presence of
K2CO3 to a€ord, respectively, 9a±d and 10.28 These
compounds were cyclised to 11a±d and 1237 by heating
at 230±240 ꢀC in the presence of 2,6-dimethylphenol.
Subsequent bromination with N-bromosuccinimide
(compounds 13a-d and 1437), treatment with CuCN (not
isolable compounds) and hydrolysis with AcOH,
H2SO4, H2O a€orded the required compounds 4a±d
and 5.
A rigid analogue of FAA (i.e. 10-oxo-10,11-dihydro-in-
deno-[1,2-b]-chromen-6-yl-acetic acid (6)) was also pre-
pared in which a ®ve membered ring is introduced in
order to ®x the position of the benzene ring in position 2
on the ¯avone moiety, closely resembling the structure
of xanthenone-4-acetic acid (2).
Compound 6 was prepared following Scheme 2. The
2-hydroxy-3-allylacetophenone was condensed with o-
phthalaldehyde using 50% KOH solution at room tem-
perature to a€ord 15, which was cyclised to 16 by
re¯uxing with MeOH±HCl (g). Oxidation of 16 with
KMnO4 a€orded 6.
Biological evaluation
It resembles particularly 5,6-dimethylxanthenone-4-
acetic acid (3), as the A, B, and C rings can mimic the
xanthenone moiety and the D ring can mimic the
substituents in positions 5 and 6 included in a cyclic
structure.
We have studied direct in vitro toxicity of FAA and
XAA analogues towards tumoral cell lines and also the
ability of these compounds to stimulate mouse perito-
neal macrophages in culture to become tumoricidal
(indirect toxicity).
Direct in vitro toxicity of the synthesised compounds
(tested as racemates) was evaluated towards four
tumoral cell lines and the ability of these compounds
to stimulate mouse peritoneal macrophages in culture
to become tumoricidal (indirect toxicity) was also
studied.
The direct e€ect of the tested compounds was deter-
mined on four human cell lines: two of these, LoVo S and
LoVo R arise from colon adenocarcinoma, while 2008
and C13* from ovarian adenocarcinoma. LoVo R cells
di€er from the parental LoVo S line because they are
resistant to doxorubicin and express MDR, while C13*
cells appear to be 10-fold more resistant to cisplatin
than the original 2008 line, and furthermore show
reduced cell membrane permeability to passive di€u-
sion38 and mitocondrial membrane functionality.39,40
Results and Discussion
Chemistry
The studied compounds 4a±d and 5 were prepared
according to Scheme 1. The substituted 7-hydroxy-
Direct toxicity. Data obtained for the tested compounds
are summarised in Table 1.

P. Valenti et al. / Bioorg. Med. Chem. 8 (2000) 239±246
241
Scheme 2. Reagents: (a) KOH 50%, room temperature, 12 h; (b) HCl(g), MeOH re¯ux 2 h; (c) KMnO4, 0±5 ꢀC, 6 h.
On LoVo S cells XAA showed toxic e€ect similar to
FAA, in fact at the highest doses tested (250 and
500 mM) XAA was able to induce 28 and 44% reduction
of cell growth, against 24 and 42% obtained with FAA.
On this cell line all the synthesised compounds appeared
more active than the two references, showing a sig-
ni®cant cytotoxic e€ect, about 30%, at the lowest con-
centration tested (100 mM). Nevertheless, it should be
stressed that their direct toxicity on LoVo S cells at
higher concentrations was not dose-dependent. Con-
sidering the IC50 values and the potency ratio (P.R.)
versus the two references, the new analogues appeared
1.4±2.4 times more potent than FAA and 1.8±3 times
more potent than XAA. In particular, 5 and 4c were the
two most toxic compounds.
four cell lines used showed a di€erent response to FAA:
generally, ovarian cells were more sensitive than colon
cells. On the contrary, the new derivatives appeared to
be more active on colon cells with respect to ovarian
cells. In particular, all analogues were signi®cantly more
potent than FAA itself on LoVo S.
Indirect toxicity. The immune system plays an essential
role in the physiological defence against tumoural cell
proliferation and several lymphocyte subpopulations
are involved in immunosurveillance against tumour
cells.41,42 Natural killer (NK) cells are a subpopulation
of large granular lymphocytes that display spontaneous
cytotoxic activity against tumour cells without restric-
tion by molecule of the major histocompatibility com-
plex.43 The lytic activity of NK lymphocytes can be
regulated by di€erent cytokines, such as interferon a,
interferon b and interleukin-2.44±47
On LoVo R cells, both FAA and XAA proved to be
more cytotoxic than on the parental cell line, whereas
the analogues showed toxic e€ect similar to the refer-
ence compounds (except for 6, more active) but lower
than their e€ect on LoVo S cells.
Flavone-8-acetic acid seems to act by an indirect
mechanism, involving the stimulation of NK cell activ-
ity,13,14 as well as the increase of macrophage-mediated
cytotoxicity.48 Furthermore, FAA induces interferon a,
b, g and TNF.49 In order to evaluate the true biological
meaning of the changes in the chemical structure of
FAA or XAA it is interesting to consider the ability of
the new derivatives to stimulate mouse peritoneal mac-
rophages in culture to become tumoricidal, evaluated
by measuring the cytotoxicity induced on C13* cells
co-cultivated with macrophages pre-treated with the
reference compounds or one of the new analogues. The
results were collected in Table 2.
Fully di€erent were the results obtained on the two
ovarian cell lines, especially on 2008 cells. In fact, not
only was FAA the most active compound on this line,
but among the new derivatives only 4b and 6 showed
the same cytotoxic activity. The other compounds were
quite inactive and a signi®cant cell growth reduction
(about 35%) was observed, only with the 500 mM dose.
Generally, the cell line bearing resistance (C13*) was
more sensitive to cytotoxic e€ect of the new derivatives
than the parental one, even if the best results were
obtained at the highest concentration tested (500 mM).
In these experiments FAA was the most active com-
pound on both cell lines and was more cytotoxic on
resistant C13* cells. XAA showed toxic e€ect similar to
FAA on 2008 cells, while it was 2.7 times less potent
than FAA on C13* cells. Among the analogues, only 6
showed toxic e€ect similar to FAA both on 2008 and
C13* cells, whereas the other compounds were inactive
or less potent.
FAA seems unable to stimulate lytic activity of macro-
phages. Comparing the IC50 values obtained on C13*
cells for direct e€ects, no appreciable di€erence was seen
(Table 2). On the contrary, XAA was the most active
compound, in fact its indirect toxicity was three times
more than its direct e€ect. Comparing the indirect e€ect
of the new derivatives with their direct activity, a gen-
eral increase of toxicity was observed, that for 4a, 4c
and 4d was about 1.7±2.6 times. Interesting were also
the results obtained with compound 5, which was inac-
tive on C13* cells, but was able, like XAA, to stimulate
lytic activity of macrophages.
In conclusion, these analogues were able to induce
direct cytotoxicity only at very high concentrations. The

242
P. Valenti et al. / Bioorg. Med. Chem. 8 (2000) 239±246
Table 2. IC50, 95% con®dence limits and potency ratio (PR) versus
FAA and versus XAA for each derivative on co-cultured macro-
phages+C13* cells and PR for IC50 of each compound obtained in
co-culture macrophages+C13* versus its direct toxicity on C13* cells
Compd
IC50 mM
PR versus
FAA
PR versus
XAA
PR versus
C13*
FAA
XAA
285 (211±386)
219 (164±293)
1
Ð
Ð
1
0.8 (0.5±1.2)
3.0 (2.2±4.2)a
4a
4b
4c
4d
5
288 (201±414) 1.0 (0.6±1.6) 0.8 (0.2±1.3) 1.7 (1.3±2.0)a
481 (317±730) 0.6 (0.3±1.0)b 0.4 (0.3±0.6)b 1.2 (0.7±2.1)
275 (209±362) 1.0 (0.7±1.5) 0.7 (0.5±0.9) 2.0 (1.4±2.7)a
240 (179±322) 1.2 (0.8±1.8) 0.9 (0.5±2.0) 2.6 (1.7±3.7)a
304 (211±439) 0.9 (0.6±1.5) 0.7 (0.4±1.1) Ð
293 (232±373) 1.0 (0.7±1.4) 0.7 (0.5±1.0) 1.0 (0.7±1.3)
6
aMore potent.
bLess potent.
Conclusion
Some furochromene and furoxanthene carboxylic acids
(4a±d and 5) in which the acetic chain of FAA and XAA
is locked in a rigid structure and a rigid analogue of
FAA (compound 6) were synthesized. Direct cytotoxic
activity, evaluated on four human cell lines, was
obtained only at very high concentrations. Generally,
FAA was more cytotoxic than XAA and ovarian cells
were more sensitive than colon cells to FAA activity. On
the contrary, the new derivatives appeared to be more
active on colon cells with respect to ovarian cells. In
particular, all analogues were signi®cantly more potent
than FAA itself on LoVo S.
All compounds showed an interesting indirect mechan-
ism of antitumour action. In particular, compound 4d
was able to signi®cantly increase the macrophage lytic
properties and has been selected for further investigations.
Experimental
Chemistry
A Buchi apparatus and open glass capillaries were used
to determine all melting points (mp), which are not
corrected. Wherever analyses are only indicated with
element symbols, analytical results obtained for those
1
elements are within 0.4% of the theoretical values. H
NMR were obtained on Gemini 300 spectrometer.
Chemical shifts are reported in parts per million (ppm)
relative to tetramethylsilane (TMS). Mass spectra were
recorded on a V.G. 7070 E spectrometer.
7-Hydroxy-2-thenoylchromone (7c). To a solution of
resacetophenone (15.2 g, 0.1 mol), pyridine (23.7 g,
0.3 mol), and anhydrous ether (100 mL) at 0 ꢀC (ice
bath), 2-thiophenecarbonyl chloride (43.8 g, 0.3 mol)
was added, dropwise, over 1 h. After the addition, the
mixture was stirred at 0 ꢀC for 1 h and then at room
temperature for 1 h. The solid was collected by ®ltration
and washed with ether (200 mL). The ®ltrate was
washed with water, 10% aq HCl, 10% aq NaHCO3,
and saturated brine solution and dried (Na2SO4). The

P. Valenti et al. / Bioorg. Med. Chem. 8 (2000) 239±246
243
®ltered solution was evaporated to give 26 g (70%) of
product mp 120±123 ꢀC (EtOH).
CH3), 3.3 (s, 2H, CH2), 6.75 (s, 1H, CH), 6.85±8.1 (m,
7H, aromatic).
A suspension of this product and K2CO3 (28 g, 0.2 mol)
in acetone (200 mL) was stirred and heated under re¯ux
in presence of nitrogen for 2 days. After cooling the
yellow solid was collected by ®ltration and washed with
toluene and then with water. The solid was stirred with
10% aq HCl and collected by ®ltration. The desired
product, 20.8 g (80%), mp 144±147 ꢀC was obtained.
The same procedure was used to prepare the following
compounds.
8,8-Dimethyl-2-(40-¯uoro)phenyl-8,9-dihydrofuro-[2,3-h]-
chromen-4-one (11b). This compound was obtained in
65% yield, mp 204±206 ꢀC (toluene). Anal. (C19H15
FO3): C, H. 1H NMR (CDCl3) d 1.55 (s, 6H, CH3), 3.25
(s, 2H, CH2), 6.7 (s, 1H, CH), 6.8±8.1 (m, 6H, aromatic).
A yellow suspension of this compound, glacial acetic
acid (200 mL) and anhydrous sodium acetate (50 g) was
re¯uxed under nitrogen for 16 h. The reaction mixture
was cooled and water was added until no further white
precipitate was formed. The solid was collected by ®l-
tration to yield 10.2 g (60%) of product mp 290 ꢀC
(dec.) (EtOH). Anal. (C13H8O3S): C, H. 1H NMR
(CDCl3) (6.7 ds, 1H, CH-3), 7.5±8.4 (m, 6H, aromatic).
8,8-Dimethyl-2-thienyl-8,9-dihydrofuro-[2,3-h]-chromen-
4-one (11c). This compound was obtained in 55%
yield, mp 219±221 ꢀC (toluene). Anal. (C17H14O3S): C,
H. H NMR (CDCl3) d 1.55 (s, 6H, CH3), 3.25 (s, 2H,
CH2), 6.6 (s, 1H, CH), 6.8±8.1 (m, 5H, aromatic).
1
8,8-Dimethyl-2,3-diphenyl-8,9-dihydrofuro-[2,3-h]chromen-
4-one (11d). This compound was obtained in 55% yield,
mp 245±247 ꢀC (toluene). Anal. (C25H20O3): C, H. H
1
7-ꢀ-Methylallyl¯avone (9a). A mixture of 7-hydroxy-
¯avone (9.52 g, 0.04 mol), K2CO3 (7 g, 0.05 mol) and
methylallylchloride (7.2 g, 0.08 mol) in acetone (100 mL)
was re¯uxed, with stirring, for 24 h and hot ®ltered. The
solvent was evaporated and the residue was taken up in
CH2Cl2. The organic layer was washed with 5% NaOH
solution, H2O and dried (Na2SO4). Evaporation of the
solvent gave a residue which was crystallised from
EtOH to give 8.2 g (70%) of product mp 118±120 ꢀC.
Anal. (C19H16O3): C, H. 1H NMR (CDCl3) d 1.9 (s, 3H,
CH3), 4.55 (s, 2H, CH2), 5.05 (s, 1H, CH2), 5.15 (s, 1H,
CH2), 6.7 (s, 1H, CH), 6.95±8.15 (m, 8H, aromatic).
NMR (CDCl3) d 1.6 (s, 6H, CH3), 3.25 (s, 2H, CH2),
6.8±8.1 (m, 12H, aromatic).
9-Bromo-8,8-dimethyl-8,9-dihydro-2-phenylfuro-[2,3-h]-
chromen-4-one (13a).
A mixture of 11a (2.92 g,
0.01 mol), N-bromosuccinimide (1.78 g, 0.01 mol) and a
catalytic amount of benzoyl peroxide in CCl4 (50 mL)
was re¯uxed for 3 h and then hot ®ltered. The ®ltrate
was evaporated to dryness and the residue on crystal-
lising from toluene gave 1.8 g (50%) of product mp
218±220 ꢀC. Anal. (C19H15BrO3): C, H. 1H NMR
(CDCl3) d 1.55 (s, 3H, CH3), 1.85 (s, 3H, CH3), 5.7 (s,
1H, CHBr), 6.8 (s, 1H, CH), 6.9±8.2 (m, 7H, aromatic).
The same procedure was used to prepare the following
compounds.
The same procedure was used to prepare the following
compounds.
7-ꢀ-Methylallyl-40-¯uoro¯avone (9b). This compound
was obtained in 80% yield, mp 136±137 ꢀC (EtOH).
1
Anal. (C19H15FO3): C, H. H NMR (CDCl3) d 1.9 (s,
3H, CH3), 4.55 (s, 2H, CH2), 5.05 (s, 1H, CH2), 5.15 (s,
1H, CH2), 6.6 (s, 1H, CH), 6.95±8.15 (m, 7H, aromatic).
9-Bromo-8,8-dimethyl-8,9-dihydro-2-(4-¯uoro)phenylfuro-
[2,3-h]-chromen-4-one (13b). This compound was
obtained in 60% yield, mp 242±246 ꢀC (toluene). Anal.
1
(C19H14BrFO3): C, H. H NMR (DMSO-d6) d 1.5 (s,
3H, CH3), 1.7 (s, 3H, CH3), 5.2 (s, 1H, CHBr), 6.35 (s,
1H, CH), 7.0±8.3 (m, 6H aromatic).
7-ꢀ-Methylallyl-2-thienylchromone (9c). This compound
was obtained in 65% yield, mp 139±141 ꢀC (EtOH).
1
Anal. (C17H14O3S): C, H. H NMR (CDCl3) d 1.9 (s,
3H, CH3), 4.55 (s, 2H, CH2), 5.05 (s, 1H, CH2), 5.15 (s,
1H, CH2), 6.6 (s, 1H, CH), 6.95±8.2 (m, 6H, aromatic).
9-Bromo-8,8-dimethyl-8,9-dihydro-2-thienylfuro-[2,3-h]-
chromen-4-one (13c). This compound was obtained in
50% yield, mp 170±176 ꢀC (toluene). Anal. (C17H13
1
7-ꢀ-Methylallyl-2,3-diphenylchromone (9d). This com-
pound was obtained in 75% yield, mp 106±108 ꢀC
BrO3S): C, H. H NMR (DMSO-d6) (1.3 (s, 3H, CH3),
1.45 (s, 3H, CH3), 5.15 (s, 1H, CHBr), 6.8 (s, 1H, CH),
6.9±8.05 (m, 5H aromatic).
1
(EtOH). Anal. (C25H20O3): C, H. H NMR (CDCl3) d
1.9 (s, 3H, CH3), 4.55 (s, 2H, CH2), 5.1 (d, 2H, CH2),
6.95±8.2 (m, 13H, aromatic).
9-Bromo-8,8-dimethyl-8,9-dihydro-2,3-diphenylfuro-[2,3-h]-
chromen-4-one (13d). This compound was obtained in
55% yield, mp 197±200 ꢀC (toluene). Anal. (C25H19BrO3):
8,8-Dimethyl-2-phenyl-8,9-dihydrofuro-[2,3-h]-chromen-
4-one (11a). Compound 9a (1.46 g, 0.005 mol) was
heated at 230±240 ꢀC with 1.5 g of 2,6-dimethylphenol
for 8 h. After cooling the reaction mixture was taken up
in diethyl ether. The organic layer was washed with
10% NaOH solution, H2O, dried (Na2SO4) and evap-
orated to dryness. The residue on crystallizing from
ligroin a€orded 1.0 g (70%) of product mp 184±186 ꢀC.
1
C, H. H NMR (CDCl3) (1.55 (s, 3H, CH3), 1.9 (s, 3H,
CH3), 5.65 (s, 1H, CHBr), 6.9±8.3 (m, 12H aromatic).
2,2-Dimethyl-1,2-dihydro-6H-furo-[2,3-c]-xanthen-6-on-
1-carboxylic acid (5). A solution of 1-bromo-2,2-di-
methyl-1,2-dihydro-6H-furo-[2,3-c]-xanthen-6-one37 (14,
1.72 g, 0.005 mol) and CuCN (0.9 g, 0.01 mol) in DMF
(20 mL) was re¯uxed for 20 h under N2 atmosphere.
1
Anal. (C19H16O3); C, H. H NMR (CDCl3) (1.6 (s, 6H,

244
P. Valenti et al. / Bioorg. Med. Chem. 8 (2000) 239±246
After cooling, the reaction mixture was poured into
H2O. The separated solid was collected by ®ltration,
washed with H2O and dried. This solid was re¯uxed for
2 h with a mixture of AcOH (5 mL), H2SO4 (5 mL) and
H2O (5 mL). After cooling the reaction mixture was
poured into H2O. The separated solid was collected by
®ltration, washed with H2O and suspended in a satu-
rated NaHCO3 solution. After ®ltration the solution
was acidi®ed with dil HCl: the separated solid was col-
lected and crystallised from EtOH to give 0.31 g (20%)
(g) was re¯uxed for 2 h and then evaporated to dryness.
The residue was crystallised from EtOH to give 1.8 g
(95%) of 16: mp 155±158 ꢀC. Anal. (C19H14O2): C, H.
1H NMR (CDCl3) (3.8 (m, 2H, CH2CHˆCH2), 3.9 (s,
2H, CH2), 5.2 (m, 2H, CH2ˆCH-), 6.1 (m, 1H,
CH2ˆCH-), 7.4±8.25 (m, 7H, Ar).
(10-Oxo-10,11-dihydro-indeno-[1,2-b]-chromen-6-yl)ace-
tic acid (6). To a cold solution of 16 (1.37 g, 5 mmol) in
AcOH (20 mL), acetone (20 mL) and H2O (10 mL) solid
KMnO4 (1.58 g, 10 mmol) was added in portions in 6 h.
The reaction mixture was stirred for 1 h at room temp
and poured into Na2S2O8 solution. The separated solid
was collected by ®ltration and crystallised from EtOH to
give 1.02g (70%) of 6: mp 250 ꢀC (dec). Anal. (C18H12O4):
of product mp 268±270 ꢀC. Anal. (C18H14O5): C, H. H
1
NMR (DMSO-d6) d 1.5 (s, 3H, CH3), 1.6 (s, 3H, CH3),
4.3 (s, 1H, CH), 6.95±8.2 (m, 6H, aromatic). MS: m/z
(relative abundance): 310 (M+, 37), 265 (100), 31 (29).
1
The same procedure was used to prepare the following
compounds.
C, H. H NMR (DMSO-d6) d 3.8 (s, 2H, CH2COOH),
4.0 (m, 2H, CH2), 7.45±8.05 (m, 7H, Ar) MS: m/z (relative
abundance): 292 (M+, 100), 278 (20), 247 (18), 189 (19).
8,8-Dimethyl-8,9-dihydro-2-phenylfuro-[2,3-h]-chromen-
4-on-9-carboxylic acid (4a). This compound was
obtained in 30% yield, mp 198±202 ꢀC (EtOH/H2O).
Cell lines
1
Anal. (C20H15O5): C, H. H NMR (DMSO-d6) d 1.5 (s,
The human colon adenocarcinoma cells LoVo R (doxo-
rubicin resistant and Multidrug Resistant cells) and
LoVo S (sensitive cells), kindly supplied by the Centro
di Riferimento Oncologico (Aviano, Pordenone-Italy),
were cultured in Ham F 12, plus 10% heat-inactivated
fetal calf serum, 1% antibiotics (all products of Bio-
chrom KG Seromed) and 1% 200 mM glutamine
(Merck). For LoVo R cells the medium was supple-
mented by 100 ng/mL of doxorubicin.
3H, CH3), 1.6 (s, 3H, CH3), 4.4 (s, 1H, CHCOOH), 6.95
(s, 1H, CH), 7.0±8.05 (m, 7H, aromatic). MS: m/z
(relative abundance): 336 (M+, 56), 291 (100), 292 (34).
8,8-Dimethyl-8,9-dihydro-2-(40-¯uoro)phenylfuro-[2,3-h]-
chromen-4-on-9-carboxylic acid (4b). This compound
was obtained in 30% yield, mp 260±262 ꢀC (EtOH).
Anal. (C20H14FO5): C, H. 1H NMR (DMSO-d6) d 1.5 (s,
3H, CH3), 1.65 (s, 3H, CH3), 4.45 (s, 1H, CHCOOH),
6.95 (s, 1H, CH), 7.0±8.1 (m, 6H, aromatic). MS: m/z
(relative abundance): 354 (M+, 76), 309 (100), 296 (72).
The human ovarian adenocarcinoma cell line 2008 and
the cis-DDP-resistant subline C13*, kindly supplied by
Professor G. Marverti, (Department of Biomedical
Sciences, University of Modena) were maintained in
RPMI 1640 supplemented with 10% heat-inactivated
FCS, 1% antibiotics (all products of Biochrom KG
Seromed) and 2 mM l-glutamine (Merck).
8,8-Dimethyl-8,9-dihydro-2-thienylfuro-[2,3-h]-chromen-
4-on-9-carboxylic acid (4c). This compound was
obtained in 25% yield, mp 151±153 ꢀC (EtOH). Anal.
1
(C18H14O5S): C, H. H NMR (DMSO-d6) d 1.5 (s, 3H,
CH3), 1.65 (s, 3H, CH3), 4.35 (s, 1H, CHCOOH), 6.85
(s, 1H, CH), 7.0±8.0 (m, 5H, aromatic). MS: m/z (rela-
tive abundance): 342 (M+, 38), 297 (64), 58 (100).
Direct cytotoxicity
Tetrazolium salts assay (MTT). The cells were seeded in
96-well tissue plates (Falcon) and treated 24 h later with
each agent at di€erent concentrations. Viable cell
growth was determined by MTT reduction assay after
24 h of incubation.50
8,8-Dimethyl-8,9-dihydro-2,3-diphenylfuro-[2,3-h]-chromen-
4-on-9-carboxylic acid (4d). This compound was
obtained in 40% yield, mp 292±294 ꢀC (EtOH). Anal.
(C26H20O5): C, H. H NMR (DMSO-d6) d 1.5 (s, 3H,
CH3), 1.6 (s, 3H, CH3), 4.4 (s, 1H, CHCOOH), 7.0±8.0
(m, 12H, aromatic). MS: spectrum not registered.
1
Twenty microlitres of MTT solution (5 mg/mL in PBS)
were added to each well, and plates were incubated for
4 h at 37 ꢀC. DMSO (150 mL) was added to all wells and
mixed thoroughly to dissolve the dark blue crystals. The
absorbance was measured on a micro-culture plate
reader (Titertek Multiscan) using a test wavelength of
570 nm and a reference wavelength of 630 nm.
(3-Allyl-2-hydroxyphenyl)-(3-hydroxy-1H-inden-2-yl)-meth-
anone (15). To a mixture of 2-hydroxy-3-allylacetoph-
enone (4.27 g, 24 mmol) and o-phthalaldehyde (3.22 g,
24 mmol) in EtOH (45 mL) 50% KOH solution (10 mL)
was added dropwise. The reaction mixture was stirred
for 12 h, poured into ice and acidi®ed with dil HCl. The
separated solid was collected by ®ltration and crystallised
from EtOH to give 2.12g (30%) of 15: mp 134±136 ꢀC.
Indirect cytotoxicity
1
Anal. (C19H16O3): C, H. H NMR (CDCl3) d 3.5 (m,
The ability of the new derivatives to stimulate mouse
macrophages in culture to become tumoricidal was
evaluated using resident peritoneal macrophages.51
2H, CH2CHˆCH2), 4.05 (s, 2H, CH2), 5.15 (m, 2H, CH2
ˆCH-), 6.05 (m, 1H, CH2ˆCH-), 6.95±7.9 (m, 7H, Ar).
6-Allyl-11H-indeno-[1,2-b]-chromen-10-one (16). A solu-
tion of 15 (2 g, 7 mmol) in MeOH saturated with HCl
Resident peritoneal cells were isolated by two injections
of 5 mL PBS containing 10 U/mL of eparin into the

P. Valenti et al. / Bioorg. Med. Chem. 8 (2000) 239±246
245
peritoneal cavity. The cavity was gently massaged for
2 min and the cells removed by drawing ¯uid out with a
syringe.52 The recovered cell suspension was centrifuged
and the pellet was washed twice in sterile PBS. The cells
were then re-suspended in RPMI 1640 plus 5% FCS
and plated in a culture ¯ask left to adhere at 37 ꢀC.
After 2 h medium and non-adherent cells were dis-
carded, the ¯ask washed with sterile PBS, cells adhering
(macrophages) were resuspended, centrifuged, counted
using 0.5% Trypan blue, re-suspended in complete
medium and then plated in 96-well plates (Falcon) at a
concentration of 1Â104 cells/well in presence of di€erent
concentrations of FAA and analogues, using triplicate
wells per drug dose. After 24 h the medium was dis-
carded and the C13* (2Â103 cells/well) cells were plated
as above. The optimal macrophages/C13* cells ratio has
been determined in preliminary experiments (results not
reported). The cells were co-cultivated for 24 h. Lysis of
C13* cells was assessed by MTT test53 and the percen-
tages of speci®c cytotoxicity were calculated as follows:
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OD ꢀmacrophages ‡ C13Æ OD ꢀmacrophages†
OD ꢀC13Æ
Statistical analysis. For each assay three di€erent
experiments were performed in triplicate. The results
were statistically evaluated by Student's t-test.54 The
IC50, 95% con®dence limits and the potency ratio
between FAA and each analogue (IC50FAA/IC50der-
ivative) were estimated using the Litch®eld and Wil-
coxon method.54
Acknowledgement
28. Rewcastle, G. W.; Atwell, G. J.; Zhuang, L.; Baguley, B.
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D. W.; Baguley, B. C.; Denny, W. A. J. Med. Chem. 1991, 34,
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This work was supported by a grant from Ministero
dell'Universita e della Ricerca Scienti®ca e Tecnologica
(MURST).
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