Synthesis and antitumor activity of new derivatives of xanthen-9-one-4-acetic acid

Article abstract of DOI:10.1021/jm020929p

Xanthen-9-one-4-acetic acid (XAA) analogues in which the substituents in positions 5 and 6 are included in cyclic structures are described. Direct in vitro toxicity of the synthesized compounds against four tumor cell lines was evaluated, and their abilit

Full text of DOI:10.1021/jm020929p

J . Med. Chem. 2002, 45, 4931-4939
4931
Syn th esis a n d An titu m or Activity of New Der iva tives of Xa n th en -9-on e-4-a cetic
Acid
Silvia Gobbi,† Angela Rampa,† Alessandra Bisi,† Federica Belluti,† Piero Valenti,*,† Anna Caputo,‡
Antonella Zampiron,‡ and Maria Carrara‡
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy, and Department of
Pharmacology and Anaesthesiology, University of Padova, Largo Meneghetti 2, 35131 Padova, Italy
Received May 7, 2002
Xanthen-9-one-4-acetic acid (XAA) analogues in which the substituents in positions 5 and 6
are included in cyclic structures are described. Direct in vitro toxicity of the synthesized
compounds against four tumor cell lines was evaluated, and their ability to stimulate mouse
peritoneal macrophages and human monocytes in culture to become tumoricidal (indirect
toxicity) was also studied. Despite low direct toxicity, almost all the compounds proved to be
able to significantly enhance the lytic properties of both murine macrophages and human
monocytes as well as the parent compound XAA and its most active derivative DMXAA taken
as reference. In particular, compounds 4a , 5a , 7a , 13a ,b, and 16a ,b showed higher activity
than the lead compound on human monocytes, compound 7a being 2.5 times more active than
DMXAA, which is the most potent compound synthesized so far. Moreover, compounds 4a , 5a ,
7a , 13a , 16a , and 16b proved to be able to induce TNF production in human immune cells.
Ch a r t 1. Reference Compounds
In tr od u ction
Flavone-8-acetic acid1 (FAA, 1; Chart 1) is a synthetic
flavonoid that was identified as a lead anticancer
compound among a broad series of molecules evaluated
for antitumor activity on several murine models for
some peculiar features. Actually, its main characteris-
tics were low activity against fast-growing tumors such
as leukemia, but striking, broad activity against slow-
growing solid tumors2 mostly insensitive to cytotoxic
drugs, and absence of myelosuppression, usually ob-
served with conventional antitumor agents.3 On the
other hand, its potency appeared to be low, since high
doses and long exposure times were required for a direct
cytotoxic effect.4 On this basis, FAA was selected for
further evaluation, but unfortunately the good activity
seen both in in vitro and in vivo experiments was not
confirmed in clinical trials.5
The unique pharmacological profile of 1 suggested an
indirect effect of the drug, rather than direct toxicity,
involving both a vascular component and the host
immune system. FAA was shown to induce reduction
of tumor blood flow and rapid hemorrhagic necrosis6,7
due to vascular collapse in subcutaneous tumors. More-
over, this drug is now believed to act as a biological
response modifier, since it stimulates the activity of NK
cells8 and enhances the lytic potential of macrophages
in mice.9 These effects are known to be mediated by the
induction of different cytokines10,11 such as tumor
necrosis factor R (TNF-R) and interferons. There is also
evidence for a dose-dependent increase in plasma nitrite
plus nitrate (NO2-/NO3-) concentrations in mice follow-
ing administration of FAA and related drugs,12 which
could contribute to tumor cell death by alteration of
blood flow and direct cell killing.
Structurally related to FAA is xanthen-9-one-4-acetic
acid13 (XAA, 2; Chart 1), where the xanthenone moiety
can be regarded as a “fused” flavone. XAA showed
higher potency than the parent compound and was
taken as a new lead for the development of new
derivatives. Structure-activity relationships of this
molecule were amply investigated, and from studies on
mono- and disubstituted XAA analogues,14-16 it was
concluded that activity was primarily dependent on the
position of the substituents rather than on their nature,
positions 5 and 6 being the most favorable for substitu-
tion, especially with small lipophilic groups.
5,6-Dimethyl-xanthen-9-one-4-acetic acid (DMXAA, 3;
Chart 1) proved to be the most potent XAA derivative,
being able to induce cytokines (especially TNF) in
human cells, an effect that was not seen with FAA.17,18
DMXAA was then selected for further evaluation, and
phase I clinical trials have now been completed.19
Extensive studies have been carried out to elucidate
the mechanism of action of FAA at the molecular level.
It was shown20 that FAA and analogues can be decar-
boxylated by radicals (among them nitrogen oxides)21
via one-electron oxidation leading to a benzyl type
radical, which would rapidly react with oxygen to give
peroxyl radicals, inducing cellular damage. Recent stud-
ies postulate a role for oxidative stress in the activity
of this drug,22 while its biological target is still under
investigation.23
* To whom correspondence should be addressed. Phone: +39 051
2099701. Fax: +39 051 2099734. E-mail: pvalenti@alma.unibo.it.
† University of Bologna.
‡ University of Padova.
10.1021/jm020929p CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/24/2002

4932 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22
Ch a r t 2. Structures of Target Compounds
Gobbi et al.
Sch em e 1a
a
Reagents: (i) Cu, CuI, K2CO3, pyridine, reflux, 2h; (ii) H2SO4,
100 °C, 1 h; (iii) NaOMe, dioxane, reflux, 100 h; (iv) NBS, benzoyl
peroxide, CCl4, reflux, 5 h; (v) NaCN, EtOH-H2O, reflux, 20 h;
(vi) AcOH, H2O, H2SO4, reflux, 2 h; (vii) pyridine‚HCl, 220-230
°C, 2.5 h; (viii) MeOH, H+, reflux, 12 h.
methoxyxanthen-9-one (9) was prepared by heating the
6-chloro derivative13 with NaOMe in dioxane. Monobro-
mination with N-bromosuccinimide using benzoyl per-
oxide as catalyst in CCl4 gave the bromomethyl deriva-
tive, which was converted to cyanide and hydrolyzed to
the desired xanthen-9-one-4-acetic acid (10).13 This
compound was demethylated to 11 by heating to 220-
230 °C with pyridine hydrochloride and finally refluxed
in MeOH in the presence of acid to afford 12.
The final compounds were then prepared as shown
in Scheme 2. The key intermediate 12 was treated with
the selected alkyl halide to afford 13a , 14a , 15a , and
16a . These esters were hydrolyzed to the corresponding
acids 13b, 14b, and 16b (compound 15b could not be
obtained pure enough to be characterized and tested)
or cyclized by heating, under different conditions, to
afford 4a , 5a , and 6a , which were then also hydrolyzed
to 4b, 5b, and 6b. Only 16a did not cyclize under any
of the conditions used, and compounds 7 and 8 were
prepared using an alternative route. Compound 12 was
heated in pyridine with 3-methyl-2-butenal to afford 7a ,
which was hydrolyzed to 7b or hydrogenated (Pd/C) to
give 8a , which was then hydrolyzed to 8b.
Continuing our SAR studies on this class of antitumor
compounds,24 in this paper we describe the synthesis
of new XAA derivatives in which the substituents in
positions 5 and 6 were included in cyclic structures, both
saturated and unsaturated, to further investigate struc-
tural requirements and steric limitations in this part
of the molecule. Since substitution with methoxy groups
still maintained the same activity seen for 3,12 different
oxygenated cycles, synthetically more convenient than
nonoxygenated ones, were introduced in the lead struc-
ture. The synthetic intermediates, bearing different
bulky alkoxy groups in position 6 of the xanthenone
nucleus (“open intermediates”), were also taken into
account and tested for their antitumor activity, since
this position is considered one of the most favorable for
substitution. Structures of the synthesized compounds
are shown in Chart 2.
Biologica l Assa ys
Since one of the peculiar features of the parent
compound flavone-8-acetic acid was its remarkable
preferential activity on solid tumors, all the target
compounds 4-8a ,b and 13-16a ,b were tested for direct
cytotoxicity in a preliminary in vitro assay against four
human tumor cell lines: LoVo S and LoVo R, arising
from human colon adenocarcinoma, and 2008 and C13*,
arising from human ovarian adenocarcinoma. LoVo R
cells differ from the parental line (LoVo S) 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,
they show reduced cell membrane permeability to
passive diffusion25 and mitocondrial membrane func-
tionality.26
Ch em istr y
The key intermediate for the synthesis was the
methyl ester of 6-hydroxyxanthen-9-one-4-acetic acid
(12). This compound was prepared as outlined in
Scheme 1 following standard procedures. 4-Methyl-6-
The antitumor effects shown by both flavone-8-acetic
acid and 5,6-dimethylxanthen-9-one-4-acetic acid seem

Derivatives of Xanthen-9-one-4-acetic Acid
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22 4933
Sch em e 2a
a
Reagents: (i) R′X, K2CO3, reflux, 24 h; (ii) KOH, reflux, 4 h; (iii) 230-240 °C, 2,6-dimethylphenol, 8 h; (iv) 250-260 °C, 3 h; (v) CsF,
N,N-diethylaniline, reflux, 4 h; (vi) 3-methyl-2-butenal, pyridine, 140 °C, 9 h; (vii) H2, Pd/C.
to deeply involve the immune system by different
pathways, including stimulation the activity of NK cells,
increase in macrophage-mediated cytotoxicity, and in-
duction of different cytokines. Thus, it was of primary
importance to evaluate the indirect effects of the syn-
thesized compounds to fully understand the biological
meaning of the structural changes that had been
introduced. Cytotoxicity induced on C13* cells cocul-
tured with murine macrophages pretreated with the
new compounds was therefore measured to test the new
derivatives for their ability to improve lytic properties
of those immune cells. Since studies performed in order
to evaluate the effects of DMXAA on the human immune
system gave stimulating results,27 the ability of the
synthesized compounds to enhance the lytic potential
of human monocytes was also assessed. This model
appeared particularly interesting because of the pres-
ence in these cells of the TLR2 receptor, which is
involved in antiinflammatory cytokine activation,28 and
also because of the evidence of the role played by
cytokines in the antitumor effects of this class of drugs.27
Tumor necrosis factor R (TNF -R) appears to play a
pivotal role in the antitumor effects of DMXAA in mice,
and a strong correlation was observed between elevation
of serum TNF and antitumor potential.29 Moreover,
Philpott’s results27 suggested that in vitro analysis of
the response of human peripheral blood leucocytes to
DMXAA could be a useful indicator of the activity of
this class of agents. Therefore, the effects of some of the
new compounds, i.e., 4a , 5a , and 7a among cyclic
compounds and 13a , 16a and 16b among 6-alkoxy
analogues, on TNF production by human peripheral
blood mononuclear cells (HPBMC) were investigated.
The responses obtained with these agents were also
compared to those obtained with LPS, a known inducer
of TNF synthesis.30
Resu lts a n d Discu ssion
Dir ect Toxicity. The cytotoxic effect of the tested
compounds on the selected tumor cell lines (LoVo S,
LoVo R, 2008, and C13*) was generally quite low, as
expected from the results obtained with DMXAA, taken
as reference. Both the reference compound DMXAA and
the new derivatives (except for 4b, 6a ,b, 16a ,b, which
resulted inactive) were able to inhibit cell growth in a
dose-dependent manner. However, at least 250 µM was
necessary to obtain significant cytotoxic activity, 4a
being the only active compound at 100 µM. The dose
requested against C13* cells was even higher (500 µM).
No other significant difference was seen in the activity
against the selected cell lines (data not shown). From
these data, it can be concluded that direct toxicity could

4934 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22
Gobbi et al.
Ta ble 1. Cytotoxicity on C13*cells, Toxicity Induced in Murine Macrophages, and Human Monocytes of 6-Alkoxy Derivatives (PR )
Potency Ratio)
murine
macrophages,
IC50 (µM)
human
monocytes
IC50 (µM)
C*13 cells,
IC50 (µM)
PR vs
DMXAA
PR vs
macrophages
compound
DMXAA
PR vs DMXAA
1
614.7
211.4
134.6
1
1.6a
(511.1-805.7)
(155.4-287.6)
(105.0-172.6)
13a
13b
14a
14b
15a
16a
16b
488.2
b
84.6
1.6a
1.6a
0.8
(384.2-620.4)
443.1
(347.8-564.5)
508.6
(382.6-676.0)
510.4
(419.3-611.2)
410.2
(362.1-465.1)
702.5
(538.6-916.4)
626.1
(67.8-105.7)
86.2
(69.2-107.3)
113.8
(92.9-139.5)
b
77.3
(57.5-104.1)
61.6
(36.8-103.1)
217.6
(142.8-331.6)
371.8
(256.4-539.0)
223.8
(168.3-297.6)
264.1
2.7a
3.4a
1.0
0.6
1.0
0.8
0.9
0.5c
b
73.6
(55.0-98.6)
64.2
1.8a
2.1a
3.0a
4.1a
(475.7-816.2)
(211.9-329.1)
(48.4-85.3)
a
b
More potent. Not detected. c Less potent.
Ta ble 2. Cytotoxicity on C13*cells, Toxicity Induced in Murine Macrophages, and Human Monocytes of Cyclic Derivatives (PR )
Potency Ratio)
murine
macrophages,
IC50 (µM)
human
monocytes,
IC50 (µM)
C*13 cells,
IC50 (µM)
PR vs
DMXAA
PR vs
macrophages
compound
DMXAA
PR vs DMXAA
1
614.7
(511.1-805.7)
548.7
(442.0-681.3)
603.9
(446.2-817.5)
211.4
(155.4-287.6)
201.3
(81.6-496.4)
219.5
(77.2-62.0)
82.8
(59.1-116.1)
157.2
(113.6-217.6)
80.9
(56.7-115.4)
42.0
(23.2-76.1)
30.5
134.6
(105.0-172.6)
73.7
(60.2-90.2)
172.3
(131.4-225.8)
67.3
(56.2-80.7)
177.9
(126.7-241.0)
1
1.6a
2.7a
1.3
4a
4b
5a
5b
6a
6b
7a
7b
8b
1.0
1.8a
0.8
2.0a
0.7
0.9
b
2.5a
1.3
1.2
711.4
(509.0-994.2)
0.9
b
2.6a
5.0a
6.9a
4.7a
2.9a
b
b
b
b
53.5
(46.0-62.2)
258.6
(138.1-484.3)
107.9
2.5a
0.5c
1.2
0.6
0.2c
0.7
(16.5-56.5)
45.1
(29.5-69.0)
71.5
725.8
(543.4-969.3)
b
(46.5-109.9)
(81.4-143.2)
a
b
More potent. Not detected. c Less potent.
be obtained with these compounds only with doses that
are much higher than the usual doses of chemothera-
peutic drugs.
The new compounds showed activity comparable to or
higher than the reference compound (compounds 13b
and 14a , 2.7 and 3.4 times, respectively). In particular,
it can be seen that insertion of an unsaturated, linear
or branched alkoxy chain led to an increase of activity
but the presence of a triple bond was less favorable,
since compound 15a showed slightly lower activity than
DMXAA. For all compounds except 14b and 15a , the
activity was maintained on human monocytes, deriva-
tives 13a ,b and 16a ,b with branched unsaturated side
chains proved to be 1.6, 1.6, 1.8, and 2.1 times more
potent than DMXAA, respectively. Going from murine
macrophages to human monocytes, the activity seemed
to be lost for compounds 14b and 15a , but it is
interesting to note that compound 13a showed higher
activity than DMXAA in the human model while it
proved to be inactive on murine macrophages.
In dir ect Toxicity. Tu m or icidal Activity. The evalu-
ation of indirect cytotoxic effects on C13* cells cocultured
with macrophages or monocytes provided more interest-
ing results, which are summarized in Table 1 for the
6-alkoxy derivatives 13-16a ,b and in Table 2 for the
cyclic compounds 4-8a ,b, taking in both cases DMXAA
as the reference compound. Generally speaking, a
remarkable increase in antitumor activity in comparison
to direct cytotoxicity was seen with all tested com-
pounds. It should also be noted that doses could be
reduced to 50% (i.e., 25, 50, and 100 µM) when testing
the compounds on cells cocultured with human mono-
cytes with respect to murine macrophages.
Considering the 6-alkoxy derivatives (13-16a,b, Table
1), both DMXAA and the new derivatives proved to be
able to significantly enhance the lytic properties of
murine macrophages and showed an important increase
in antitumor activity with respect to their direct toxicity.
All cyclic compounds (4-8a ,b, Table 2) showed re-
markable activity on murine immune cells, generally
higher than their direct toxicity. Compounds bearing
an ester function seemed to be more active than the

Derivatives of Xanthen-9-one-4-acetic Acid
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22 4935
corresponding acids, and both five- and six-membered
rings seemed to lead to increased activity. In particular,
compounds 6b and 7a proved to be the most active
derivatives, being 5.0 and 6.9 times more active than
DMXAA, respectively. Interestingly, compounds 5a , 6a ,
6b, 7a , and 8b were able to induce indirect toxicity, even
though no direct cytotoxicity was detected. Activity on
human monocytes was maintained for almost all tested
derivatives except 6a ,b, which proved to be inactive. The
introduction of a cyclic moiety therefore seemed to be
favorable for activity on human immune cells also.
Compounds 4a , 5a , and 7a seem to be of particular
interest, being 1.8-, 2.0-, and 2.5-fold more active than
DMXAA, respectively.
The results obtained can be summarized as follows.
(1) All the new compounds, except for 13a , were able to
enhance the lytic properties of murine macrophages. In
particular, the introduction of a five- or six-membered
ring seemed to be the most favorable, since almost all
the compounds in this series were significantly more
active than DMXAA, particularly compounds 6a ,b, 7a ,b,
and 8b, whereas most of them did not show any direct
cytotoxicity versus C*13 cells. Among 6-alkoxy deriva-
tives, 13b and 14a proved to be 2.7 and 3.4 times more
active than the reference compound.
(2) Most of the new derivatives except for 6a ,b, 14b,
and 15a showed significant toxicity on C*13 cells co-
cultured with human monocytes. Among 6-alkoxy deriv-
atives, 13a ,b and 16a ,b with branched side chains
proved to be 1.6, 1.6, 1.8, and 2.1 times more active than
DMXAA, respectively. Considering the cyclic deriva-
tives, it should be noted how esters seemed to be more
active than free acids, since compounds 4a , 5a , and 7a
showed activity up to 2.5 times higher than DMXAA;
however, this could partly be due to better cell uptake
of these molecules with respect to the corresponding free
acids.
F igu r e 1. Effect of DMXAA and analogues tested at different
concentrations on TNF-R production by human monocytes. The
first two bars represent medium from unstimulated cells.
(3) By comparison of the activities on murine mac-
rophages and human monocytes, a certain degree of
species specificity could be noted. In particular, com-
pound 13a , bearing a branched side chain, was able to
stimulate the lytic potential of human immune cells
while it did not show any such activity on murine
macrophages. Compound 4a , with a saturated five-
membered ring, and 16a ,b, also with a branched side
chain, also proved to be respectively 2.7, 3.0, and 4.1
times more active on human cells with respect to murine
cells. On the other hand, compounds 6a ,b, with an
unsaturated five-membered ring, and 14b and 15a , with
a linear side chain, were active only on murine cells.
in response to 13a were 60% of those obtained in res-
ponse to LPS (59.5 pg/mL versus 100.2 pg/mL). LPS is
known to form complexes with LPS binding proteins
(LBP), which interact with the CD14 receptor, a mem-
brane-bound glycoprotein32 linked to the TNF and IL-1
kinase cascades by the TLR2 protein on the plasma
membrane,28,33 inducing NF-κB activity.28 Because TNF
is synthesized by human peripheral blood mononuclear
cells in response to the tested compounds, it is possible
that the activation pathways are the same for the two
agents.
Tu m or Necr osis F a ctor r (TNF -r) P r od u ction .
The effects of the selected compounds (13a , 16a ,b, 4a ,
5a , 7a ), tested at three different concentrations, on
TNF-R production by HPBMC are shown in Figures 1
and 2. The results showed that no significant cytokine
production was stimulated by DMXAA alone, as ex-
pected from Philpott’s results,31 at any of the concentra-
tions tested. On the contrary, although the response of
HPBMC to the new derivatives was at all times lower
than the response to LPS, after 4 h of incubation the
levels of TNF-R obtained in response to 100 µM of all
tested compounds (and especially 13a ) were signifi-
cantly larger than the level of the negative control
(Figure 1). In particular, the levels of TNF-R obtained
Con clu sion s
A new series of derivatives of the drug xanthen-9-one-
4-acetic acid (XAA), bearing a rather large lypophilic
substituent in position 6 or a cyclic moiety in positions
5 and 6, was synthesized. The compounds were tested
both for direct cytotoxicity on four human tumor cell
lines and for indirect activity, evaluating their ability
to stimulate mouse peritoneal macrophages and human
monocytes in culture to become tumoricidal. Despite
quite low direct toxicity, almost all the compounds were
able to induce an increase in the lytic properties of
murine macrophages in the same way as that for the
reference compound DMXAA. Most of the new deriva-
tives showed remarkable activity on human monocytes

4936 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22
Gobbi et al.
4-Meth yl-6-m eth oxyxa n th en -9-on e (9). A mixture of 2,4-
dichlorobenzoic acid (85 g, 0.4 mol), o-cresol (104 g, 0.8 mol),
K2CO3 (110.4 g, 0.8 mol), pyridine (15.8 g, 0.2 mol), Cu (2 g),
and CuI (2 g) in water (250 mL) was refluxed for 2 h. The
reaction mixture was basified with Na2CO3 and washed with
ether and then acidified with HCl, and the resulting precipitate
was filtered and resuspended in dilute NaOH. The solution
was filtered and acidified with acetic acid. The separated solid
was then collected by filtration and crystallized from MeOH/
H2O to give 73.5 g (70%) of 4-chloro-2-(2-methylphenoxy)-
benzoic acid, mp 151-154 °C (lit. mp13 158-159 °C). 1H NMR
(CDCl3): δ 2.2 (s, 3H, CH3), 6.6-8.2 (m, 7H, aromatic).
The compound (70 g, 0.27 mol) was then added portionwise
to concentrated H2SO4 (700 mL), and the solution was heated
to 100 °C for 1 h. The reaction mixture was then poured into
ice and the separated solid was filtered, washed with water,
dried, and crystallized from EtOH to give 39.5 g (60%) of
4-methyl-6-chloroxanthen-9-one, mp 148-149 °C (lit. mp13
1
145-146 °C). H NMR (CDCl3): δ 2.5 (s, 3H, CH3), 7.3-8.25
(m, 6H, aromatic).
A suspension of the above 4-methyl-6-chloroxanthen-9-one
(20 g, 0.08 mol) in dioxane (400 mL) was added to a solution
of Na (38 g) in MeOH (500 mL), and the mixture was refluxed
for 100 h. After removal of the solvent, the residue was taken
up in water and filtered to give 18.2 g (95%) of 9, mp 152-
155 °C (EtOH) (lit. mp13 151-152 °C). 1H NMR (CDCl3): δ
2.5 (s, 3H, CH3), 3.95 (s, 3H, CH3O), 6.9-8.25 (m, 6H,
aromatic). Anal. (C15H12O3) C, H.
6-Meth oxyxa n th en -9-on e-4-a cetic Acid (10). A mixture
of 9 (10 g, 0.04 mol), N-bromosuccinimide (7.6 g, 0.04 mol),
and a catalytic amount of benzoyl peroxide in CCl4 (300 mL)
was refluxed under intense illlumination for 5 h. The mixture
was then hot-filtered, and the precipitate was filtered and
crystallized from toluene to give 10 g of 4-(bromomethyl)-6-
methoxyxanthen-9-one (82%), mp 177-180 °C. 1H NMR
(CDCl3): δ 3.95 (s, 3H, CH3O), 4.85 (s, 2H, CH2Br), 6.95-8.4
(m, 6H, aromatic).
To a solution of this compound (4 g, 13 mmol) in EtOH (400
mL) NaCN (0.66 g, 13 mmol) in water (10 mL) was added,
and the mixture was refluxed for 20 h. After evaporation of
the solvent, the residue was resuspended in water and
extracted with CH2Cl2. The organic layer was then dried over
Na2SO4 and was evaporated to dryness, and the resulting solid
was crystallized from toluene to give 2.75 g (80%) of 6-meth-
oxyxanthen-9-one-4-acetonitrile, mp 208-210 °C. 1H NMR
(CDCl3): δ 3.95 (s, 3H, CH3O), 4.05 (s, 2H, CH2CN), 6.9-8.35
(m, 6H, aromatic).
F igu r e 2. Effect of DMXAA and analogues tested at different
concentrations on TNF-R production by human monocytes. The
first two bars represent medium from unstimulated cells.
as well, compounds 13a ,b and 16a ,b, with branched side
chains, and 4a , 5a , and 7a , esters of cyclic compounds,
being significantly more potent than DMXAA. Since the
antitumor effects of DMXAA are known to be associated
with TNF production and since the TLR2 receptor for
the induction of inflammatory cytokines was found in
lymphoid tissues with the highest expression in periph-
eral blood leukocytes,28 studies were carried out to
investigate if compounds 4a , 5a , 7a , 13a , 16a , and 16b
were able to induce TNF production in HPBMC cul-
tures. For all tested compounds, a significant response
could be noticed after 4 h of incubation. These com-
pounds proved to be particularly promising and were
therefore selected for further evaluation.
The above acetonitrile (1.5 g, 6 mmol) was dissolved in a
mixture of acetic acid (6 mL), water (6 mL), and concentrated
H2SO4, and the mixture was refluxed for 2 h. Water was then
added, and a precipitate was formed, which was filtered and
dissolved in aqueous NaHCO3. The solution was filtered and
acidified with HCl, and the separated solid was filtered, dried,
and crystallized from EtOH to give 1.25 g (73%) of 10, mp 196-
1
197 °C (lit. mp13 205-207 °C). H NMR (DMSO-d6): δ 3.95 (s,
3H, CH3O), 4.0 (s, 2H, CH2COOH), 6.95-8.1 (m, 6H, aromatic).
Anal. (C16H12O5) C, H.
6-Hyd r oxyxa n th en -9-on e-4-a cetic Acid (11). A mixture
of 6-methoxyxanthen-9-one-4-acetic acid13 10 (1 g, 3.5 mmol)
and dry pyridine hydrochloride was heated to 210-220 °C for
2.5 h. After cooling, the reaction mixture was poured into H2O
and the separated solid was collected by filtration and dried
to give 0.85 g (90%) of solid, mp 283-285 °C (EtOH) (lit. mp
303-305 °C). 1H NMR (DMSO-d6): δ 3.95 (s, 2H, CH2COOH),
6.9-8.05 (m, 6H, aromatic), 11.0 (s, 1H, OH). Anal. (C15H10O5)
C, H.
Exp er im en ta l Section
Ch em istr y. Gen er a l Meth od s. All melting points were
determined in open glass capillaries using a Bu¨chi apparatus
and are uncorrected. 1H NMR spectra were recorded in
solution on a Varian Gemini 300 spectrometer with Me4Si as
the internal standard. Mass spectra were recorded on a V.G.
7070 E spectrometer. Silica gel (Merck, 230-400 mesh) was
used for purification with flash chromatography. Wherever
analyses are only indicated with element symbols, analytical
results obtained for those elements are within 0.4% of the
theoretical values. The names of compounds were obtained
using AUTONOM, PC software for nomenclature in organic
chemistry from Beilstein-Institut and Springer.
Meth yl 6-Hyd r oxyxa n th en -9-on e-4-a ceta te (12). A solu-
tion of 11 (0.65 g, 2.4 mmol) in MeOH (20 mL) and some drops
of H2SO4 was refluxed for 12 h and then was evaporated to
dryness. The residue, on crystallizing from MeOH, afforded
1
0.5 g (70%) of product, mp 239-240 °C. H NMR (DMSO-d6):
δ 3.65 (s, 3H, CH3), 4.0 (s, 2H, CH2COO-), 6.9-8.5 (m, 6H,
aromatic), 11.0 (s, 1H, OH). Anal. (C16H12O5) C, H.

Derivatives of Xanthen-9-one-4-acetic Acid
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22 4937
Gen er a l P r oced u r e for th e Syn th esis of Meth yl-6-
a lk yloxyxa n th en -9-on e-4-a ceta tes (13a -16a ). A mixture
of 12 (5 mmol), K2CO3 (6 mmol), and the selected alkyl halide
(7 mmol) in acetone was refluxed for 24 h and was hot-filtered.
The solvent was then evaporated, and the residue was dis-
solved in CH2Cl2, washed with 5% NaOH solution and with
H2O, dried over Na2SO4, and evaporated to dryness. The
resulting solid was crystallized from toluene.
13a : starting from 12 and 3-chloro-2-methylpropene, yield
60%, mp 85-87 °C. 1H NMR (CDCl3): δ 1.9 (s, 3H, CH3), 3.75
(s, 3H, COOCH3), 4.0 (s, 2H, CH2COOH), 4.6 (s, 2H, CH2O),
5.1 (s, 1H), 5.2 (s, 1H) [CH2d], 6.9-8.3 (m, 6H, aromatic). 13C
NMR (CDCl3): δ 19.4, 35.2, 52.2, 72.2, 101.1, 113.4, 113.6,
115.6, 122.0, 123.2, 123.4, 125.9, 128.1, 135.5, 139.6, 154.1,
157.4, 164.1, 171.0, 175.0. MS m/z (rel abundance): 338 (M+,
9.78), 91 (100), 56 (53.30). Anal. (C20H18O5) C, H.
14a : starting from 12 and allyl bromide, yield 95%, mp 97-
100 °C. 1H NMR (CDCl3): δ 3.75 (s, 3H, COOCH3), 4.0 (s, 2H,
CH2COOH), 4.7 (m, 2H, CH2OAr), 5.4 (m, 2H, CH2dCH), 6.1
(m, 1H, CH2dCH-CH2), 6.9-8.25 (m, 6H, aromatic). 13C NMR
(CDCl3): δ 35.2, 52.1, 69.2, 101.0, 113.6, 115.5, 118.3, 121.9,
123.1, 123.4, 125.8, 128.1, 131.9, 135.5, 154.0, 157.4, 163.8,
170.9, 174.6. MS m/z (rel abundance): 324 (M+, 100), 283
(41.46), 265 (38.75). Anal. (C19H16O5) C, H.
15a : starting from 12 and propargyl bromide, yield 100%,
mp 167-169 °C. 1H NMR (CDCl3): δ 2.6 (m, 1H, CH), 3.75 (s,
3H, COOCH3), 4.0 (s, 2H, CH2COOH), 4.8 (m, 2H, CH2O), 7.0-
8.3 (m, 6H, aromatic). 13C NMR (CDCl3): δ 35.2, 52.2, 56.2,
76.4, 77.3, 101.5, 113.5, 116.1, 121.9, 123.2, 123.5, 125.9, 128.3,
135.6, 154.1, 157.3, 162.7, 171.0, 176.0. MS m/z (rel abun-
dance): 322 (M+, 100), 294 (26.32), 262 (44.26). Anal. (C19H14O5)
C, H.
8.35 (m, 5H, aromatic). 13C NMR (CDCl3): δ 14.2, 36.1, 52.3,
100.2, 108.7, 116.7, 118.4, 121.6, 122.1, 123.6, 123.7, 126.2,
135.5, 150.5, 153.9, 156.5, 158.6, 171.1, 176.7. MS m/z (rel
abundance): 322 (M+, 78.43), 263 (100), 59 (10.75). Anal.
(C19H14O5) C, H.
Meth yl (3,3-Dim eth yl-7-oxo-3H,7H-4,12-d ioxa ben zo[a ]-
a n th r a cen -10-yl)a ceta te (7a ). 3-Methyl-2-butenal (1 mL, 10
mmol) was slowly added to a suspension of 12 (2 g, 7 mmol)
in anhydrous pyridine (2 mL, 25 mmol) that had been heated
to 140 °C, and the mixture was then heated for 3 h. Another
1 mL of 3-methyl-2-butenal was added, and heating was
continued for another 6 h. The solvent was then evaporated,
and the residue was separated by flash chromatography
(toluene/ethyl acetate 7:3) to give 7a (10%), mp 138-140 °C
(toluene). 1H NMR (CDCl3): δ 1.52 (d, 6H, CH3), 3.7 (s, 3H,
COOCH3), 3.95 (s, 2H, CH2COO-), 5.75-8.25 (m, 7H, olefinic
and aromatic). 13C NMR (CDCl3): δ 28.4, 36.1, 52.3, 78.1,
109.1, 114.2, 115.0, 115.1, 121.9, 123.6, 124.6, 126.1, 127.3,
130.1, 135.6, 151.1, 153.6, 158.2, 171.4, 174.5. MS m/z (rel
abundance): 350 (M+, 8.84), 335 (100), 276 (19.38). Anal.
(C21H18O5) C, H.
Meth yl (3,3-Dim eth yl-7-oxo-2,3-d ih yd r o-1H,7H-4,12-d i-
oxa ben zo[a ]a n th r a cen -10-yl)a ceta te (8a ). A solution of 7a
(0.15 g, 0.43 mmol) in THF was hydrogenated at room temper-
ature and pressure over Pd/C. The reaction mixture was filter-
ed, and the solvent was evaporated. The residue was hydro-
lyzed without further purification.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
4b-8b a n d 13b-16b. A mixture of the methyl ester (0.8
mmol) in ethanol and KOH (1.2 mmol) in H2O was refluxed
for 4 h. The solvent was evaporated, and the residue was
dissolved in H2O and acidified with HCl. The precipitate was
then collected by filtration and crystallized from toluene.
4b: yield 51%, mp 235-236 °C. 1H NMR (DMSO-d6): δ 1.55
(s, 6H, CH3), 3.2 (s, 2H, CH2Ar), 3.9 (s, 2H, CH2COOH), 6.92-
8.05 (m, 5H, aromatic). 13C NMR (DMSO-d6): δ 28.0, 35.2, 90.4,
107.7, 113.1, 114.8, 120.8, 123.5, 124.6, 124.8, 127.6, 136.0,
152.6, 153.4, 164.4, 171.7, 174.5. MS m/z (rel abundance): 324
(M+, 3.53), 91 (100), 65 (12.64). Anal. (C19H16O5) C, H.
16a : starting from 12 and 3,3-dimethylallyl bromide, yield
75%, mp 95-96 °C. 1H NMR (CDCl3): δ 1.85 (d, 6H, 2 × CH3),
3.75 (s, 3H, COOCH3), 4.0 (s, 2H, CH2COOH), 4.68 (d, 2H,
CH2O), 5.55 (m, 1H, dCH-), 6.95-8.3 (m, 6H, aromatic). 13C
NMR (CDCl3): δ 18.2, 25.8, 35.2, 52.2, 65.5, 100.9, 113.9, 115.4,
118.6, 122.1, 123.2, 123.5, 126.0, 128.1, 135.5, 139.3, 154.2,
157.6, 164.4, 171.1, 176.1. MS m/z (rel abundance): 352 (M+,
4.70), 284 (100), 69 (86.79). Anal. (C20H20O5) C, H.
1
5b: yield 10%, mp 238-240 °C. H NMR (DMSO-d6): δ 1.5
(d, 3H, CH3), 2.95-3.55 (m, 2H, CH2Ar), 3.9 (s, 2H, CH2-
COOH), 5.25 (m, 1H, CH), 6.95-8.05 (m, 5H, aromatic). 13C
NMR (DMSO-d6): δ 21.6, 32.7, 35.2, 82.3, 107.6, 113.4, 115.0,
120.9, 123.1, 123.6, 124.7, 127.7, 136.1, 153.5, 157.1, 162.3,
171.9, 174.7. MS m/z (rel abundance): 310 (M+, 69.66), 309
(100), 77 (47.22). Anal. (C18H14O5) C, H.
Meth yl (2,2-Dim eth yl-6-oxo-1,2-d ih yd r o-6H-3,11-d ioxa -
cyclop en ta [a ]a n th r a cen -10-yl)a ceta te (4a ). A mixture of
13a (0.8 g, 2.3 mmol) and 2,6-dimethylphenol (0.7 g, 5.5 mmol)
was heated to 230-240 °C for 8 h. After cooling, the mixture
was dissolved in diethyl ether, washed with 10% NaOH
solution and with H2O, dried over Na2SO4, and evaporated to
dryness. The residue was crystallized from ligroin to give 4a
6b: yield 15% (ligroin), mp 275-276 °C (dec). 1H NMR
(DMSO-d6): δ 2.55 (s, 3H, CH3), 4.0 (s, 2H, CH2COOH), 6.92
(s, 1H, CHAr), 7.45-8.15 (m, 5H, aromatic). 13C NMR (DMSO-
d6): δ 14.2, 36.4, 100.3, 108.5, 116.5, 118.2, 121.4, 122.2, 123.0,
123.9, 126.2, 135.4, 150.1, 153.5, 156.6, 158.4, 171.3, 176.6.
MS m/z (rel abundance): 308 (M+, 94.76), 263 (100), 131 (9.54).
Anal. (C18H12O5) C, H.
1
(52%), mp 189-190 °C. H NMR (CDCl3): δ 1.6 (s, 6H, CH3),
3.2 (s, 2H, CH2Ar), 3.7 (s, 3H, COOCH3), 3.9 (s, 2H, CH2COO-
), 6.8-8.3 (m, 5H, aromatic). 13C NMR (CDCl3): δ 28.4, 35.8,
39.4, 52.2, 90.2, 108.0, 112.5, 114.6, 120.7, 123.2, 123.5, 126.2,
128.6, 135.4, 152.7, 153.5, 163.8, 173.9, 174.6. MS m/z (rel
abundance): 338 (M+, 100), 278 (35.20), 263 (43.90). Anal.
(C20H18O5) C, H.
1
7b: yield 35%, mp 202-204 °C. H NMR (DMSO-d6): δ 1.5
(s, 6H, CH3), 3.97 (s, 2H, CH2COOH), 5.95-8.1 (m, 7H, olefinic
and aromatic). 13C NMR (DMSO-d6): δ 27.8, 35.6, 78.0, 108.6,
113.8, 114.0, 114.8, 120.7, 123.7, 124.5, 125.0, 126.6, 130.7,
136.2, 151.0, 153.5, 157.7, 171.7, 174.7. MS m/z (rel abun-
dance): 336 (M+, 5.62), 321 (100), 276 (10.06). Anal. (C20H16O5)
C, H.
Meth yl (2-Meth yl-6-oxo-1,2-d ih yd r o-6H-3,11-d ioxa cy-
clop en ta [a ]a n th r a cen -10-yl)a ceta te (5a ). Compound 14a
(0.5 g, 1.5 mmol) was heated to 250-260 °C for 3 h. The residue
was dissolved in CH2Cl2, washed with dilute NaOH, dried over
Na2SO4, evaporated to dryness, and crystallized from ethanol
1
to give 5a (41%), mp 134-136 °C. H NMR (CDCl3): δ 1.6 (d,
1
3H, CH3), 3.00-3.55 (m, 2H, CH2Ar), 3.7 (s, 3H, COOCH3),
3.9 (s, 2H, CH2COO-), 5.2 (m, 1H, CH), 6.8-8.3 (m, 5H,
aromatic). 13C NMR (CDCl3): δ 21.9, 29.7, 33.6, 35.8, 52.2, 82.3,
107.8, 113.1, 116.0, 122.0, 123.3, 123.6, 126.2, 128.7, 135.5,
154.8, 157.6, 162.0, 171.4, 174.9. MS m/z (rel abundance): 324
(M+, 89.55), 265 (100), 59 (39.12). Anal. (C19H16O5) C, H.
Met h yl (2-Met h yl-6-oxo-6H-3,11-d ioxa cyclop en t a [a ]-
a n th r a cen -10-yl)a ceta te (6a ). A mixture of 15a (0.7 g, 2
mmol), CsF (0.43 g, 3 mmol), and N,N-diethylaniline (10 mL)
was refluxed under N2 for 4 h. The reaction mixture was then
diluted with diethyl ether and filtered. A precipitate was
formed, which was crystallized from toluene to give 6a (66%),
8b: yield 30%, mp 208-210 °C. H NMR (DMSO-d6): δ 1.5
(s, 6H, CH3), 1.95 (m, 2H, CH2CH2Ar), 2.95 (m, 2H, CH2CH2-
Ar), 4.0 (s, 2H, CH2COOH), 6.9-8.1 (m, 5H, aromatic). 13C
NMR (DMSO-d6): δ 12.9, 27.2, 35.4, 48.4, 73.7, 108.3, 113.6,
114.6, 120.5, 123.6, 124.2, 125.1, 126.2, 130.6, 151.3, 154.0,
157.8, 171.6, 174.5. MS m/z (rel abundance): 338 (M+, 94.37),
283 (100), 255 (96.03). Anal. (C20H18O5) C, H.
13b: yield 68%, mp 217-218 °C. 1H NMR (CDCl3): δ 1.8 (s,
3H, CH3), 4.0 (s, 2H, CH2COOH), 4.52 (s, 2H, CH2O), 5.05-
5.12 (d, 2H, CH2d), 6.92-8.25 (m, 6H, aromatic). 13C NMR
(DMSO-d6): δ 19.1, 34.6, 71.5, 101.2, 112.6, 114.0, 114.6, 121.0,
123.6, 124.5, 124.6, 127.4, 136.2, 140.0, 153.7, 157.0, 163.7,
171.7, 174.7. MS m/z (rel abundance): 324 (M+, 53.11), 309
(14.18), 55 (100). Anal. (C19H16O5) C, H.
1
mp 187-189 °C. H NMR (CDCl3): δ 2.58 (s, 3H, CH3), 3.75
(s, 3H, COOCH3), 4.0 (s, 2H, CH2COO-), 6.8 (s, 1H, CH), 7.4-

4938 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 22
Gobbi et al.
14b: yield 33%, mp 195-197 °C. 1H NMR (DMSO-d6): δ
3.95 (s, 2H, CH2COOH), 4.78 (m 2H, CH2O), 5.4 (m, 2H, CH2d
CH-), 6.1 (m, 1H, CH2dCH-CH2-), 7.1-8.1 (m, 6H, aro-
matic). 13C NMR (DMSO-d6): δ 34.6, 69.0, 101.1, 114.1, 114.7,
118.1, 121.1, 123.6, 124.6, 124.7, 127.5, 132.7, 136.3, 153.8,
157.1, 163.8, 171.8, 174.9. MS m/z (rel abundance): 310 (M+,
100), 77 (16.50). Anal. (C18H14O5) C, H.
16b: yield 75%, mp 197-200 °C. 1H NMR (DMSO-d6): δ
1.78 (d, 6H, CH3), 3.98 (s, 2H, CH2COOH), 4.72 (d, 2H, CH2O),
5.5 (m, 1H, CH), 7.05-8.1 (m, 6H, aromatic). 13C NMR (DMSO-
d6): δ 18.0, 25.4, 34.6, 65.4, 100.8, 114.2, 114.4, 118.8, 121.0,
123.5, 124.5, 124.6, 127.3, 136.1, 138.1, 153.6, 157.0, 164.0,
171.7, 174.7. MS m/z (rel abundance): 338 (M+, 12.99), 270
(45.88), 69 (100). Anal. (C19H18O5) C, H.
Biologica l Assa ys. Com p ou n d s. The compounds were
dissolved in DMSO and stored in the dark as stock solutions
(1000 µM) at -20 °C. For experimental use, all compounds
were prepared from stock solutions, diluted with growth
medium, filtered, sterilized, and used immediately.
Cell Lin es. The human colon adenocarcinoma cells LoVo
R (doxorubicin-resistant and multidrug-resistant cells) and
LoVo S (sensitive cells), kindly supplied by the Centro di
Riferimento Oncologico (Aviano, Pordenone, Italy), were cul-
tured in Ham F 12, plus 10% heat-inactivated fetal calf serum,
1% antibiotics (all products of Biochrom KG Seromed), and
1% 200 mM glutamine (Merck). For LoVo R cells, the medium
was supplemented with 100 ng/mL doxorubicin.
Hu m a n Mon on u clea r Cells. Human peripheral blood
mononuclear cells (HPBMC) were isolated from heparinized
whole blood by centrifugation over Ficoll-Paque (Pharmacia),
plated in 96-well plates, and allowed to adhere at 37 °C. After
2 h, medium and nonadherent cells were discarded and the
plates were vigorously washed three times with RPMI 1640
medium supplemented with 5% FCS and further incubated
in complete medium in the presence of different concentrations
(25, 50, 100 µM) of DMXAA and analogues, using triplicate
wells per drug dose. After 24 h, the medium was discarded
and the C13* cells (2 × 103 cells/well) were plated as above.
The cells were cocultured for 24 h and then assessed by MTT
test.
Qu a n tifica tion of TNF -r P r od u ction . Human peripheral
blood mononuclear cells (HPBMC) were isolated as described
above and treated with culture medium or DMXAA or the
selected compounds at the concentrations of 25, 50 and 100
µM. LPS (lipopolysaccharide from E. coli serotype 0127;B8,
SIGMA) was used as a positive control at the final concentra-
tion of 1 µg/mL. After 4 and 24 h incubations, the medium
was carefully collected and stored at -70 °C until assayed.
Commercially available enzyme-linked immunosorbent assay
(ELISA) kits were used to determine the concentration of
TNF-R (Biotrak ELISA System, Amersham Pharmacia Bio-
tech) according to the manufacturer’s instructions.
St a t ist ica l An a lysis. For each assay, three different
experiments were performed in triplicate. The results were
statistically evaluated by the Student’s t test.38 The IC50, 95%
confidence limits, and the potency ratio between DMXAA and
each analogue (IC50(DMXAA)/IC50(derivative)) were estimated
using the Litchfield and Wilcoxon method.38
The human ovarian adenocarcinoma cell line 2008 and the
cis-DDP-resistant subline C13*, kindly supplied by Prof. 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).
Refer en ces
Dir ect Cytotoxicity. The cells were seeded in 96-well
tissue plates (Falcon) and treated 24 h later with each agent
at different concentrations. Viable cell growth was determined
by tetrazolium salts reduction assay (MTT) after 24 h of
incubation.34
An amount of 20 µL of MTT solution (5 mg/mL in PBS) was
added to each well, and plates were incubated for 4 h at 37
°C. DMSO (150 µL) was added to all wells and mixed
thoroughly to dissolve the dark-blue crystals. The absorbance
was measured on a microculture plate reader (Titertek Mul-
tiscan) using a test wavelength of 570 nm and a reference
wavelength of 630 nm.
In d ir ect Toxicity. Mu r in e Resid en t Ma cr op h a ges. The
ability of the new derivatives to stimulate mouse macrophages
in culture to become tumoricidal was evaluated using resident
peritoneal macrophages.35 Resident peritoneal cells were
isolated by two injections of 5 mL of PBS containing 10 U/mL
heparin into the peritoneal cavity. The cavity was gently
massaged for 2 min, and the cells were removed by drawing
fluid out with a syringe.36
The recovered cell suspension was centrifuged, and the pel-
let was washed twice in sterile PBS. The cells were then re-
suspended in RPMI 1640 plus 5% FCS and plated in a culture
flask left to adhere at 37 °C. After 2 h, medium and nonadher-
ent cells were discarded, the flask was washed with sterile
PBS, adhering cells (macrophages) were resuspended, centri-
fuged, counted using 0.5% trypan blue, resuspended in com-
plete medium, and then plated in 96-well plates (Falcon) at a
concentration of 1 × 104 cells/well in the presence of different
concentrations of DMXAA and analogues, using triplicate wells
per drug dose. After 24 h, the medium was discarded and the
C13* cells (2 × 103 cells/well) were plated as above. The opti-
mal macrophages/C13* cells ratio was determined in prelimi-
nary experiments (results not reported). The cells were cocul-
tured for 24 h. Lysis of C13* cells was assessed by the MTT
test,37 and the percentages of specific cytotoxicity were calcu-
lated as follows:
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OD(macrophages+C13*) - OD(macrophages)
OD(C13*)

Derivatives of Xanthen-9-one-4-acetic Acid
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