Synthesis of xanthones and benzophenones as inhibitors of tumor cell growth

Article abstract of DOI:10.2174/157018010791526250

The synthesis of two new sulfated xanthones and two O-substituted benzophenones was carried out to evaluate for their inhibitory effect on growth of human tumor cell lines. The sulfation of 3,6-(2) and 3,4-dihydroxyxanthone (3) was accomplished using sulfur trioxide-pyridine to give, respectively, xanthone-3,6-O,O-bis(sulfate) (4) and xanthone- 3,4-O,O-bis(sulfate) (5). Treatment of 2,2',4,4'-tetrahydroxybenzophenone (6) with acetic acid and prenyl bromide furnished 2,2',4,4'- tetraacetoxybenzophenone (7) and (5-hydroxy-2,2-dimethylchroman-6-yl)(2'-hydroxy-4'-(tertpentyloxy) phenyl)methanone (8), respectively. Compounds 2-8 were tested for their effect on the in vitro growth of four representative human tumor cell lines: MCF-7 ER(+) (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), SF-268 (glioma), and A375-C5 (melanoma). Compounds 2 and 7-8 showed an inhibitory activity in the μM range.

Full text of DOI:10.2174/157018010791526250

Letters in Drug Design & Discovery, 2010, 7, 487-493
487
Synthesis of Xanthones and Benzophenones as Inhibitors of Tumor Cell
Growth
Elisangela Costa1,2, Emília Sousa*,1,2, Nair Nazareth2,3, Maria S.J. Nascimento2,3 and
Madalena M.M. Pinto1,2
1Department of Chemistry, Laboratory of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy, University of
Porto, Rua Aníbal Cunha, 164, 4050-047 Porto, Portugal
2Research Center of Medicinal Chemistry - University of Porto (CEQUIMED-UP), Portugal
3Department of Microbiology, Faculty of Pharmacy, University of Porto, Portugal
Received January 14, 2010: Revised April 14, 2010: Accepted May 12, 2010
Abstract: The synthesis of two new sulfated xanthones and two O-substituted benzophenones was carried out to evaluate
for their inhibitory effect on growth of human tumor cell lines. The sulfation of 3,6-(2) and 3,4-dihydroxyxanthone (3)
was accomplished using sulfur trioxide-pyridine to give, respectively, xanthone-3,6-O,O-bis(sulfate) (4) and xanthone-
3,4-O,O-bis(sulfate) (5). Treatment of 2,2’,4,4’-tetrahydroxybenzophenone (6) with acetic acid and prenyl bromide fur-
nished 2,2’,4,4’- tetraacetoxybenzophenone (7) and (5-hydroxy-2,2-dimethylchroman-6-yl)(2’-hydroxy-4’-(tert-
pentyloxy)phenyl)methanone (8), respectively. Compounds 2-8 were tested for their effect on the in vitro growth of four
representative human tumor cell lines: MCF-7 ER(+) (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer),
SF-268 (glioma), and A375-C5 (melanoma). Compounds 2 and 7-8 showed an inhibitory activity in the ꢀM range.
Keywords: Antitumor, Benzophenones, Prenylated, Sulfated, Xanthones.
INTRODUCTION
Benzophenones are biosynthetic blocks as well as inter-
mediates in many synthetic pathways of xanthones and con-
stitute another interesting class of tumor cell growth inhibi-
tors. One of the most interesting antiproliferative benzophe-
nones is benzophenone-4,4ꢁ-O,O-bis-sulfamate (1, Ben-
zomate), which was obtained during an attempt to design
nonsteroidal steroid sulfatase (STS) inhibitors [12]. Struc-
ture-activity relationship studies revealed that the presence of
the carbonyl group is essential for the activity whereas the
bis-sulfamate moiety is required for the irreversible mecha-
nism [12]. Interestingly, it was found that the conformational
flexibility of the molecule is not pivotal for this activity. This
fact led us to attempt to investigate a more rigid but similar
structure by using the xanthonic scaffold to obtain potential
STS inhibitors. It is well established that steroid sulfatase
(STS) is one of the key enzymes of estrogen biosynthesis;
thus blocking this enzyme will result in inhibition of estro-
gen production. Consequently, the hormone receptors will
receive fewer growth signals and thus the growth of the can-
cer cells will slow down or stop. Interestingly, Di et al. [13]
have reported the isolation of sulfonated xanthones from
Hypericum sampsonii, which exhibited a significant cytotox-
icity against cancer cell lines. These findings, together with
the structural feature of Benzomate (1), led us to hypothetize
the sulfation of the xanthone scaffold as a strategy of obtain-
ing new xanthone derivatives as potential inhibitors of the
cancer cells growth.
Many naturally occurring and synthetic xanthones have
been found to possess interesting biological and pharmacol-
ogical activities [1-5]. Their chemotherapeutic potential
seems rather attractive, since many xanthone derivatives are
endowed with potent cytotoxic properties [4]. Consequently,
we have focused our attention on the synthesis of xanthone
derivatives and their capacity to inhibit the in vitro growth of
some tumor cell lines, especially MCF-7 ER(+) (breast ade-
nocarcinoma estrogen receptor positive) [6-9]. It was pro-
posed that the growth inhibitory effect of xanthones was
associated not only to their tricyclic scaffold, but also that
this effect strongly depends on the nature and/or position of
the different substituents. Particularly, in the field of antican-
cer drugs, isopropylfuran and dimethylpyran units fused onto
xanthone cores appear interesting, with the remarkable ex-
ample of a natural furanoxanthone, psorospermin, that was
used as model for further chemical and biological develop-
ments [10]. Particularly, we have recently synthesized a se-
ries of dihydropyranoxanthones to evaluate their effect on
the in vitro growth of three human tumor cell lines and we
have found that some of these derivatives were not only po-
tent growth inhibitors but also selective against the MCF-7
ER(+) cells [6,11]. Besides these derivatives, we have found
that bromoalkoxyxanthones and xanthonolignoids also
showed selectivity against the growth inhibition of the MCF-
7 ER(+) human tumor cells [7,9].
On the other hand, the growth inhibitory effect described
for prenylated benzophenones [14-18], and particularly, for
natural benzophenones with a biciclo 3.3.1-nonane-2,4,9
trione system [19-22], are well recognized. Interestingly, for
O-substituted benzophenones, only one geranyloxybenzo-
phenone has been investigated for its growth inhibitory ac-
tivity showing the IC50<10μg/mL against breast tumor cell
*Address correspondence to this author at the Research Center of Medicinal
Chemistry - University of Porto (CEQUIMED-UP), Department of Chemis-
try, Laboratory of Organic and Pharmaceutical Chemistry, Faculty of Phar-
macy, University of Porto, Rua Aníbal Cunha, 164, 4050-047 Porto, Portu-
gal; Tel: +351 222078984; Fax: +351 222 003 977; E-mail: esousa@ff.up.pt
1570-1808/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

488 Letters in Drug Design & Discovery, 2010, Vol. 7, No. 7
Costa et al.
O
O
9
6'
3'
6
1
8
1
1'
2'
R1
8a
9a
4a
5'
R3
5
7
2
6
R3
2
3
R4
4
4'
O
R1
R3
3
10a
4
R2
5
R2
R1 and R2
R3 and R4
R1
R2
OSO2NH2
OH
1
6
H
OH
OH
OH
H
OH
H
OH
H
2
3
OCOCH3
OCOCH3
7
-
-
-
4
5
OSO3
OSO3
OSO3
H
-
OSO3
OH
O
OH
4
2'
1'
5
6
3'
3
4'
7
O
1
O
6'
5'
2
8
8
Fig. (1). Structures of Benzomate (1) and benzophenones (6-8)/xanthones (2-5) investigated.
line MCF-7 [14,15,23]. However, a problem associated to
benzophenones activity is that many of them also exhibit
estrogenic activity which can interfere with the evaluation of
their antiproliferative effect, especially on the MCF-7 ER(+)
cells. Thus, is important to dissociate the estrogenic activity
from the antiproliferative effect of these compounds. It is
well known that a phenolic moiety is important for estro-
genicity [24,25]. Additionally, it has been previously shown
for a large, structurally diverse group of chemicals that the
substitution of the hydroxyl by an alkoxyl group such as a
methoxyl group significantly decreases the affinity for the
ER [26]. Based on these facts, we have carried out a molecu-
lar modification of benzophenones by introducing a hydro-
phobic prenyl group to the benzophenone scaffold to block
phenolic hydroxyl groups, eventually associated to a estro-
genic activity.
man-6-yl)(2’-hydroxy-4’-(tert-pentyloxy)phenyl)methanone
(8) were also obtained (Fig. 1). Compounds 4-5 and 7-8,
along with their respective precursors (2-3 and 6), were
evaluated for their in vitro growth inhibitory effect on human
tumor cell lines: MCF-7 ER(+), NCI-H460, SF-268, and
A375-C5 cell lines.
RESULTS AND DISCUSSION
3,4-Dihydroxyxanthone (3) was obtained according to
the previously described procedure (65%) [8]. Attempts to
synthesize 3,6-dihydroxyxanthone (2) were based on base-
catalyzed cyclization reactions, by oxidative or dehydrative
processes [5]. The dehydrative process was performed by
heating 2,2’,4,4’-tetrahydroxybenzophenone (6) in furnace
(180ºC) to afford 3,6-dihydroxyxanthone (2) in excellent
yield (85%) (Fig. 2A).
Consequently, the synthesis of analogues of benzophe-
none-4,4ꢀ-O,O-bis-sulfamate (1, Benzomate) with a xan-
thone/benzophenone sacaffold was planned in order to ob-
tain a series of molecules and to evaluate their effect on the
in vitro growth of tumor cell lines. The molecular modifica-
tions were carried out by the introduction of sulfate groups
into the xanthone scaffold for potential reversible STS in-
hibitors while acetyl/prenyl groups were introduced into the
benzophenone scaffold to dissociate the potential estrogenic
activity from the antiproliferative activity.
Sulfated xanthonic derivatives 4-5 were successfully ob-
tained, in the presence of sulfur trioxide(SO3)-pyridine ad-
duct [27], from dihydroxyxanthones 2-3, respectively, in
moderate yields (28-49%) (Fig. 2B). Sulfur trioxide–pyridine
has been used extensively for sulfating alcohols, sterols,
phenols and carbohydrates and normally the reactions run
under moderate temperatures, usually below 120º C in the
presence of pyridine excess [28]. However, in the case of the
sulfated xanthones 4-5, better yields were obtained when
dimethylacetamide (DMA) was used as solvent.
Herein, we report the synthesis of the rigid analogues of a
potent STS inhibitor Benzomate (1): xanthone-3,6-O,O-
bis(sulfate) (4) and xanthone-3,4-O,O-bis(sulfate) (5). Other
two analogues of Benzomate (1), namely 2,2’,4,4’-tetra-
acetoxybenzophenone (7) and (5-hydroxy-2,2-dimethylchro-
In the dehydrative process of 6, with the presence of ace-
tic anhydride, 2,2’,4,4’-tetraacetoxybenzophenone (7) was
obtained in moderate yield (30%) (Fig. 2C). The prenylated
benzophenone 8 was obtained (6%) by the one-pot reaction

Xanthones and Benzophenones Inhibitors of Tumor Cell Growth
Letters in Drug Design & Discovery, 2010, Vol. 7, No. 7 489
A)
O
O
HO
OH
furnace,180˚ C , overnight
O
HO
OH
HO
OH
6
2
(85%)
SO3
:Pyridine
O
B)
O
4 mol/OH
DMA, 65˚C, 4 days
HO
O
OH
-
-O3SO
OSO3
O
4
2
(49%)
O
O
O
SO3:Pyridine
4 mol/OH
OH
DMA, 65˚C, 4 days
-
O
OSO3
OH
-
OSO3
5
3
(32%)
OCOCH3 O
OCOCH3
C)
O
HO
OH
, reflux, 1day
(CH3CO)2O/Na2CO3
OCOCH3
OH
HO
OCOCH3
7
6
(30%)
OH
O
OH
D)
OH
O
OH
K10 clay, prenyl bromide,
CHCl3, MW, 250 W, 1 h
O
O
HO
8
OH
6
(6%)
Fig. (2). Reagents and conditions for the synthesis of A) 3,6-dihydroxyxanthone (2), B) xanthone-3,6-O,O-bis(sulfate) (4) and xanthone-3,4-
O,O-bis(sulfate) (5), C) 2,2’,4,4’- tetraacetoxybenzophenone (7), and D) (5-hydroxy-2,2-dimethylchroman-6-yl)(2’-hydroxy-4’-(tert-
pentyloxy)phenyl)methanone (8).
of benzophenone 6 and prenyl bromide (Fig. 2D), applying
the Montmorillonite K10 clay-catalyzed condensation, as
recently described by Castanheiro et al. [11]. The formation
of this unexpected asymmetrical product (8) observed under
microwave irradiation, might be favored by the temperature
regime that is often described to change selectivities as com-
pared to conventional heat [29,30]. Compound 8 was hy-
pothesized to be formed through a cascade sequence involv-
ing clays-induced aldol type reaction of benzophenone 6
with prenyl bromide as previously proposed by Jeso and
Nicolaou [31].
Structure Elucidation
The structure of compound 2 was established by com-
1
parison of its H and 13C NMR data with those reported in
the literature [32] while the structures of compounds 4-5 and
7-8 were elucidated using IR, HRMS, in addition to NMR
techniques, and these data are reported in the experimental
section.
The structures of the sulfated xanthones 4 and 5 were es-
tablished not only by comparison of their proton and carbon
chemical shift values with those of their respective precursor

490 Letters in Drug Design & Discovery, 2010, Vol. 7, No. 7
Costa et al.
xanthones (2 and 3), but also by the IR and HRMS spectra.
The IR spectra of the sulfated derivatives 4 and 5 revealed
two strong bands, characteristic of the S=O (1178 cm-1 and
1184 cm-1) and the C-O-S (1067 cm-1 and 1004 cm-1) groups,
respectively. The HRMS allowed determining the number of
sulfate groups for compounds 4 and 5 as two groups.
MCF-7 ER(+) (breast adenocarcinoma), NCI-H460 (non-
small cell lung cancer), SF-268 (glioma), and A375-C5
(melanoma) after a continuous exposure of 48 hours and
results are shown in Table 1. Compound 3 has previously
shown a moderate growth inhibitory effect on human tumor
cell lines [8].
The multiplicity and coupling constants of the protons
observed in the 1H NMR spectrum of 2,2’,4,4’- tetraacetoxy-
benzophenone (7) showed, besides the existence of two
symmetrical 1,2,4-trisubstituted benzene rings, the signals of
the protons of the acetoxyl groups at ꢀ1.84 and 2.31, respec-
tively. The 13CNMR spectrum showed, besides the carbon
signals of the aromatic rings and the carbonyl carbon of ben-
zophenone (ꢀ190.3), the carbonyl and methyl groups of the
acetates (ꢀ117.3 and 20.0, respectively).
The growth inhibitory effect for compounds 2 and 7-8
was in general moderate but was shown to be dose-
dependent (see Supportive/Supplementary Material) and due
to growth arrest and not to cell death, as inferred from the
sulforhodamine B (SRB) assay. 3,6-Dihydroxyxanthone (2)
and benzophenones 6-8 were found to be active in the four
human tumor cell lines but with benzophenones 7 and 8
showing a significant inhibitory activity against an estrogen
dependent MCF-7 ER(+) cell line (GI50 < 30 μM, Table 1).
However, no activity was observed for the sulfated xan-
thones 4 and 5 on the growth of the human cancer cell lines
tested (GI50>100 μM, Table 1).
In turn, the 1H NMR spectrum of (5-hydroxy-2,2-di-
methylchroman-6-yl)(2’-hydroxy-4’-(tert-pentyloxy)phenyl)
methanone (8) showed the proton signals of two methylene
groups (ꢀ1.83t, J=6.8 and 2.73t, J=6.8) and two methyl
groups (ꢀ1.37s), characteristic of the fused dihydropyran
ring. This was corroborated by the chemical shift values ob-
served in the 13C NMR spectrum (ꢀ16.9CH2, 26.9CH3,
32.7CH2, 67.4C). The fusion of the dihydropyran ring with
C3-C4 of the aromatic ring of the benzophenone is evi-
denced by the lack of H-3 signal and the presence of two
ortho coupled aromatic protons, corresponding to H-7 and
H-8. O-Prenylation also occurred on the other aromatic ring
since its pattern of substitution was maintained as can be
observed by the multiplicities and coupling constants of its
protons. The presence of 4’-(tert-pentyloxy) substituent on
this ring was confirmed by an observation of the proton
(ꢀ1.26s, 1.28t, 1.36m) and carbon signals (ꢀ21.7CH3,
It can be inferred that the introduction of hydrophobic
groups, a hindrance of the hydroxyl groups, led to the en-
hancement of the inhibitory growth effects on MCF-7 ER(+)
when compared the growth inhibitory effects exhibited by O-
acetylated (7) and O-prenylated (8) benzophenones, with that
of their precursor, 2,2’,4,4’-tetrahydroxybenzophenone (6),
(Table 1). Nonetheless, the selectivity shown by compounds
7 and 8 for MCF-7 cells over the other cells was not evident
and therefore modulation of the estrogenic pathway by these
compounds is very unlikely. Since compounds 7 and 8 are
Benzomate (1) analogues, should be interesting to further
investigate if their antiproliferative mechanism of action is
related to the interaction of compound 7 and 8 with steroid
sulfatase (STS).
1
27.1CH3, 54.8CH3 and 69.9C) from the H and 13C NMR
spectra. The proposed structure was in agreement with the
result obtained from the high-resolution mass spectrometry
(HRMS).
MATERIALS AND METHODS
General Methods
Purifications of compounds were performed by flash
chromatography using Merck silica gel 60 (0.040-0.063 mm)
and preparative thin layer chromatography (TLC) using
Merck silica gel 60 (GF254) plates. Melting points were ob-
tained in a Köfler microscope. IR spectra were measured on
an ATI Mattson Genesis series FTIR (software: WinFirst
Effect on the Growth of Tumor Cell Lines
The hydroxy- (6) acetoxy- (7) and prenyloxy-(8) benzo-
phenone derivatives, as well as 3,6-dihydroxyxanthone (2)
and the sulfated xanthones 4 and 5 were evaluated for their
effect on the in vitro growth of four human tumor cell lines:
Table 1. GI50 Values of Benzophenones 6, 7, and 8, Xanthone 2 and Sulfated Xanthone Derivatives 4 and 5 on the In Vitro Growth
of Human Tumor Cell Lines
Compounds
GI50 (μM)a
MCF-7 R(+) (breast)
NCI H460 (lung)
A375-C5 (melanoma)
SF-268 (glioma)
2
74.6b
118.5b
31.1b
145.0b
ND
140.0b
149.1b
> 150
4
5
> 150
> 150
> 150
> 150
6
66.1 ± 3.8
27.4b
105.7b
ND
142.3b
20.1b
7
41.0 ± 1.2
23.0 ± 1.0
35.6 ± 1.6c
ND
8
20.3 ± 0.7
43.3 ± 2.6c
22.5 ± 0.4
130.2 ± 10.1c
ND
Doxorubicin
94.0 ± 7.0c
aResults expressed as GI50, concentrations of the compound that cause 50% inhibition of cell growth, are mean ±SEM of 3-5 independent experiments performed in duplicate carried
out independently. ND= not determined. bData based on two independently run duplicate experiments. cGI50 values are expressed in nM for the positive control doxorubicin.

Xanthones and Benzophenones Inhibitors of Tumor Cell Growth
Letters in Drug Design & Discovery, 2010, Vol. 7, No. 7 491
v.2.10) spectrophotometer in KBr microplates (cm-1). 1H and
13CNMR spectra were taken in CDCl3 or DMSO-d6 at room
temperature, on Bruker Avance 300 instrument (300.13 MHz
zophenone (7, 418 mg, 30%); mp 184-185ºC; IR (KBr) ꢁmax :
1
1762; 1661; 1605; 1372; 1198; 1140; H NMR (CDCl3,
300.13 MHz) ꢀ: 7.60 (1H, d, J = 8.5 Hz, H-6; H-6’), 7.24
(2H, dd, J = 8.5; 2.2 Hz, H-5; H-5’), 7.17 (2H, d, J = 2.0 Hz,
H-3;H-3’), 2.31 (6H, s, 2-OCOCH3, 2’-OCOCH3), 1.84 (6H,
s, 4-OCOCH3, 4’-OCOCH3); 13C NMR (CDCl3, 75.47 MHz)
ꢀ: 190.3 (CO), 153.8 (C-2,C-2’,C-4,C-4’), 120.0 (C-1; C-1’),
117.3 (C-3,C-3’,C-5,C-5’, 2-OCOCH3, 2’-OCOCH3, 4-
OCOCH3, 4’-OCOCH3 ), 131.5 (C-6, C-6’), 20.0 (2-
OCOCH3, 2’-OCOCH3, 4-OCOCH3, 4’-OCOCH3); HRMS:
1
for H and 75.47 MHz for 13C). Chemical shifts are ex-
pressed in ꢀ (ppm) values relative to tetramethylsilane
(TMS) as an internal reference. HRMS results were obtained
in the services of C.A.C.T.I., Vigo, Spain. 3,6-
Dihydroxyxanthone (2) [33] and 3,4-dihydroxyxanthone (3)
[8] were synthesized according to described methods. The
following materials were synthesized and purified by the
described procedures.
415.1027 [M+H]+, C21H19O9 ; calcd 415.1029.
+
Xanthone-3,6-O,O-bis(sulfate) (4)
5-hydroxy-2,2-dimethylchroman-6-yl)(2’-hydroxy-4’-(tert-
pentyloxy)phenyl)methanone (8)
A mixture of 3,6-dihydroxyxanthone (2, 285 mg; 1.25
mmol), DMA (4 mL), and SO3-pyridine adduct (10 mmol;
1.59 g) was kept under reflux for 4 days. The reaction mix-
ture (pH 4) was basified with triethylamine to pH 7, and 10
mL of acetone was added. After cooling, the brown oil
formed was washed with acetone and ether and suspended in
aqueous solution of sodium acetate 30% (2mL). Absolute
ethanol (50 mL) was added to the suspension and brown oil
was formed after cooling. The crude oil (500 mg) was puri-
fied by solid phase extraction with a cation exchange car-
tridge Discovery® DSC-SCX, with a sulfonic acid moiety
following the steps: condition with methanol (5 mL); loading
[(500 mg of sample; 2 mL (1:1 MeOH: CH3COOH 1% in
water)]; elution with CH3COOH (15 mL; 1% in water); the
acidic fractions were collected and the solvent evaporated
under reduced pressure to afford a yellow solid correspond-
ing to xanthone-3,6-O,O-bis(sulfate) (4, 250 mg; 49%); mp
> 300 ºC; IR (KBr) ꢁmax : 1632; 1533; 1484, 1178; 1067; 1H
NMR (DMSO, 300.13 MHz) ꢀ: 7.99 (2H, d, J=8.6, H-1; H-
8), 6.87 (2H, dd, J=8.6 and 2.2, H-2; H-7), 6.83 (2H, d,
J=2.2, H-4, H-5); 13C NMR (DMSO, 75.47 MHz) ꢀ: 174.6
(CO), 163.4 (C-3 and C-6), 157.5 (C-4a and C-10a), 127.9
(C-1 and C8), 114.0 (C-8a and C-9a), 114.0 (C-2 and C-7),
102.2 (C-4 and C-5); HRMS: 479.07387, C13H9O10 S2.5H2O;
calcd 479.01652.
A mixture of 2,2’,4,4’-tetrahydroxybenzophenone (6,
0.11 g; 0.39 mmol), prenyl bromide (0.2 mL; 1.64 mmol),
and Montmorillonite K10 Clay (2.04 g), in a 12 ml closed
microwave reactor under stirring was irradiated at 250 W for
3 ꢀ 20 min at a final temperature of 90ºC. After cooling, the
solid was filtered and washed and the solvent removed under
reduced pressure to furnish a brown oil. This crude product
was
purified
by
liquid
chromatography
(SiO2;
CHCl3/hexane, 70:30) yielding an impure solid. The purifi-
cation was then carried out by preparative TLC (SiO2; hex-
ane/AcOEt, 80:20) affording a white compound correspond-
ing to (5-hydroxy-2,2-dimethylchroman-6-yl)(2’-hydroxy-
4’-(tert-pentyloxy)phenyl)methanone (8, 10 mg, 6%); mp
238-240ºC, IR (KBr) ꢁmax :2922; 1609, 1585, 1484, 1461,
1
1361, 1259, 1227, 1154, 1116, 879, 790, 757, H NMR
(CDCl3, 300.13 MHz) ꢀ: 7.50 (1H, d, J=8.6, H-6’), 7.38 (1H,
d, J=9.0, H-7), 6.46 (1H, s, H-3’), 6.43 (1H, d, J=8.6, H-5’),
6.35 (1H, d, J=9.0, H-8), 2.73 (2H, t, J = 6.8, H-4), 1.83 (2H,
t, J=6.8, H-3), 1.37 (6H, s, CH3-1’’), 1.36 (m, CH2-3’’’),
1.28 (t, CH3-4’’’), 1.26 (6H, s, CH3-1’’’); 13C NMR (CDCl3,
125.77 MHz) ꢀ: 199.1 (CO), 164.4 (C-2’), 162.2 (C-4’),
161.9 (C-4), 160.8 (C-2), 134.3 (C-6’), 129.1 (C-7), 113.9
(C-1’), 113.5 (C-3), 112.6 (C-6), 107.3 (C-5’), 105.2 (C-8),
103.9 (C-3’), 69.9 (C-2’’’), 67.4 (C-2), 54.8 (C-3’’’), 32.7
(C-3), 27.1 (C-1’’’), 26.9 (C-1’’), 21.7 (C-4’’’), 16.9 (C-4);
HRMS: 383.18499, C23H28O5 calcd 383.18530.
Xanthone-3,4-O,O-bis(sulfate) (5)
A mixture of 3,4-dihydroxyxanthone (3, 285 mg; 1.25
mmol), DMA (4 mL) and SO3-pyridine adduct (10 mmol;
1,59 g) was kept under reflux for 4 days. The same proce-
dure used above for compound 4 was followed in the work-
up. The acidic fractions were collected and the solvent
evaporated under reduced pressure to afford a yellow solid
corresponding to xanthone-3,4-O,O-bis(sulfate) (5, 170 mg,
32%); mp > 300ºC, IR (KBr) ꢁmax : 1630; 1531; 1481; 1184;
Tumor Cell Growth Assay
The effects of 2–8 on the in vitro growth of human tumor
cell lines were evaluated according to the procedure adopted
by the National Cancer Institute (NCI, USA) in the ‘In vitro
Anticancer Drug Discovery Screen’ that uses the protein-
binding dye sulforhodamine B to assess cell growth.[34,35]
Briefly, exponentially, cells growing in 96-well plates were
then exposed for 48 h to five serial concentrations of each
compound (1:3 to 1:2 dilutions), starting from a maximum
concentration of 150 μM. Following this exposure period
adherent cells were fixed, washed, and stained. The bound
stain was solubilized and the absorbance was measured at
492 nm in a plate reader (Bio-Tek Instruments Inc., Power-
wave XS, Wincoski, USA). For each test compound (2-8)
and cell line, a dose–response curve was obtained (see sup-
portive/supplementary material). The growth inhibition of
50% (GI50), corresponding to the concentration of the com-
pounds that inhibited 50% of the net cell protein increase in
control cells during compounds incubation, was calculated in
terms of %T/C [(OD of treated cells/OD of control
1
1004; H NMR (DMSO, 300.13 MHz) ꢀ: 8.16 (1H, dd ,
J=8.0,1.5, H-8), 7.84 (1H, dd, J=8.0, 8.0, 1.5, H-6), 7.64
(1H, dd, J=8.0, H-5), 7.57 (1H, d, J=8.8, H-1), 7.45 (1H,
ddd, J=8.0, 8.0, 1.5, H-7), 6.95 (1H, d, J=8.8, H-2); HRMS:
479.07403, C13H9O10 S2.5H2O; calcd 479.01652.
2,2’,4,4’- Tetraacetoxybenzophenone (7)
A mixture of 2,2’,4,4’-dihydroxybenzophenone (6, 1 g;
4.06 mmol) and acetic anhydride (20 mL) was refluxed at
140ºC, for 2 days. After the reaction was finished, aqueous
sodium bicarbonate 10% (10 mL) was added to the reaction
mixture, which was then poured onto 50 g of crushed ice.
The solid thus obtained was filtered, washed with water and
dried to furnish a white solid of 2,2’,4,4’- tetraacetoxyben-

492 Letters in Drug Design & Discovery, 2010, Vol. 7, No. 7
Costa et al.
growth of human cancer cell lines and on the proliferation of hu-
man lymphocytes in vitro. Helv. Chim. Acta, 2002, 85, 2862-2876.
Nguyen, H.T.; Lallemand, M.-C.; Boutefnouchet, S.; Michel, S.;
Tillequin, F. Antitumor Psoropermum xanthones and Sarcomeli-
cope Acridones: privileged structures implied in DNA alkylation. J.
Nat. Prod., 2009, 72, 527-539.
Castanheiro, R.A.P.; Pinto, M.M.; Cravo, S.M.M.; Pinto,
D.C.G.A.; Silva, A.M.S.; Kijjoa, A. Improved methodologies for
synthesis of prenylated xanthones by microwave irradiation and
combination of heterogeneous catalysis (K10 clay) with microwave
irradiation. Tetrahedron, 2009, 65, 3848-3857.
cells)ꢀ100)] as described elsewhere [34]. Doxorubicin was
used as a positive control and tested in the same manner.
[10]
[11]
CONCLUSION
The synthesis of two new sulfated xanthones was suc-
cessfully achieved by using sulfur trioxide-pyridine adduct
in dimethylacetamide. Though, these new compounds did
not exhibit an in vitro growth inhibitory effect on the human
tumor cell line tested, they represent the new class of xan-
thonic derivatives waiting for other models of biological
evaluation, such as anticoagulant activity in which sulfated
small molecules have promising representatives [36].
[12]
Hejaz, H.A.; Woo, L.W.; Purohit, A.; Reed, M.J.; Potter, B.V.
Synthesis, in vitro and in vivo activity of benzophenone-based in-
hibitors of steroid sulfatase. Bioorg. Med. Chem., 2004, 12, 2759-
2772.
[13]
[14]
Di, H.; Feng, Y.; Lihong H.; Ping L. Sulfonated xanthones from
Hypericum sampsonii. Phytochemistry, 2004, 65, 2595–2598.
Epifano, F.; Genovese, S.; Menghini, L. Chemistry and pharmacol-
ogy of oxyprenylated secondary metabolites. Phytochemistry,
2007, 68, 939-953.
Pecchio, M.; Solis, P.N.; Lopez-Perez, J.L.; Vasquez, Y.; Rodri-
guez, N.; Olmedo, D.; Correa, M.; San Feliciano, A.; Gupta, M.P.
Cytotoxic and antimicrobial benzophenones from the leaves of To-
vomita longifolia. J. Nat. Prod., 2006, 69, 410-413.
Kralj, A.; Kehraus, S.; Krick, A.; Eguereva, E.; Kelter, G.; Maurer,
M.; Wortmann, A.; Fiebig, H.H.; König, G.M. Arugosins G and H:
Prenylated polyketides from the marine-derived fungus Emericella
nidulans var. acristata. J. Nat. Prod., 2006, 69, 995-1000.
Tanaka, N.; Takaishi, Y.; Shikishima, Y.; Nakanishi, Y.; Bastow,
K.; Lee, K-H.; Honda, G.; Ito, M.Takeda, Y.; Kodzhimatov, O.K.;
Ashurmetov, O. Prenylated Benzophenones and Xanthones from
Hypericum scabrum. J. Nat. Prod., 2004, 67, 1870-1875.
Chaturvedula, V.S.P.; Schilling, J.K.; Kingston, D.G.I. New cyto-
toxic coumarins and prenylated benzophenone derivatives from the
bark of Ochrocarpus punctatus from the Madagascar rainforest. J.
Nat. Prod., 2002, 65, 965-972.
Santos, M.H.; Nagem, T.J.; Oliveira, T.T.; Braz-Filho, R. 7-
Epiclusianone, the new tetraprenylated benzophenone and others
chemical constituents from the fruits of Rheedia gardneriana.
Quím. Nova, 1999, 22, 654-660.
Diaz-Carballo, D.; Malak, S.; Freistühler, M.; Elmaagacli, A.;
Bardenheuer, W.; Reusch, H.P. Nemorosone blocks proliferation
and induces apoptosis in leukemia cells. Int. J. Clin. Pharmacol.
Ther., 2008, 46, 428-439.
Díaz-Carballo, D.; Malak, S.; Bardenheuer, W.; Freistuehler, M.;
Reusch, H.P. Cytotoxic activity of nemorosone in neuroblastoma
cells. J. Cell. Mol. Med., 2008, 12, 2598-2608.
Cuesta-Rubio, O.; Frontana-Uribe, B.A.; Ramirez-Apan, T.;
Cardenas, J. Polyisoprenylated benzophenones in Cuban propolis;
biological activity of nemorosone. Z. Naturforsch. [C], 2002, 57,
372-378.
On the contrary, introduction of the hydrophobic groups
to the benzophenone scaffold (7 and 8) was found to enhance
the in vitro growth inhibitory effect, in a micromolar range,
on the human tumor cells, especially the MFC-7 ER(+). The
activity exhibited by these two derivatives provides an inter-
esting clue for further molecular modifications to be per-
formed in order to improve their potency.
[15]
[16]
[17]
[18]
[19]
[20]
ACKNOWLEDGEMENTS
We thank Fundação para a Ciência e a Tecnologia (FCT)
for the financial support to this work (I&D 4040/2007) and
for the PhD grant to Elisangela Costa (SFRH/BD/30615/
2006). We thank Sara Cravo for technical support.
SUPPLEMENTARY MATERIAL
Supplementary material is available on the publishers
Web site along with the published article.
REFERENCES
[1]
Pinto, M.M.M.; Sousa, E.; Nascimento, M.S.J. Xanthone derivati-
ves: new insights in biological activity. Curr. Med. Chem., 2005,
12, 2517-2538.
[21]
[22]
[2]
[3]
Fotie, J.; Bohle, S. Pharmacological and biological activities of
xanthones. Curr. Med. Chem. - Anti-Infect. Agents., 2006, 5, 15-31.
Pinto, M.M.M.; Castanheiro, R. In: Natural Products Chemistry,
Biochemistry and Pharmacology; Brahmachari, Ed.; Narosa Pub-
lishing House PVT. LTD.: West Bengal, India, 2009, Vol. 17, pp.
520-675.
[23]
[24]
Bohlmann, F.; Subita, A. Neue Phloroglucin-Derivate aus Leon-
tonyx-Arten sowie weitere Verbindungen aus Vertretern der Tribus
inuleae. Phytochemistry, 1978, 17, 1929-1934.
[4]
[5]
Pouli, N.; Marakos, P. Fused xanthone derivatives as antiprolifera-
tive agents. Anti Cancer Agents Med. Chem., 2009, 9, 77-98.
Franklin, G.; Conceição, L.F.R.; Kombrink, E.; Dias, A.C.P.
Xanthone biosynthesis in Hypericum perforatum cells provides an-
tioxidant and antimicrobial protection upon biotic stress. Phyto-
chemistry, 2009, 70, 65-73.
Anstead, G.M.; Carlson, K.E. The estradiol pharmacophore: ligand
structure-estrogen receptor binding affinity relationships and a
model for the receptor binding site. Steroids, 1997, 62, 268-303.
Duax, W.L.; Weeks, C.M. In: Estrogens in the Environment;
McLachlan, Ed.; North Holland: New York, 1980, pp. 11-31.
Blair, R.M.; Fang, H.; Branham, W.S.; Hass, B.S.; Dial, S.L.; Mo-
land, C.L.; Tong, W.; Shi, L.; Perkins, R.; Sheehan, D.M. Estrogen
receptor relative binding affinities of 188 natural and xenochemi-
cals: structural diversity of ligands. Toxicol. Sci., 2000, 54, 138-
153.
Pinto, M.M.; Sousa, E.; Correia-da-Silva, M.; Marques, F.; Car-
valho, F. Xantonas sulfatadas e análogos xantónicos glicosilados
sulfatados com actividade anticoagulante e processos para a sua
preparação. Portuguese Patent 104739, March 09, 2011.
Everett, G.E. The reactions of sulfur trioxide, and of its adducts,
with organic compounds. Chem. Rev., 1962, 62, 549-589.
Kappe, C.O.; Murphree, S.; Dallinger, D. Practical Microwave
Synthesis for Organic Chemists: Strategies, Instruments, and Pro-
tocols; Wiley-Vch Verlag GmbH & Co. KGaA: Weinheim, 2009.
Pinto, M.M.M.; Castanheiro, R. Synthesis of Prenylated Xantho-
nes: An Overview. Curr. Org. Chem., 2009, 13, 1215-1240.
Jeso, V.; Nicolaou, K.C. Total synthesis of tovophyllin B. Tetra-
hedron Lett., 2009, 50, 1161-1163.
[25]
[26]
[6]
Castanheiro, R.A.P.; Pinto, M.M.; Silva, A.M.S.; Cravo, S.M.M.;
Gales, L.; Damas, A.M.; Nazareth, N.; Nascimento, M.S.J.; Eaton,
G. Dihydroxyxanthones prenylated derivatives: synthesis, structure
elucidation and growth inhibitory activity on human tumor cell
lines with improvement of selectivity for MCF-7. Bioorg. Med.
Chem., 2007, 15, 6080-6088.
[27]
[7]
[8]
[9]
Sousa, E.; Paiva, A.; Nazareth, N.; Gales, L.; Damas, A.M.; Nas-
cimento, M.S.J.; Pinto, M.M. Bromoalkoxyxanthones as promising
antitumor agents: synthesis, crystal structure and effect on human
tumor cell lines. Eur. J. Med. Chem., 2009, 44, 3830-3835.
Pedro, M.; Cerqueira, F.; Sousa, M.E.; Nascimento, M.S.J.; Pinto,
M.M. Xanthones as inhibitors of growth of human cancer cell lines
and their effects on the proliferation of human lymphocytes in vi-
tro. Bioorg. Med. Chem., 2002, 10, 3725-3730.
[28]
[29]
Sousa, E.; Silva, A.M.S.; Pinto, M.M.M.; Pedro, M.M.; Cerqueira,
F.A.M.; Nascimento, M.S.J. Isomeric kielcorins and dihydroxyxan-
thones: synthesis, structure elucidation and inhibitory activities of
[30]
[31]

Xanthones and Benzophenones Inhibitors of Tumor Cell Growth
Letters in Drug Design & Discovery, 2010, Vol. 7, No. 7 493
[32]
Westerman, P.W.; Gunasekera, S.P.; Uvais, M.; Sultanbawa, S.;
Kazlauskas, R. Carbon-13 N.m.r. Study of Naturally Occurring
Xanthones. Org. Magn. Reson., 1977, 9, 631-635.
Meyer, R.; Conzetti, A. Ueber 3.6-Dioxyxanthon. Ber. Dtsch.
Chem. Ges., 1897, 30, 969-973.
Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.;
Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vaigrowolff, A.;
Graygoodrich, M.; Campbell, H.; Mayo, J.; Boyd, M. Feasibility of
a high-flux anticancer drug screen utilizing a diverse panel of hu-
man tumor cell lines in culture. J. Natl. Cancer Inst., 1991, 83,
757-766.
Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J.T.; Bokessch, H.; Kenney, S.; Boyd, M.R.
New colorimetric cytotoxicity assay for anticancer-drug screening.
J. Natl. Cancer Inst., 1990, 82, 1107-1112.
Sousa, M.E.; Correia-da-Silva, M.; Pinto, M.M.M. In: Natural
Products Chemistry, Biochemistry and Pharmacology; Brah-
machari, Ed.; Narosa Publishing House PVT. LTD.: West Bengal,
India, 2009; Vol. 15, pp. 392-416.
[35]
[36]
[33]
[34]