Xanthones as inhibitors of growth of human cancer cell lines and their effects on the proliferation of human lymphocytes in vitro

Article abstract of DOI:10.1016/S0968-0896(02)00379-6

Twenty-seven oxygenated xanthones have been assessed for their capacity to inhibit in vitro the growth of three human cancer cell lines, MCF-7 (breast cancer), TK-10 (renal cancer) and UACC-62 (melanoma). The effect of these xanthones on the proliferation

Full text of DOI:10.1016/S0968-0896(02)00379-6

Bioorganic & Medicinal Chemistry 10 (2002) 3725–3730
Xanthones as Inhibitors of Growth of Human Cancer Cell
Lines and Their Effects on the Proliferation of Human
Lymphocytes In Vitro
Madalena Pedro,a Fatima Cerqueira,a Maria Emılia Sousa,a,b
Maria Sao Jose Nascimentoa and Madalena Pintoa,*
a
´
Centro de Estudos de Quımica Orgaˆnica, Fitoquımica e Farmacologia da Universidade do Porto, Faculdade de Farmacia,
´
´
´
Rua Anıbal Cunha, 164, 4050-047 Porto, Portugal
bInstituto Superior das Cieˆncias da Sau´de-Norte, Rua Central da Gandra, 1317, 4580 Gandra, Portugal
Received 21June 2002; accepted 9 August 2002
Abstract—Twenty-seven oxygenated xanthones have been assessed for their capacity to inhibit in vitro the growth of three human
cancer cell lines, MCF-7 (breast cancer), TK-10 (renal cancer) and UACC-62 (melanoma). The effect of these xanthones on the
proliferation of human T-lymphocytes was also evaluated. Differences on their potency towards the effect on the growth of the
human cancer cell lines as well as on the proliferation of human T-lymphocytes can be ascribed to the nature and positions of the
substituents on the xanthonic nucleus.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
and investigated their potential antitumor and immu-
nomodulatory activity.
The xanthone structure is a very interesting scaffold for
different groups in different positions leading to a large
variety of pharmacological activities reported1ꢀ15
namely inhibitors of growth of a variety of tumor cell
lines6ꢀ11 as well as modulators of PKC isoforms.12
Although the anti-inflammatory activity of this group of
compounds is documented,13,14 little information has
been accumulated about its interference with the effectors
cells of the specific immunity.15
Consequently, this work reports the synthesis and the
study of the in vitro effect of twenty seven xanthone
derivatives on the growth of three human cancer cell
lines, MCF-7 (breast cancer), TK-10 (renal cancer) and
UACC-62 (melanoma) and on the mitogenic response
of human lymphocytes to phytohemaglutinin (PHA).
The aspect of structure–activity relationship within this
class of compounds is also tentatively discussed.
As xanthones from natural origin are relatively limited
in type and position of the substituents imposed by the
biosynthetic pathways, the syntheses of new compounds
can afford to enlarge the possibilities of having different
nature and positions of the substituents on the xanthonic
nucleus that will allow us to rationalize and characterize
the structure features that are important to their activity.
Results and Discussion
Chemistry
Chemically xanthones (9H-xanthen-9-ones) are hetero-
cyclic compounds with the dibenzo-g-pyrone framework
(Fig. 1). The xanthone nucleus is numbered according
to a biosynthetic convention with carbons 1–4 being
assigned to acetate-derived ring A and carbons 5–8 to
the shikimate-derived ring B. The other carbons are
indicated as 4a, 4b, 8a, 9 and 9a for structure elucida-
tion purposes.
Based on these considerations we have synthesized a
series of mono-, di- and trioxygenated simple xanthones
*Corresponding author. Tel.: +351-222-057358; fax: +351-222-
057358; e-mail: madalena@ff.up.pt
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00379-6

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M. Pedro et al. / Bioorg. Med. Chem. 10 (2002) 3725–3730
given in concentrations that were able to cause 50% of
cell growth inhibition (GI50), are summarized in Table
1. The growth inhibition effects presented by these xan-
thones cannot be attributed to a toxic effect, as inferred
from the sulforhodamine B (SRB) assay (data not
shown).
Figure 1. Nucleus of xanthone.
The standard methods for the synthesis of xanthones
include the via benzophenone (I) and the via diaryl
ether (II) intermediates (Scheme 1). In the Grover, Shah
and Shah classic method the xanthones are obtained by
condensation between an ortho-oxygenated benzoic acid
and an activated phenol, in phosphorous oxychloride
and zinc chloride (a).16 The intermediate benzophenone
derivatives (I) are also accessible through condensation
by Friedel–Crafts acylation of appropriately substituted
benzoyl chlorides with phenolic derivatives (b).17 While
the benzophenone method involves the cyclization of
2,20-dioxygenated benzophenones (I) through a dehy-
drative or oxidative process (c),17 the diaryl method
(Ullman synthesis)18 was carried out via suitable biphe-
nyl ether intermediate (II) with the ring formation by
one-step conversion by lithium diisopropylamide (d)19
or by acetyl chloride (e).20
No capacity to inhibit the growth of the human cancer
cell lines was observed with xanthone (1) even when
tested at concentrations higher than 200 mM, but the
introduction of oxygenated groups in the xanthonic
nucleus led, in general, to the appearance of a dose
dependent growth inhibitory effect. Although this effect
showed to be not very strong (GI50>50 mM) for the
majority of the xanthone derivatives, some xanthones
exhibited interesting growth inhibitory effects on the
human cancer cell lines tested with GI50 values ꢁ20 mM.
Although there was, in general, no discernible cell-type
selectivity for the xanthones studied, it was found that
1,2-dihydroxyxanthone (10), 3,4-dihydroxyxanthone (13),
and 2,3-dihydroxy-4-methoxyxanthone (25) were signif-
cantly (p<0.01) much more active to the melanoma
UACC-62 cell line than to MCF-7 and TK-10.
In the mono-oxygenated xanthones with substituents on
C(2) or C(4) it is possible to observe drastic changes in
the potency of the growth inhibitory activity: substituting
OH by OCH3 on C(2) leads to the appearance of activity
while the opposite phenomenon occurs with the same
change on C(4).
The compounds 2, 6 and 24 were synthesized by via (a),
while the compounds 3, 4, 7, 8, 13, 14, 17, 18, 20–23,
25–27 were obtained by via (b). The diaryl process has
led to the synthesis of the compounds 10 and 15 by via
(d) and the compounds 5, 9, 12 and 16 by via (e),
respectively (see Experimental protocols).
The nature of the substituents also seems to influence the
growth inhibitory effect of the dioxygenated xanthones.
The dihydroxylation at 1,2 (10), 2,3 (12), 3,4 (13), and
3,5 (14) positions of the xanthonic nucleus was asso-
ciated with compounds that showed growth inhibitory
effects that were significantly (p<0.05) stronger to all
the three cell lines than those presented by 1,2- (15), 2,3-
(16), 3,4- (17), and 3,5-dimethoxyxantones (18). Simi-
larly, the hydroxy and methoxy substituents at 2,1( 19),
4,3 (22), and 5,3 (23) positions were associated with a
For compounds 10, 12, 13, and 16 are indicated, for the
first time, spectral data from INEPT, HETCOR, COSY
or HMBC experiments in order to clarify the structural
elucidation of these compounds.
Effect on the growth of human cancer cell lines
The effects of the 27 xanthones on the growth of three
human cancer cell lines, MCF-7, TK-10, UACC-62,
Scheme 1. General methods for the synthesis of xanthones.

M. Pedro et al. / Bioorg. Med. Chem. 10 (2002) 3725–3730
3727
Table 1. Effects of xanthones on the growth of human cancer cell lines and proliferation of human lymphocytes
GI50 (mM)a
IC50 (mM)a
No. Compounds
MCF-7b (breast cancer)
TK-10b (renal cancer)
UACC-62b (melanoma)
Human lymphocytesc
1 Xanthone
>200
>200
>200
>200
2 1-Hydroxy
3 2-Hydroxy
4 3-Hydroxy
5 4-Hydroxy
>200
>200
69.3ꢂ10.9
>200
25.7142.9ꢂ20.11
>200
ꢂ21.7
>200
>200
125.5ꢂ23.8
125.8ꢂ7.9
117.9ꢂ9.4
80.2ꢂ9.2
53.8ꢂ5.0
66.0ꢂ2.7
120.8ꢂ12.1
6 1-Methoxy
7 2-Methoxy
8 3-Methoxy
9 4-Methoxy
94.4ꢂ8.2
87.4ꢂ8.5
>200
106.2ꢂ10.2
118.7ꢂ14.7
94.4ꢂ14.7
>200
78.1ꢂ14.5
59.0ꢂ8.2
73.8ꢂ12.1
>200
180.5ꢂ34.8
142.5ꢂ23.2
53.5ꢂ5.1
>200
>200
10 1,2-Dihydroxy
11 1,7-Dihydroxy
12 2,3-Dihydroxy
13 3,4-Dihydroxy
14 3,5-Dihydroxy
38.4ꢂ2.7
57.1ꢂ11.0
40.6ꢂ1.3
40.5ꢂ1.5
146.5ꢂ13.1
4.0
65.8ꢂ5.11
60.1ꢂ9.6
61.4ꢂ4.3
59.2a
ꢂ0.3
51.3ꢂ9.6
31.7ꢂ3.7
21.6ꢂ2.6
ꢂ9.8
73.3ꢂ2.2
20.2ꢂ0.2
31.3ꢂ1.5
12.2ꢂ1.3
180.2ꢂ5.1
101.6ꢂ9.177.5
15 1,2-Dimethoxy
16 2,3-Dimethoxy
17 3,4-Dimethoxy
18 3,5-Dimethoxy
>200
174.1ꢂ13.8
>200
>200
174.1ꢂ14.1
130.1ꢂ11.3
>200
136.7ꢂ16.6
125.9ꢂ21.1
162.1ꢂ22.2
>200
>200
59.9ꢂ18.5
42.1ꢂ6.3
>200
>200
19 2-Hydroxy-1-methoxy
20 3-Hydroxy-4-methoxy
21 3-Hydroxy-5-methoxy
22 4-Hydroxy-3-methoxy
23 5-Hydroxy-3-methoxy
24.1ꢂ3.0
>200
>200
35.2ꢂ7.6
>200
49.6ꢂ16.8
72.7ꢂ9.2
30.9ꢂ2.3
39.3ꢂ10.6
>200
36.9ꢂ20.2
78.5ꢂ11.9
37.7ꢂ6.7
11.6ꢂ0.8
178.5ꢂ8.9
>200
108.8ꢂ22.7
92.8ꢂ11.8
66.1ꢂ12.6
111.2ꢂ8.3
24 1,3-Dihydroxy-2-methyl
21.9ꢂ0.4
37.2d
34.3ꢂ3.8
76.6ꢂ7.0
73.1ꢂ9.0
128.1ꢂ14.3
20.0ꢂ0.5
19.8ꢂ1.6
66.7ꢂ8.5
59.3ꢂ7.5
84.4ꢂ12.4
17.4ꢂ2.7
80.7ꢂ11.7
19.2ꢂ3.3
25 2,3-Dihydroxy-4-methoxy
26 1-Formyl-4-hydroxy-3-methoxy
27 2-Formyl-3-hydroxy-4-methoxy
75.3ꢂ8.6
82.7 ꢂ14.8
aResults are expressed as GI50 (concentrations of compounds that cause 50% inhibition of cancer cell growth) or IC50 (concentrations that cause
50% inhibition of lymphocytes proliferation) and show meansꢂSEM of 3–6 independent observations performed in duplicate.
bDoxorubicin was used as positive control in cancer cell lines growth (GI50 MCF-7=42.8ꢂ8.2 nM; GI50 TK-10=548.0ꢂ60.0 nM; GI50 UACC-
62=94.0ꢂ9.4 nM).
cCyclosporin A was used as positive control in lymphocytes proliferation (IC50=0.34ꢂ0.04 mM).
dData based on two independently run duplicate experiments.
significant (p<0.05) increase of activity when compared
with dimethoxyderivatives (15, 17, 18) in the same
positions. Interestingly the hydroxy-methoxylation at
3,4 (20) caused a complete loss of activity of the com-
pound. The improvement of the activity from
3-hydroxy-4-methoxyxanthone (20) to 3,4-dihydroxy-
xantone (13) is also remarkable especially for UACC
cell line. When 4-hydroxy-3-methoxyxanthone (22) and
3-hydroxy-4-methoxyxanthone (20) were compared for
their activity, it was observed that the shift of the sub-
stituents lead to a complete lost of activity in all the cell
lines.
Xanthone (1) showed to be devoid of activity. But the
mono-, di- and tri-substitution of the xanthonic nucleus
was shown to be, in general, associated with the
appearance of a dose-dependent antiproliferative effect.
Although the majority of the xanthone derivatives have
exhibited a weak inhibitory effect (IC50>50 mM), some
oxygenated xanthones, namely 1,7-dihydroxyxanthone
(11), 3,4-dihydroxyxanthone (13), 2-hydroxy-1-methoxy-
xanthone (19), 2,3-dihydroxy-4-methoxyxanthone (25),
and 2-formyl-3-hydroxy-4-methoxyxanthone (27) were
found to possess a pronounced inhibitory activity on the
mitogenic response of human lymphocytes to PHA
(IC50ꢁ20 mM). No lymphocytoxicity was observed
when the human lymphocytes were exposed to the IC50
concentrations of these xanthones (cell viability
>70%), which leads to the conclusion that their inhibi-
tory activity was associated with cell proliferation rather
than to a toxic effect (data not shown).
In the case of the trisubstituted xanthones tested, the
number of compounds is not sufficient to draw any
conclusion. However, it is interesting to point out that
1,3-dihydroxy-2-methylxanthone (24) was found to
have the best inhibitory activity to all the three cell lines.
Table 1showed that the mono-oxygenated xanthones
exhibited in generally weaker antiproliferative effect
on T-lymphocytes than di- and trioxygenated xan-
thones. However, there are interesting structural fea-
tures within the mono-oxygenated xanthones on their
activity.
Effect on human lymphocytes proliferation
The effects of the 27 xanthones against the proliferation
of human lymphocytes to PHA, given in concentrations
that were able to cause 50% inhibition of proliferation
(IC50), are also shown in Table 1.

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M. Pedro et al. / Bioorg. Med. Chem. 10 (2002) 3725–3730
The nature of the substituents on C(2) and C(4) of the
xanthonic nucleus showed to be very decisive for the
antiproliferative effect of these compounds. Thus,
2-hydroxyxanthone (3) and 3-hydroxyxanthone (4)
which OH groups on C(2) and C(3) were inactive while
the presence of OCH3 group in the same positions cau-
ses the appearance of the activity in 2-methoxy (7) and
3-methoxyxanthone (8). In contrast, the activity was
lost when substituting OH group on C(4) in 4-hydroxy
xanthone (5) by OCH3 in 4-methoxyxanthone (9).
Experimental
Chemistry
Purifications of compounds were performed by column
chromatography (CC) using Merck silica gel 60 (0.50–
0.20 mm), flash chromatography using Merck silica gel
60 (0.040–0.063 mm) and preparative thin layer chro-
matography (TLC) using Merck silica gel 60 (GF254).
Melting points were obtained in a Kofler microscope
and are uncorrected. IR spectra were recorded on a
1
Perkin–Elmer 257 in KBr. H and 13C NMR spectra
In the dioxygenated xanthones, the nature and positions
of the substituents also played an important role in their
activity. Thus 1,2-dihydroxyxanthone (10) (IC50=73.3
mM) is more than 6-fold less active than 2-hydroxy-1-
methoxyxanthone (19) (IC50=11.6 mM) when OH was
replaced by OCH3 on C(1) (p<0.01). Interestingly, the
complete lost of activity was observed when both OH
groups were replaced by OCH3 in 1,2-dimethoxy deri-
vative (15). The effects of OH and OCH3 substituents
were also observed for activity of the xanthones with
these substituents on the positions 3,4. As a result, 3,4-
dihydroxyxanthone (13) (IC50=12.2 mM) was more
active than 3,4-dimethoxyxanthone (17) (IC50=42.1
mM) and even more than 3-hydroxy-4-methoxy-
xanthone (20) (IC50=178.5), respectively.
were taken in CDCl3 or DMSO-d6 at room temperature,
on Bruker AC 200, DRX 300 and DRX 500 instruments.
Chemical shifts are expressed in d (ppm) values relative
to tetramethylsilane (TMS) as an internal reference. MS
spectra were recorded as EI (electronic impact) mode on
a Hitachi Perkin–Elmer. Xanthone was purchased from
Sigma Chemical Co., St. Louis, USA.
The following xanthone derivatives were synthesized
and purified by the described procedures.
The synthesis of the following xanthones has already
been described according to the references cited below.
1-Hydroxyxanthone (2) (48%), 2-hydroxyxanthone (3)
(74%), 3-hydroxyxanthone (4) (92%), 4-hydroxy-
xanthone (5) (59%), 1-methoxyxanthone (6) (86%), 2-
methoxyxanthone (7) (66%), 3-methoxyxanthone (8)
(45%), 4-methoxyxanthone (9) (14%), 3,4-dimethoxy-
xanthone (17) (61%), 3,5-dihydroxyxanthone (14) (55%),
3,5-dimethoxyxanthone (18) (35%), 3-hydroxy-5-meth-
oxyxanthone (21) (42%), 3-hydroxy-4-methoxyxanthone
(20) (51%) and 4-hydroxy-3-methoxyxanthone (22) (39%)
were obtained according to the described procedure.4 1,2-
Dimethoxyxanthone (15) (74%) was obtained21 and
characterized21,22 according to the described procedures.
Two of the trisubstituted xanthones 2,3-dihydroxy-4-
methoxyxanthone (25) and the 2-formyl-3-hydroxy-4-
methoxyxanthone (27) were found to be the most active.
The presence of OH or CHO on C(2) seemed to be
responsible for the ten-fold increase of the anti-
proliferative activity of these compounds when com-
pared to 3-hydroxy-4-methoxyxanthone (20).
Conclusion
In summary, from all these results it can be inferred that
the effects of oxygenated xanthones on the growth of
the human cancer cell lines as well as on the prolifera-
tion of human T-lymphocytes can be ascribed to the
nature, positions and number of substituents on the
xanthonic nucleus.
2-Hydroxy-1-methoxyxanthone (19) and 1,7-dihydroxy-
xanthone (11). These xanthones 2-hydroxy-1-methoxy-
xanthone (19) and 1,7-dihydroxyxanthone (11) were
isolated from Calophyllum teysmannii var. inophylloide
(Guttiferae)15 and Cratoxylum maingayi (Guttiferae),23
respectively.
Concerning the effect on the growth of human cancer
cell lines, 1,3-dihydroxy-2-methylxanthone (24) was
found to have the best inhibitory activity to all the three
cell lines, while some selectivity for UACC-62 cell line
was found for 1,2-dihydroxy (10), 3,4-dihydroxy (13),
and 2,3-dihydroxy-4-methoxyxanthones (25).
1,2-Dihydroxyxanthone (10). The compound 10 was
obtained by demethylation of 1,2-dimethoxyxanthone (15)
according to the described procedure (48%).24,25 Mp 163–
165ꢃC, methylene chloride/hexane (lit.,26 166–167 ꢃC, eth-
anol aq); UV (EtOH): 261, 243, 232, 203 (33,300, 28,800,
14,200, 11,000); UV (EtOH+NaOH): 311, 236, 222
(76,000, 23,900, 17,000); UV (EtOH+AlCl3): 305, 283,
266, 204 (33,000, 22,900, 87,00); IR (KBr) umax: 3467,
The dihydroxylation at all the positions of the xanthone
derivatives tested was associated with stronger inhibi-
tion to all the three cell lines than those presented by the
corresponding dimethoxy analogues.
1606, 1475, 1365, 1288, 1045, 748 cmꢀ1 1H NMR
.
(DMSO-d6, 300.13 MHz) d:27,28 12.45 (1-OH), 9.42
(2-OH), 8.16 (1H, dd, J=7.9, and 1.6 Hz, H-8), 7.81
(1H, ddd, J=8.4, 7.0, and 1.6 Hz, H-6), 7.60 (1H, dd,
J=8.4, and 0.8 Hz, H-5) 7.46 (1H, ddd, J=7.9, 7.0, and
0.8 Hz, H-7), 7.32 (1H, d, J=9.0 Hz, H-4), 6.96 (1H, d,
J=9.0 Hz, H-3); 13C NMR (DMSO-d6, 75.47 MHz) d:
182.3 (C-9), 155.9 (C-4b), 148.3 (C-4a), 147.6 (C-1),
140.2 (C-2), 136.4 (C-6), 125.4 (C-8), 124.5 (C-3), 124.2
(C-7), 119.3 (C-8a), 118.0 (C-5), 108.8 (C-9a), 106.2
For the effect on human lymphocytes proliferation the
dioxygenated derivatives 2-hydroxy-1-methoxyxanthone
(19) and 3,4-dihydroxyxanthone (13) showed the most
interesting results for the inhibitory response.
Based on the described results further modifications are
underway to improve the activity of these compounds.

M. Pedro et al. / Bioorg. Med. Chem. 10 (2002) 3725–3730
3729
(C-4); EIMS, m/z (% rel. int.): 230 (1, [M+2]+), 229 (1,
[M+1]+), 228 (70, [M+]), 199 (13), 126 (5), 115 (4), 78
(95), 63 (100).
formamide (DMF), (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT), dimethylsulfoxide
(DMSO), doxorubicin hydrochloride, gentamicin,
l-glutamine, Histopaque-1077, phytohemagglutinin
(PHA), sodium lauryl sulfate (SDS), and sulforhod-
amine B (SRB) were purchased from Sigma Chemical
Co. National Cancer Institute (Bethesda, MD, USA)
kindly provided the tumor cell lines.
2,3-Dihydroxyxanthone (12). The compound 12 was
obtained by demethylation of 2,3-dimethoxyxanthone
(17) according to the described procedure (91%).25 Mp
>330 ꢃC, acetone/hexane (lit.25 >350 ꢃC ethyl acetate,
lit.29 293–295 ꢃC chloroform/methanol); UV, IR and
EIMS data accord with lit.29 The unequivocal assignments
of carbon resonances, mainly by using 1D selective
INEPT and 2D HETCOR experiments, allowed us to
correct some 1H and 13C resonances of 12,29 namely the
assignment of the proton resonances of H-6 to 7.78 (1H,
ddd, J=8.4, 6.8, and 1.7 Hz) and H-5 to 7.58 (1H, d,
J=8.4 Hz), and the carbon resonances of C-2 (144.1),
C-3 (151.3), and C-4a (154.2).
Stock solutions of xanthones were prepared in DMSO and
stored at ꢀ20 ꢃC providing uniform samples for re-tests.
These frozen concentrates were then diluted to the
desired final concentrations with the appropriate solvents
immediately prior the biological assays. Final con-
centrations of DMSO showed no interference with the
biological activities tested.
Cell growth assay. The effects of compounds on the
growth of human cancer cell lines were evaluated
according to the procedure adopted in the NCI’s in
vitro anticancer drug screening that uses the SRB assay
to assess growth inhibition.35 This colorimetric assay
estimates cell number indirectly by staining cellular
protein with the protein-binding dye SRB. Three human
tumor cell lines were used, MCF-7 (breast cancer), TK-
10 (renal cancer), UACC-62 (melanoma). Cells were
routinely maintained as adherent cell cultures in RPMI-
1640 medium containing 5% heat-inactivated fetal
bovine serum, 2 mM glutamine and 50 mg/mL gentamicin
at 37 ꢃC in a humidified air incubator containing 5%
CO2. Each cell line was plated at an apropriate density
(1.5ꢄ105 cells/mL to MCF-7 and TK-10, 1.0ꢄ105 cells/
mL to UACC-62) in 96-well plates and allowed to
attach overnight. Cells were then exposed for 48 h to
serial concentrations of compounds and to the positive
control, doxorubicin. Following this incubation period,
the adherent cells were fixed in situ washed and dyed
with SRB. The bound stain was solubilized and the
absorbance was measured at 492 nm in a microplate
reader. Growth inhibition of 50% (GI50) was calculated
as described elsewhere.36 Toxicity of xanthones was
inferred from the SRB assay by comparing, after 48 h,
the absorbance of the wells with xanthone-treated cells
with the absorvance of the wells containing untreated
cells that were fixed at time zero (time at which com-
pounds were added). Lower absorbances after 48 h of
treatment indicate occurrence of cell death instead of
growth arrest.
3,4-Dihydroxyxanthone (13).25 (78%) Mp above 330 ꢃC
(lit.,16 240–241 ꢃC, ethanol aq; lit.,29 238–240 ꢃC,
methanol); UV, IR and EIMS data accord with lit.29 the
1
analysis of the H, 13C, COSY, HETCOR and HMBC
spectra of 12 permits correction of the literature29
assignment of the proton resonances of H-6 and H-5 to
7.81(1H, ddd, J=8.6, 6.9, and 1.7 Hz, H-6), and 7.62
(1H, dd, J=8.6, and 0.9 Hz, H-5).
2,3-Dimethoxyxanthone (16). The 2-(30,40-dimethoxy-
phenoxy) benzoic acid obtained by an Ullman reac-
tion18 underwent an appropriate cyclization to 16
according to the described procedure (92%).20,30 Mp
170–171 ꢃC, methylene chloride/pentane (lit.,20 164–
165 ꢃC, acetone; lit.,29 157–158 ꢃC, chloroform; lit.,31
155–159 ꢃC, methylene chloride/heptane; lit.,32 165–
167 ꢃC, chloroform/petrol); IR and EIMS data accord
with lit.29 The unequivocal assignments of carbon reso-
nances, mainly by using 1D selective INEPT and 2D
1
HETCOR experiments, allowed us to correct some H
and 13C resonances of 16:17,29 1H NMR (DMSO-d6,
200.13 MHz) d: 8.16 (1H, dd, J=7.9 and 1.5 Hz, H-8),
7.82 (1H, ddd, J=8.4, 7.4 and 1.5 Hz, H-6), 7.60 (1H, d,
J=8.4 Hz, H-5), 7.49 (1H, s, H-1), 7.44 (1H, ddd,
J=7.9, 7.4, and 0.9 Hz, H-7), 7.20 (1H, s, H-4), 3.94 (3H,
s, 3-OCH3), 3.87 (3H, s, 2-OCH3); 13C NMR (DMSO-d6,
50.03 MHz) d: 174.8 (C-9), 155.7 (C-4b), 155.6 (C-4a),
152.0 (C-3), 146.7 (C-2), 134.8 (C-6), 125.9 (C-8), 124.3
(C-7), 120.9 (C-8a), 118.0 (C-5), 114.0 (C-9a), 104.7 (C-
1), 100.5 (C-4), 56.6 (3-OCH3), 55.9 (2-OCH3).
1,3-Dihydroxy-2-methylxanthone (24). The compound
24 was obtained (28%) and characterized according to
the described procedures.33,34
Lymphocyte assays
The effects of xanthones on the mitogenic response of
human lymphocytes to PHA (10 mg/mL) were evaluated
using a modified colorimetric MTT assay.37 This assay
was previously described by us.15 Mononuclear cells
were isolated from heparinized peripheral blood of
healthy volunteers by Histopaque-1077 density centri-
fugation. The toxicity of xanthones against human
lymphocytes was evaluated by an assay based on the
ability of viable cells to reduce the colorless tetrazolium
salt MTT to a colored formazan product. Briefly, in flat-
bottom 96-well plates, 2–3ꢄ106 mononuclear cells/mL in
RPMI-1640 medium containing 10% heat-inactivated
2,3-Dihydroxy-4-methoxyxanthone (25), 1-formyl-4-
hydroxy-3-methoxyxanthone (26), 2-formyl-3-hydroxy-4-
methoxyxanthone (27). The compounds 25 (74%), 26
(36%) and 27 (28%) were obtained according to the
described procedure.34
Pharmacology
Fetal bovine serum and RPMI-1640 medium were
obtained from Gibco BRL. Cyclosporin A, N,N-dimethyl-

3730
M. Pedro et al. / Bioorg. Med. Chem. 10 (2002) 3725–3730
fetal bovine serum, 2 mM glutamine and 50 mg/mL of
gentamicin were exposed for 24 h to the various con-
centrations of each sample. Following this incubation
period the MTT solution (1mg/mL) was added. After
incubation for 4 h the MTT formazan products were
solubilized with SDS/DMF solution (20% SDS in a
50% solution of DMF, pH 4.7) overnight at 37 ꢃC.
Absorbance (OD 550 nm) of the colored solution was
measured with a plate reader. Lymphocytoxicity, deter-
mined in terms of the percentage of viable cells, was
present when the viability of the exposed cells was less
than 70% of the non-exposed control cells.
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Acknowledgements
The authors thank FCT (I&D No. 226/94), POCTI (QCA
III), and FEDER for financial support and to PRAXIS
XXI and FCT for the PhD grants to Madalena Pedro
(SFRH/BD/1456/2000), Fatima Cerqueira (PRAXIS
XXI/BD/21801/99) and to Emılia Sousa (PRAXIS
XXI/BD/15663/98). We are indebted to the National
Cancer Institute, Bethesda, MD, USA for the generous
provision of the tumor cell lines.
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