Bromoalkoxyxanthones as promising antitumor agents: Synthesis, crystal structure and effect on human tumor cell lines

Article abstract of DOI:10.1016/j.ejmech.2009.04.011

In a study involving the synthesis of bis-intercalators, a bisxanthone and a minor product, 1-(6-bromohexyloxy)-xanthone were obtained. Although no capacity to inhibit the growth of human tumor cell lines was observed for the bisxanthone, the bromoalkoxyx

Full text of DOI:10.1016/j.ejmech.2009.04.011

European Journal of Medicinal Chemistry 44 (2009) 3830–3835
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Bromoalkoxyxanthones as promising antitumor agents:
Synthesis, crystal structure and effect on human tumor cell lines
a
,
a
b
c
,
,d
Emılia Sousa , Ana Paiva , Nair Nazareth , Luis Gales d, Ana M. Damas c
,
*
´
Maria S.J. Nascimento b, Madalena Pinto a
a
ˆ
´
´
Laborato´rio de Qu´ımica Organica, Centro de Estudos Quımica Medicinal da Universidade do Porto (CEQUIMED-UP), Faculdade de Farmacia,
Universidade do Porto, Rua An´ıbal Cunha, 164, 4050-047 Porto, Portugal
b
´
Laborato´rio de Microbiologia, Centro de Estudos Qu´ımica Medicinal da Universidade do Porto (CEQUIMED-UP), Faculdade de Farmacia,
Universidade do Porto, Rua An´ıbal Cunha, 164, 4050-047 Porto, Portugal
c
ˆ
´
ICBAS, Instituto de Ciencias Biomedicas Abel Salazar, Universidade do Porto, Largo Prof. Abel Salazar no. 2, 4099-003 Porto, Portugal
d IBMC, Instituto de Biologia Molecular e Celular, Universidade do Porto, R. do Campo Alegre no. 823, 4150 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
In a study involving the synthesis of bis-intercalators, a bisxanthone and a minor product, 1-(6-bro-
mohexyloxy)-xanthone were obtained. Although no capacity to inhibit the growth of human tumor cell
lines was observed for the bisxanthone, the bromoalkoxyxanthone revealed this biological activity. In
light of these results bromoalkylation of 3,4-dihydroxyxanthone furnished two bromohexyloxy-
xanthones that were investigated for their effect on the in vitro growth of human tumor cell lines MCF-7
(ERþ, breast), MDA-MB-231 (ERꢀ, breast), NCI-H460 (non-small lung), and SF-268 (central nervous
system). The X-ray structure of 1-(6-bromohexyloxy)-xanthone revealed that the xanthone skeleton
remains essentially planar forming a dihedral angle of 61.3(2)ꢁ with the 6-bromohexyl side chain. These
results revealed bromoalkoxyxanthones as interesting scaffolds to look for potential anticancer drugs.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Received 17 February 2009
Received in revised form
23 March 2009
Accepted 3 April 2009
Available online 14 April 2009
Keywords:
Antitumor
Bromoalkoxyxanthone
Oxygen heterocycle
X-ray diffraction
Xanthones
1. Introduction
an effect compatible with protein kinase C (PKC) activation [6,7]
and PKC inhibition [8].
Heterocycles play an important role in the design and discovery
of new pharmacologically active compounds. The xanthone
framework is an important O-heterocycle of documented relevance
for development of anticancer agents [1]. Two derivatives, dime-
thylxanthenone-4-acetic acid (DMXAA), that causes tumor necrosis,
and psorospermin, that acts by intercalation into the DNA molecule
and by the alkylation of guanine at the topoisomerase II cleavage
site, have entered clinical trials. Recently, there has been an increase
in the synthesis of new xanthonic structures [1,2], partly due to
the ability for their derivatives to bind to different classes of
receptors [1].
DNA is also an important target for prenylated and/or oxygen-
ated xanthones that due to their specific binding are capable of
a wide range of antitumor responses such as DNA breaks, DNA–
protein cross-links and DNA synthesis suppression [9–12]. The
overall results for xanthone binding studies with DNA indicate that
the planar tricycle moiety serves as an important feature for
designing new DNA intercalators. There are a number of DNA
intercalators that recognize one of the DNA groves, although the
three-dimensional structures of some complexes reveal differences
in the position of the drugs within each grove. Efforts to identify
molecules with a greater affinity for DNA have resulted in the
development of bis-intercalators in which two intercalating
ligands (hydrophilic [13–15] or lipophilic [16]) are linked by
a chain.
Due to this interest, natural and synthetic xanthones have been
reported by our group to demonstrate an antiproliferative effect on
human tumor cell lines [3–5] and on human lymphocytes [3,4] and
The primary aim of this work was to obtain potential DNA bis-
intercalators with a xanthonic framework for CNS tumors, i.e., with
suitable lipophilicity for blood–brain barrier penetration. With that
purpose, the synthesis of dimeric compounds with an alkyl bridge
was initially planned.
* Corresponding author. Tel.: þ351 222078984; fax: þ351 222003977.
E-mail address: esousa@ff.up.pt (E. Sousa).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.011

E. Sousa et al. / European Journal of Medicinal Chemistry 44 (2009) 3830–3835
3831
2. Chemistry
(GI50 ¼ 30.2 ꢃ 3.6
m
M) and NCI-H460 (GI50 ¼ 30.2 ꢃ 3.6
m
M). Curi-
ously, this molecular modification led to the appearance of
The preparation of a hexamethylenebis(oxy)-linked assembly is
illustrated in Scheme 1. The Nencki reaction [step (I) in Scheme 1]
offered a convenient method for preparing 1-hydroxyxanthone (1)
in 48% yield [17]. The intermediate benzophenone 1a was also
isolated in 5% yield and its structure was determined by X-ray
crystallography.1,10-[Hexane-1,6-diylbis(oxy)]bis[9H-xanthen-9-one]
(2, 50%) and a minor product 1-(6-bromohexyloxy)-9H-xanthen-9-
one (3, 30%) were obtained from the reaction of 1-hydroxyxanthone
(1) with dibromohexane in basic conditions [step (II) in Scheme 1].
The work proceeded with the bromoalkylation of a more potent
xanthone, 3,4-dihydroxyxanthone (4), previously found as an
inhibitor of the growth of human tumor cell lines [3]. Two bro-
mohexyloxyxanthones, 3-(60-bromohexyloxy)-4-hydroxyxanthone
(5, 40%) and 3,4-bis(60-bromohexyloxy)-xanthone (6, 10%), were
obtained in higher yields with the dropwise addition of dibromo-
hexane (Scheme 2).
a growth inhibitory activity against the estrogen receptor (ERþ)
MCF-7 (GI50 ¼ 22.7 ꢃ 1.3 M) but not against the ERꢀ MDA-MB-231
m
(GI50 > 100 mM). The growth inhibition effects presented by these
xanthones and benzophenone derivatives cannot be attributed to
a toxic effect, as inferred from the sulforhodamine B assay (data not
shown).
Similarly to bis-alkoxyxanthone 2, no relevant growth inhibition
was observed for bromoalkoxyxanthone 5 on all the tumor cell lines
tested (GI50 > 100 mM). Compound 6 was found to inhibit only the
growth of the breast adenocarcinoma cell lines (MCF-7 and MDA-
MB-231). Similarly to compound 3, bromoalkoxyxanthone 6 was
also more active than its precursor 4 against ERþ MCF-7 (P < 0.05).
On the contrary, compound 4 was much more active with glioma SF-
268, lung NCI-H460, and MDA-MB-231 cell lines than with MCF-7.
Although more studies are needed to establish a structure–activity
relationship, results showed that one free hydroxyl group in the
bromoalkoxyxanthone scaffold (compound 5) is unfavorable to the
growth inhibitory activity against ERþ MCF-7.
3. Pharmacology: in vitro
The structure of compounds 1–6 was established on the basis of
IR, MS, and NMR techniques. For compounds 1 and 4 all the data
were in accordance with the literature [8,19]. The 1H NMR and
13C NMR data of compounds 2, 3, 5, and 6 are described for the first
time (see Experimental section). The analysis of the 1H and
13C NMR spectra of compounds 2, 3, 5, and 6 allowed the assign-
ment of the AKPX spin system corresponding to the resonance of
H-5, H-6, H-7 and H-8. The assignment of the corresponding carbon
resonances was made from the correlations found in the HSQC
spectra, whereas those of the quaternary carbons of the unsub-
stituted ring were based on the connectivities found in the HMBC.
Herein, the crystal structures of the inhibitor of human tumorcell
lines 1-(6-bromohexyloxy)-9H-xanthen-9-one (3) and of 2,20,4-
trihydroxybenzophenone (1a) are also described. A perspective
view of the crystal structure of compound 3 showing the atom-
labelling scheme is presented in Fig. 1 and was obtained using
ORTEP [20]. The atoms that form thexanthone skeleton (C1–C2–C3–
C4–C4A–O4A–C10A–C5–C6–C7–C8–C8A–C9–C9A) define a plane
with an rms deviation of 0.03 Å. The C9 and the O4A atoms are the
ones that deviate more from this plane: 0.05 Å and 0.06 Å, respec-
tively; while O1 lies on the plane, O9 is 0.104 Å away and this is
Being the investigation of secondary products a strategy in drug
discovery and having a bromoimidazolxanthone revealed as having
high potency in inhibiting P450 17a-hydroxylase-17,20-lyase,
a target for the development of drugs for cancer treatment [18],
both secondary products (1a) and (3) were investigated along with
compound 2 for their effect on the in vitro growth on four human
tumor cell lines: SF-268 (central nervous system cancer), NCI-H460
(non-small lung cancer), MCF-7 (breast cancer, ERþ), and MDA-
MB-231 (breast cancer, ERꢀ). 3-(60-Bromohexyloxy)-4-hydroxy-
xanthone (5) and 3,4-bis(60-bromohexyloxy)-xanthone (6) were
further investigated for their growth inhibitory effect. The results of
this study are summarized in Table 1.
4. Results and discussion
Although for bis-alkoxyxanthone (2) and 2,20,4-trihydroxy-
benzophenone (1a) no relevant growth inhibition was observed on
these tumor cell lines (GI50 ꢂ 100
mM), the secondary product
bromoalkoxyxanthone (3) showed an interesting growth inhibitory
effect, higher than the parent compound (1), against SF-268
OH
O
OH
O
7
OH
OH
2
COOH
I
2'
1
1'
3
4
3'
4'
+
+
OH
HO
O
OH
6'
6
5'
5
1
1a
II
5'
3'
1'
Br
O
O
O
O
1
2'
2
8
4'
6'
7
6
9
+
3
O
O
O
O
O
4
5
3
2
Scheme 1. Reagents and conditions: (I) zinc chloride, 200 ꢁC/5 min–180 ꢁC/4 h; (II) potassium carbonate, dry DMF, 1,6-dibromobutane, room temperature, 24 h.

3832
E. Sousa et al. / European Journal of Medicinal Chemistry 44 (2009) 3830–3835
O
O
O
OMe
OMe
COCl
OMe
a
b
+
OMe
HO
OMe
OMe
OMe
OMe
OMe
O
O
c
d
Br
Br
O
4
OH
O
O
OH
OH
+
5
O
1'
O
O
O
Br
1''
6
Scheme 2. Reagents and conditions: (a) aluminum chloride, dry Et2O, room temperature (85%); (b) NaOH, MeOH, H2O, reflux (98%); (c) aluminum chloride, dry toluene, reflux
(75%); (d) potassium carbonate, dry DMF, 1,6-dibromobutane dropwise addition, room temperature, 24 h.
probably due to a repulsion between these two oxygen atoms. The
C10–C20–C30–C40–C50–C60–Br atoms also lie in a plane defined with
an rms deviation of 0.04 Å and the dihedral angle between both
planes is 61.3(2)ꢁ. In the crystal structure of compound 3 the packing
of the molecules is governed essentially by van der Waals forces.
While most of xanthone crystal structures show an efficient packing
into columns with the xanthone skeleton planes parallel to one
another [21], this compound reveals two stacking planes, each of
them with an intermolecular separation of 4.0 Å.
The crystal structure of compound 1a is shown in Fig. 2. The two
aromatic ring planes form an angle of 44.69(6)ꢁ and the rotation of
the three OH groups around the C–OH bound is less than 10ꢁ in
relation to the plane of the respective aromatic ring. Moreover, the
hydroxyl groups bound to C2 and C20 are hydrogen bond to O7.
Similar interactions are found in crystal structures of xanthone
derivatives when –OH are bound to C1 or C8 [21,22].
intercalative binding to DNA. They comprise a polycyclic, rigid and
planar system coupled with a flanking long chain [23]. The dihedral
angle between the polycyclic structure and the long chain is
approximately 94ꢁ in the case of the widely prescribed anthracy-
cline antitumor antibiotic daunorubicin [24]. In most cases, the
polycyclic ring binds to the minor groove of DNA via hydrophobic
and stacking effects. Interestingly, a series of substituted imidazo-
thioxanthones were studied in order to find out about their
possible role as DNA triple helix-binding ligands [25] and the
results suggested that a protonated or positively charged side chain
Several promising compounds with antitumor and antiviral
activities share a common scaffold that is pertinent to possible
Table 1
Effect of compounds on the growth of human tumor cell lines.a
Compounds
GI50 (mM)
SF-268
(glioma)
NCI-H460
(lung)
MCF-7
(breast, ERþ)
MDA-MB-231
(breast, ERꢀ)
1
1a
2
70.8 ꢃ 10.9
>100
>100
84.9 ꢃ 13.6
91.2 ꢃ 8.7
>100
>100
100
>100
66.0 ꢃ 5.4
ND
>100
3
4
5
30.2 ꢃ 3.6
30.7 ꢃ 3.2
22.7 ꢃ 1.3
52.4 ꢃ 6.3
>100
>100
22.6b
11.4b
22.8 ꢃ 4.8
>100
>100
>100
6
>100
>100
20.5 ꢃ 1.9
56.8 ꢃ 11.2
a
Results are given in concentrations that were able to cause 50% of cell growth
inhibition (GI50) after a continuous exposure of 48 h and represent means ꢃ SEM of
3–5 independent experiments performed in duplicate and carried out
independently.
b
Results of one or two independent experiments performed in duplicate. Doxo-
rubicin was used as positive control, GI50
:
MCF-7 ¼42.8 ꢃ 8.2 nM; MDA-MB-
Fig. 1. Molecular structure of compound 3 showing the atom-labelling scheme. H
atoms are depicted as spheres of arbitrary radii. Displacement ellipsoids are shown at
the 50% probability level.
231 ¼10.86 ꢃ 1.28 nM; SF-268 ¼ 94.0 ꢃ 7.0 nM; NCI-H460 ¼ 94.0 ꢃ 8.7 nM. ND ¼
not determinate.

E. Sousa et al. / European Journal of Medicinal Chemistry 44 (2009) 3830–3835
3833
24 h at room temperature and then poured into ice-water (40 mL).
The yellow pale solid thus obtained was filtered and purified by CC
[petroleum ether (313–333 K boiling fraction)/diethyl ether, several
proportions]. The collected fractions from petroleum ether/diethyl
ether (8:2) furnish a yellow solid that was crystallized from acetone
to give compound 3 (30%) and from the collected fractions eluted
with petroleum ether/diethyl ether (6:4), a white solid was crys-
tallized from ethyl acetate/petroleum ether (4:1) to give compound
2 (50%).
Compound 2 mp 76 ꢁC; 1H NMR (300.13 MHz, DMSO-d6):
d
¼ 8.26 (H-8, dd, J ¼ 8.0 and 1.7 Hz, 2H), 7.65 (H-6, ddd, J ¼ 9.3, 6.2
and 1.7 Hz, 2H), 7.40 (H-3 and H-5, m, 4H), 7.32 (H-7, ddd, J ¼ 9.5,
6.3 and 1.0 Hz,) 2H, 6.91 (H-4, dd, J ¼ 7.5 and 0.8 Hz, 2H), 6.74 (H-2,
dd, J ¼ 7.5 and 0.5 Hz, 2H), 4.17 (H-10, t, 4H), 2.04 (H-20, m, 4H),
Fig. 2. Molecular structure of compound 1a showing the atom-labelling scheme.
1.77 ppm (H-30, m, 4H); 13C NMR (75.47 MHz, DMSO-d6):
d
¼ 176.2
(C-9), 160.3 (C-1), 158.0 (C-4a), 155.0 (C-10a), 134.6 (C-3), 133.9
(C-6), 126.7 (C-8), 123.6 (C-7), 123.1 (C-8a), 117.2 (C-5), 113.2 (C-9a),
109.5 (C-4), 106.3 (C-2), 68.8 (C-10), 28.7 (C-20), 24.9 ppm (C-30); IR
is necessary for the interaction with DNA. Although the side chain
in bromoalkoxyxanthones is not positively charged, the bromine
atom could serve as an anchor for the interaction with DNA.
(KBr):
n
¼ 1658, 1598, 1458, 1354, 1274, 1081, 760 cmꢀ1; MS (EI) m/z
(%): 506 (37) [M]þ, 225 (32), 212 (100), 196 (24). HRMS-ESI m/z
calcd. for C32H26O6Na: 529.1621597, found: 529.1607320.
Compound 3 mp 83–84 ꢁC; 1H NMR (300.13 MHz, DMSO-d6):
5. Conclusion
In conclusion, the bromoalkoxyxanthone scaffold was revealed
as promising for development of new potential antitumor drugs
and led to the increase of a growth inhibitory activity against the
estrogen receptor (ERþ) MCF-7 cell line. With the structure of 1-(6-
bromohexyloxy)-xanthone (3) resolved by X-ray crystallography,
DNA unwinding experiments combined with molecular modeling
studies concerning the binding of molecule 3 to DNA or to estrogen
receptors might disclose some mechanistic aspects in the future.
d
¼ 8.30 (H-8, dd, J ¼ 8.0 and 1.7 Hz, 1H), 7.65 (H-6, ddd, J ¼ 11.7, 7.7
and 1.7 Hz, 1H), 7.58 (H-3, dd, J ¼ 8.4 and 8.5 Hz, 1H), 7.41 (H-5, dd,
J ¼ 8.0 and 0.6 Hz, 1H), 7.34 (H-7, ddd, J ¼ 7.7, 6.7 and 1.0 Hz, 1H),
7.04 (H-4, dd, J ¼ 8.4 and 0.7 Hz, 1H), 6.78 (H-2, d, J ¼ 8.3 Hz, 1H),
4.14 (H-10, t, 2H), 3.45 (H-60, t, 2H), 1.97 (H-20 and H-50, m, 4H),
1.61 ppm (H-30 and H-40, m, 4H); 13C NMR (75.47 MHz, DMSO-d6):
d
¼ 176.4 (C-9), 160.2 (C-1), 158.1 (C-4a), 155.0 (C-10a), 134.7 (C-3),
134.1 (C-6), 126.7 (C-8), 123.7 (C-7), 123.0 (C-8a), 117.2 (C-5), 112.7
(C-9a), 109.8 (C-4), 106.3 (C-2), 69.1 (C-10), 34.0 (C-60), 32.6 (C-20),
28.9 (C-50), 27.9 (C-30), 25.2 ppm (C-40); IR (KBr):
6. Experimental
n
¼ 1656, 1598,
1479, 1356, 1236, 917, 777 cmꢀ1; MS (EI) m/z (%): 378 (1) [M þ 4]þ,
377 (5) [M þ 3]þ, 376 (15) [M þ 2]þ, 375 (6) [M þ H]þ, 374 (15) [M]þ,
296 (6), 295 (21), 277 (13), 239 (32), 226 (22), 225 (100), 212 (96),
196 (26). HRMS-ESI m/z calcd for C19H19O3BrNa: 397.0409782,
found: 397.0404690.
6.1. Chemistry
Purifications of compounds were performed by column chro-
matography (CC) using Merck silica gel 60 (0.50–0.20 mm) and for
monitoring reactions TLC with Merck silica gel 60 (GF254) was used.
Melting points were obtained in a Kofler microscope and are
uncorrected. IR spectra were recorded on a Perkin Elmer 257 in KBr.
1H and 13C NMR spectra were taken in CDCl3 or DMSO-d6 at room
temperature, on a Bruker DRX 300 instrument. Chemical shifts are
6.1.3. 3-(60-Bromohexyloxy)-4-hydroxy-9H-xanthen-9-one (5)
and 3,4-bis(60-bromohexyloxy)-9H-xanthen-9-one (6)
To a solution of compound 4 (1.0 g, 4.4 mmol) in dry DMF
(15 mL) was added K2CO3 (605 mg, 4.4 mmol) and the mixture was
stirred at room temperature for 15 min. To this suspension was
added, in 30 min a solution of 1,6-dibromobutane (1.1 g, 4.4 mmol)
in dry DMF (5 mL). The reaction mixture was stirred for 24 h at
room temperature and then poured into ice-water (50 mL). The
brown solid thus obtained was filtered and purified by CC [petro-
leum ether (313–333 K boiling fraction)/diethyl ether, several
proportions]. The collected fractions from petroleum ether/diethyl
ether (55:45) furnish a yellow solid that was further isolated by
prep. TLC petroleum ether/diethyl ether (35:65) to give compound
6 (10%), from the collected fractions eluted with petroleum ether/
diethyl ether (4:6) a white solid was obtained which was crystal-
lized from ethyl acetate/petroleum ether (3:1) to give compound 5
(40%).
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. HRMS results were
obtained in the services of C.A.C.T.I., Vigo, Spain.
6.1.1. 1-Hydroxyxanthone (1), 1,1,3-trihydroxybenzophenone (1a)
and 3,4-dihydroxyxanthone (4)
The synthesis of 1-hydroxyxanthone (1) [step (I) in the Scheme 1]
was carried out according to the method of Pankajamani and
Seshadri [17] and characterized according to the described proce-
dure (48%) [19]. Crystallization of the mother-liquor from acetone
rendered compound 1a in 10% yield.
The synthesis of 3,4-dihydroxyxanthone (4) (Scheme 2) was
carried out according to the method of Gottlieb et al. [26] and
characterized according to the described procedure (70%) [4].
Compound 5 mp 173 ꢁC; 1H NMR (300.13 MHz, DMSO-d6):
¼ 8.34 (H-8, dd, J ¼ 8.0 and 1.6 Hz, 1H), 7.91 (H-1, d, J ¼ 8.9 Hz, 1H),
d
7.73 (H-6, ddd, J ¼ 7.0, 7.0 and 1.5 Hz, 1H), 7.60 (H-5, d, J ¼ 7.9 Hz,
1H), 7.38 (H-7, ddd, J ¼ 7.0, 7.0 and 1.5 Hz, 1H), 6.99 (H-2, d,
J ¼ 8.9 Hz, 1H), 5.75 (HO–C(4), s, 1H), 4.23 (H-10, t, 2H), 3.44 (H-60, t,
2H), 1.93 (H-20 and H-50, m, 4H), 1.59 ppm (H-30 and H-40, m, 4H);
6.1.2. 1,10-[Hexane-1,6-diylbis(oxy)]bis[9H-xanthen-9-one] (2)
and 1-(6-oxy)-9H-xanthen-9-one (3)
To a solution of 1-hydroxyxanthone (1) (215 mg, 1 mmol) in dry
DMF (5 mL), was added K2CO3 (138 mg, 1 mmol) and the mixture
was stirred at room temperature for 30 min. To the suspension
formed was added one portion of 1,6-dibromobutane (244 mg,
1 mmol) in dry DMF (1 mL). The reaction mixture was stirred for
13C NMR (75.47 MHz, DMSO-d6):
d
¼ 175.1 (C-9), 166.0 (C-10a),
150.3 (C-3), 144.9 (C-4a), 134.6 (C-6), 133.5 (C-4), 126.7 (C-8), 123.9
(C-7), 121.6 (C-8a), 118.2 (C-5), 118.2 (C-1), 116.6 (C-9a), 108.3 (C-2),
69.5 (C-10), 33.7 (C-60), 32.9 (C-20), 29.7 (C-50), 28.0 (C-30), 25.1 ppm

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E. Sousa et al. / European Journal of Medicinal Chemistry 44 (2009) 3830–3835
(C-40); IR (KBr):
n
¼ 1645, 1606, 1455, 1334, 1250, 1184, 1081,
6.3.2. Cell cultures
757 cmꢀ1; HRMS-ESI m/z calcd for C19H20BrO4: 391.05395, found:
391.05375.
Four human tumor cell lines, MCF-7 (breast adenocarcinoma,
estrogen-dependent ER (þ)), MDA-MD-231 (breast adenocarci-
noma, estrogen-independent ER (ꢀ)), SF-268 (CNS cancer) and
NCI-H460 (non-small cell lung cancer) were used. MCF-7 and
MDA-MB-231 were obtained from the European Collection of Cell
Cultures (ECACC, Salisbury, UK) and NCI-H460 and SF-268 were
kindly provided by the National Cancer Institute (NCI, Bethesda,
USA). They grow as monolayer and routinely maintained in RPMI-
1640 medium supplemented with 5% heat-inactivated FBS, 2 mM
glutamine and antibiotics (penicillin 100 U/mL, streptomycin
Compound 6 mp 63 ꢁC; 1H NMR (300.13 MHz, DMSO-d6):
d
¼ 8.33 (H-8, dd, J ¼ 7.9 and 1.6 Hz, 1H), 8.06 (H-1, d, J ¼ 9.0 Hz,
1H), 7.72 (H-6, ddd, J ¼ 8.5, 6.9 and 1.7 Hz, 1H), 7.53 (H-5, dd, J ¼ 8.0
and 0.6 Hz, 1H), 7.38 (H-7, ddd, J ¼ 7.6, 6.7 and 1.0 Hz, 1H), 6.99 (H-
2, d, J ¼ 9.0 Hz, 1H), 4.15 (H-10, t, 4H), 3.45 (H-60, t, 4H), 1.92 (H-20
and H-50, m, 8H), 1.60 ppm (H-30 and H-40, m, 8H); 13C NMR
(75.47 MHz, DMSO-d6):
d
¼ 176.6 (C-9), 157.3 (C-3), 156.1 (C-10a),
150.8 (C-4a), 134.4 (C-6), 133.5 (C-4), 126.6 (C-8), 123.9 (C-4), 122.1
(C-7), 121.5 (C-8a), 118.0 (C-5), 117.9 (C-1), 116.6 (C-9a), 109.5 (C-2),
73.9 (C-10), 69.0 (C-100), 33.8 (C-60), 33.6 (C-600), 32.8 (C-20), 32.6
(C-200), 29.6 (C-50), 29.0 (C-500), 28.0 (C-30), 27.8 (C-300), 25.3 (C-40),
100 m
g/mL), at 37 ꢁC in a humidified atmosphere containing 5%
CO2. Exponentially growing cells were obtained by plating
1.5 ꢄ105 cells/mL for MCF-7 and SF-268 and 0.75 ꢄ104 cells/mL for
NCI-H460, followed by 24 h of incubation. The effect of the vehicle
solvent (DMSO) on the growth of these cell lines was evaluated in
all the experiments by exposing untreated control cells to the
maximum concentration (0.5%) of DMSO used in each assay.
25.2 ppm (C-400);; IR (KBr):
n
¼ 1655, 1600, 1461, 1436, 1330, 1288,
1084, 745 cmꢀ1; MS (EI) m/z (%): 555 (1) [M]þ, 310 (26), 228 (100),
171 (10). HRMS-ESI m/z calcd for C25H31Br2O4: 553.05836, found:
553.05765.
6.3.3. Tumor cell growth assay
6.2. X-ray crystallography
The effects of 1–6 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 sulforhod-
amine B to assess cell growth [29,30]. Briefly, exponentially, cells
growing in 96-well plates were then exposed for 48 h to five serial
concentrations of each compound, starting from a maximum
6.2.1. 1,1,3-Trihydroxybenzophenone (1a)
Crystals suitable for X-ray diffraction were obtained by slow
evaporation of an acetone solution. They were monoclinic, space
group P21/c, cell volume V ¼ 1054.8(3) Å3 and unit cell dimensions
a ¼ 12.386(2) Å, b ¼ 5.1781(7) Å, c ¼ 17.064(3) Å,
b
¼ 105.47(2)ꢁ
(uncertainties in parenthesis). The calculated density was 1.450 g/L.
Diffraction data were collected at 293 K with a Gemini PX Ultra
concentration of 200 mM. Following this exposure period adherent
cells were fixed, washed, and stained. The bound stain was solu-
bilized and the absorbance was measured at 492 nm in a plate
reader (Bio-Tek Instruments Inc., Powerwave XS, Wincoski, USA).
For each test compound and cell line, a dose–response curve was
obtained and the growth inhibition of 50% (GI50), corresponding to
the concentration of the compounds that inhibited 50% of the net
cell growth, was calculated as described elsewhere [29]. Doxoru-
bicin was used as a positive control and tested in the same manner.
Unpaired Student’s t-tests were used. Differences with P values
below 0.05 were considered statistically significant.
equipped with Mo K
reflections were measured, of which 2161 were independent and
1745 were observed (I > 2 (I)). The structure was solved by direct
methods using SHELXS-97 [27] and refined with SHELXL-97 [28].
Non-hydrogen atoms were refined anisotropically and the refine-
ment converged to R (all data) ¼ 4.72% and wR2 (all data) ¼ 10.36%.
a
radiation (
l
¼ 0.71073 Å). A total of 7072
s
6.2.2. 1-(6-Bromohexyloxy)-9H-xanthen-9-one (3)
Crystals were obtained by slow evaporation of an acetone
solution. They were found to be monoclinic, space group C2/c, cell
volume V ¼ 3413.9(14) Å3 and unit cell dimensions a ¼ 27.744(4) Å,
b ¼ 11.833(3) Å, c ¼ 15.849(3) Å,
b
¼ 139.00(1)ꢁ. The calculated
Acknowledgements
density was 1.460 g/L.
Financial support from FCT (I&D no. 4040/07), FEDER, POCI, FCT
(PTDC/SAU-NEU/69123/2006), is acknowledged. The authors are
also indebted to the National Cancer Institute, Bethesda, MD, USA,
for the generous provision of the human tumor cell lines and to
Sara Cravo for technical support.
Diffraction data were collected at 292 K with a Stoe IPDS
image plate equipped with Mo K radiation (
¼ 0.71073 Å). A
total of 13 028 reflections were measured, of which 3131 were
independent and 2308 were observed (I > 2 (I)). The structure
was solved by direct methods using SHELXS-97 [27] with atomic
positions and displacement parameters refined with SHELXL-97
[28]. Non-hydrogen atoms were refined anisotropically and the
a
l
s
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