Synthesis and SAR study of prenylated xanthone analogues as HeLa and MDA-MB-231 cancer cell inhibitors

Article abstract of DOI:10.2174/157018011795906820

In order to explore structure-activity relationship (SAR) associated with xanthone framework, a series of prenylated xanthone derivatives 2-9 was synthesized from the key building block 1,3,6,8-tetrahydroxyxanthone 1 and evaluated for their in vitro growth inhibitory activities against HeLa and MDA-MB-231 human cancer cell lines. The in vitro evidence indicated that the inhibitory activity was significantly influenced by the position and number of linked group on the xanthone skeleton, and the presence of chroman-4-one moiety in the xanthone scaffold was found to be critically important for strong cytotoxic activity. The novel 2H-xanthene-3,9-dione analogues 3 and 4 were reported to elicit potent activities comparable to those of standard drugs doxorubicin and cisplatin. This preliminary investigation has highlighted the therapeutic potential of 2H-xanthene-3,9-dione derivatives to be exploitable as lead compound for the development of potent HeLa and MDA-MB-231 cancer cell inhibitors.

Full text of DOI:10.2174/157018011795906820

Letters in Drug Design & Discovery, 2011, 8, 523-528
523
Synthesis and SAR Study of Prenylated Xanthone Analogues as HeLa and
MDA-MB-231 Cancer Cell Inhibitors
Chan Kiang Lim*,a, Lai-Yeng Thoa, Cheng Hoe Lima, Yang Mooi Limb, Syed Adnan Ali Shahc and
Jean-Frederic Faizal Weberc
aDepartment of Chemical Science, Faculty of Science, Universiti Tunku Abdul Rahman, 31900 Kampar, Perak,
Malaysia
bDepartment of Pre-Clinical Sciences, Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman,
43000 Kajang, Selangor, Malaysia
cResearch Institute of Natural Products for Drug Discovery (RiND), Faculty of Pharmacy, Universiti Teknologi MARA
(UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia
Received November 07, 2010: Revised March 03, 2011: Accepted April 08, 2011
Abstract: In order to explore structure-activity relationship (SAR) associated with xanthone framework, a series of preny-
lated xanthone derivatives 2-9 was synthesized from the key building block 1,3,6,8-tetrahydroxyxanthone 1 and evaluated
for their in vitro growth inhibitory activities against HeLa and MDA-MB-231 human cancer cell lines. The in vitro evi-
dence indicated that the inhibitory activity was significantly influenced by the position and number of linked group on the
xanthone skeleton, and the presence of chroman-4-one moiety in the xanthone scaffold was found to be critically impor-
tant for strong cytotoxic activity. The novel 2H-xanthene-3,9-dione analogues 3 and 4 were reported to elicit potent activi-
ties comparable to those of standard drugs doxorubicin and cisplatin. This preliminary investigation has highlighted the
therapeutic potential of 2H-xanthene-3,9-dione derivatives to be exploitable as lead compound for the development of po-
tent HeLa and MDA-MB-231 cancer cell inhibitors.
Keywords: Cytotoxicity, 2H-xanthene-3,9-dione scaffold, 2H-xanthene-3,6,9(7H)-trione scaffold, Inhibitory activity, Synthe-
sis, Xanthone derivatives.
INTRODUCTION
xanthone analogues derived from the key building block
1,3,6,8-tetrahydroxyxanthone 1 to clarify the structure-activity
correlation underlying the compounds that can guide us to the
discovery of potent inhibitors for therapeutic use. In our study,
reaction of precursor 1 with prenyl bromide in alkaline me-
dium yielded three novel xanthone derivatives 2-4. Prenylated
derivatives 3 and 4 with a distorted nucleus are reported to
elicit a higher cytotoxic activity than the typical xanthones 5 to
9, and a comparable activity with those of doxorubicin and
cisplatin. We described herein the synthesis of xanthone ana-
logues 1 to 9 with the aim to provide comprehensive insight
into the correlation between structures and inhibitory activities
of xanthones.
Natural or synthetic xanthones, in view of their unique
structures, have been reported to exhibit a wide range of
biological profiles including cytotoxic, anti-inflammatory,
antioxidant, antimalarial, antibacterial and antifungal ac-
tivities [1-5]. The interesting structural chromophore and
biological efficacy of xanthones have attracted many re-
searchers to isolate or synthesize these compounds for de-
velopment of potential new drug leads for treatment of
cancer diseases. In the past, many xanthones isolated from
plants were found to bear prenyl moieties, and many of
which were reported to exhibit a significant inhibitory ac-
tivity against a panel of human cancer cell lines [6-9]. Pre-
vious prenylation studies on the precursors 1,3-
dihydroxyxanthone and 1,3,5-trihydroxyxanthones have
highlighted the cytotoxic activity to be substantially influ-
enced by the degree of substitution and the substitution
pattern of prenyl group at the xanthone ring [10,11]. Be-
sides that, the presence of hydroxyl group was also found
to be essential since etherification led to a decrease in the
growth inhibitory activity [12]. More interestingly, the
presence of prenyl groups in different key-positions on
xanthonic nucleus was found to give selective effect on the
human cancer cell lines [10,11], and this prompted us to
conduct a detailed SAR study on a series of prenylated
RESULTS AND DISCUSSION
Chemistry
The synthetic route toward xanthone derivatives 1-9 is out-
lined in Scheme 1. In previous study, Liu et al. reported the
synthesis of compound 1 in low yield by using polyphosphoric
acid as a catalyst [13]. In this study, the key building block 1
was synthesized by condensation of 2,4,6-trihydroxybenzoic
acid with phloroglucinol in the presence of phosphorus
pentoxide and methanesulfonic acid according to the method
described in the literature [14]. This afforded a higher yield
compared to the former synthetic method. The prenylated
xanthones 2-9 were obtained from the reaction of their corre-
sponding building block 1 with 3-methylbut-2-enyl bromide in
10% aqueous KOH. The use of aqueous KOH in the synthesis
*Address correspondence to this author at the Department of Chemical
Science, Faculty of Science, Universiti Tunku Abdul Rahman, 31900
Kampar, Perak, Malaysia; Tel: +605 4688888; Fax: +605 4661676;
E-mail: cklim@utar.edu.my
1570-1808/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

524 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 6
Lim et al.
OH
O
OH
OH
O
9
OH
1
8
8a
9a
4a
7
6
2
3
a
OH
OH
+
HO
14
OH
HO
HO
10a
OH
HO
O
OH
4
5
1
b
14
14
29'
15
13
15
15
30'
13
29
28
13
12
12'
OH
8
O
9
OH
1
28'
OH
8
O
9
OH
1
11
2
12
15'
OH
8
O
9
OH
1
11
2
12
15'
11
2
15'
26
12'
13'
12'
13'
27'
8a
9a
4a
26'
8a
9a
4a
13'
27
7
6
8a
9a
4a
30
28
29
7
6
30
27
HO
11'
7
6
11'
3
26
O
11'
3
14'
3
14'
14'
10a
+
10a
HO
O
O
O
O
4
16
10a
+
4
16
5
21
O
O
5
4
16
5
21
21
17
18
22
23
17
18
22
23
17
18
22
23
24
19
25
24
20
19
25
20
24
19
25
20
4
3
2
14
24
23
14
13
OH
OH
O
OH
1
OH
8
O
9
OH
1
OH
8
O
9
OH
1
8
11
21
11
8a
9a
4a
2
8a
9a
4a
2
9
8a
9a
4a
2
7
6
13
7
6
15
15
12
7
25
22
HO
12
3
3
6
3
10a
+
+
10a
HO
O
OH
15
10a
HO
25
O
OH
20
4
5
O
OH
4
16
5
4
16
5
16
11
21
12
13
17
18
17
18
22
23
17
18
7
6
5
14
19
20
19
19
24
14
13
20
OH
8
O
9
OH
1
OH
O
OH
1
8a
9a
4a
2
11
8
7
6
8a
9a
4a
8
2
9
7
6
15
3
12
10a
+
3
HO
O
OH
4
5
10a
HO
O
OH
4
5
11
12
13
9
15
14
Scheme 1. Synthetic route for xanthone derivatives 1-9. Reagents and conditions: (a) P2O5-CH3SO3H, 80 oC, 0.5 h, 62%; (b) 3-methylbut-2-
enyl bromide, 10% aqueous KOH, rt, overnight, 2% for 2, 1% for 3, 2% for 4, 3% for 5, 4% for 6, 4% for 7, 4% for 8, 5% for 9.
was found to produce C-prenylated derivatives by sup-
pressing the formation of O-prenylated derivatives [15].
The suppression of O-prenylation is critically important to
maintain the phenolic hydroxyl groups in xanthones which
were found to be crucial to the cytotoxic activities [12].
Prenylation of building block 1 was reported to give gem-
diprenylated xanthones 2-4 and this is in agreement with
the previous study on acylphloroglucinols which reacted
under the same conditions to give gem-diprenylated po-
ducts [16].
compound 1 suggesting that the cytotoxicity of xanthones can
be largely amplified by the presence of prenyl functionality,
and this result was in agreement with the previous reports that
prenyl group plays a significant role in biological activities
[10,11]. From the structural point of view, the prenylated de-
rivatives obtained in this study can be classified into three
main groups based on the variation in their xanthonic core, that
are, compound 2 with 2H-xanthene-3,6,9(7H)-trione scaffold;
compounds 3-4 with 2H-xanthene-3,9-dione scaffold; com-
pound 5-9 with the typical xanthonic nucleus. The cytotoxic
assay data of 2H-xanthene-3,9-dione analogues indicated that
the presence of prenyl substituent at the C7 position was found
to induce a highly potent growth inhibitory activity against the
two cell lines tested. Compound 3 bearing the C-7 prenyl
group as compared with compound 4 without the group, was
found to exert a more potent antiproliferative activity towards
MDA-MB-231 cells and, however, a less cytotoxic potency for
HeLa cells. In comparison with doxorubicin and cisplatin as
positive control, compound 4 was found to exhibit at least 3-
fold much higher activity than both the standard drugs used in
the HeLa assay, while for MDA-MB-231 cells, compound 3
Growth Inhibition
The inhibitory activities of the parent xanthone 1 and
its prenylated derivatives 2-9 towards HeLa (cervical car-
cinoma) and MDA-MB-231 (human estrogen receptor
negative breast cancer) cancer cell lines were evaluated by
the method described in the literature [17] with some
modifications. The obtained IC50 values for the compounds
are shown in Table 1.
Under our assay conditions, prenylated xanthones 2-9
showed much higher inhibitory activities than the parent

Synthesis and Cytotoxic Activity of Prenylated Xanthones
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 6 525
was found to give a comparable activity with the standard
drugs.
pounds, compounds 3-4 were found to exhibit outstanding
activity suggesting that the novel 2H-xanthene-3,9-dione scaf-
fold could serve as a potential drug lead in the future drug de-
velopment. However, there is one drawback in this study due
to the low yields of compounds obtained, and currently our
team is working on a modified synthetic approach to improve
the yield of compounds 3 and 4. At the meantime, compounds
3 and 4 are aimed for apoptosis signalling pathways study to
provide insights into the mechanism of action underlying the
two cancer cell lines, which will be reported in due course.
Table 1. In Vitro Inhibitory Activities of Synthesized Com-
pounds Against HeLa and MDA-MB-231 Human
Cancer Cell Lines
Compound
IC50 (ꢀM)
HeLa
MDA-MB-231
1
> 60
52.4
11.7
3.8
> 60
46.4
10.0
60.1
32.3
40.9
20.2
73.1
54.9
11.0
16.7
2
EXPERIMENTAL
3
Melting points were determined on the Stuart SMP10 melt-
ing point apparatus and are uncorrected. UV and IR spectra
were recorded on Perkin-Elmer Lambda 35 UV/VIS and
Perkin-Elmer Spectrum RXI FTIR spectrometers, respectively.
NMR spectra were measured on a Bruker AV-III 500 MHz
NMR or JEOL JNM-ECX 400 MHz FTNMR spectrometer in
CDCl3 or acetone-d6 with TMS as the internal standard.
Chemical shifts (ꢁ) are reported in ppm and coupling constants
(J) are expressed in Hertz. EIMS and HREIMS were measured
on Agilent 5975C MSD mass spectrometer and Thermo Finni-
gan MAT95XL mass spectrometers, respectively. All reagents
were of analytical quality and used without further purification
unless otherwise specified. Column chromatography (CC) was
performed with silica gel 60 (230-300 mesh, Silicycle) and
Sephadex LH-20. Analytical TLC was performed on precoated
silica gel 60 F254 (Merck; Germany).
4
5
30.2
28.0
12.6
115.8
47.2
12.9
13.3
6
7
8
9
Doxorubicin
Cisplatin
aValues reported are an average of at least two independent experiments in triplicate.
For the group of compounds 5-9 with typical xanthonic
nucleus, some apparent trends were observed for their in-
hibitory activities against the two cell lines. The results
clearly indicated that the position of prenyl groups imparts
determinant effect on inhibitory activities of the typical
xanthone derivatives. In general, di-prenyl substituted xan-
thones were found to display more potent activity than
mono- and tri-prenyl substituted xanthones against HeLa
and MDA-MB-231 cells. Compound 7 bearing prenyl
groups at C4 and C5 showed the highest activity as com-
pared to other members in the group. The attempt to re-
move C4 or C5 prenyl group from compound 7 (as exem-
plified by compounds 8 and 9), or introduce C2 or C7
prenyl group to compound 7 (as exemplified by compound
6), both were found to decrease the inhibitory activity to-
wards the two cell lines suggesting that appropriate intro-
duction of a prenyl group to the xanthonic nucleus plays a
substantial role in antiproliferative activities.
Synthesis of Building Block 1,3,6,8-Tetrahydroxy-9H-
xanthen-9-one (1)
A mixture of 2,4,6-trihydroxybenzoic acid monohydrate
(9.40 g, 50 mmol) and phloroglucinol (6.30 g, 50 mmol) was
added slowly to 120 mL of Eaton’s reagent (P2O5/CH3SO3H,
Acros). The mixture was heated at 80 oC for 30 minutes, under
stirring. After cooling to room temperature, the reaction mix-
ture was poured onto crushed ice and stirred for 1 hour. The
resulting solid was collected by filtration and dried in the oven
o
at 50 C overnight. The solid was purified on a silica gel (40-
63 ꢂm) column with a mixture of hexane-ethyl acetate (9:1) as
eluent to give 1.
1,3,6,8-Tetrahydroxy-9H-xanthen-9-one (1)
62% (8.1 g) as a yellow solid, mp 210-212 oC ; UV ꢃmax nm
(EtOH) (log ꢄ): 233, 246, 334; IR ꢅmax (KBr) cm-1 : 3468,
3414, 2930, 2815, 2731, 2650, 1637, 1618, 1522, 1458, 1385,
Compound
2
with 2H-xanthene-3,6,9(7H)-trione
framework showed the highest number of attached prenyl
groups which principally is expected to give a better activ-
ity than the former two groups of compounds but it is the
other way round. This finding helps to explain that the
presence of chroman-4-one moiety in xanthone nucleus is
important for its cytotoxic activity as this was evident by a
much higher inhibitory activity elicited by the analogues 3-
7 bearing an intact chroman-4-one moiety in their nuclei.
The impairment of conjugated ꢀ-system on both benzene
rings in the nucleus as exemplified by compound 2 was
found to destroy the chroman-4-one moiety and hence
weaken the inhibitory activity. Apart from the discussion
above, compounds 8 and 9 were found to be much less
potent suggesting that a minimum of two prenyl groups
have to be present together with chroman-4-one moiety in
order to elicit a potent activity. Among all the test com-
1
1321, 1278, 1183, 1159, 1069, 833, 759; H NMR (500 MHz,
acetone-d6): 11.93 (2H, s, 1- OH & 8- OH ), 6.31 (2H, d, J =
1.8 Hz, H-4 & H-5), 6.19 (2H, d, J = 1.8 Hz, H-2 & H-7); 13
C
NMR (125 MHz, acetone-d6): 183.1 (C-9), 165.8 (C-3 & C-6),
163.0 (C-1 & C-8), 157.8 (C-4a & C-10a), 101.1 (C-8a & C-
9a), 98.5 (C-2 & C-7), 94.3 (C-4 & C-5); EIMS m/z (rel. int.):
260 ([M]+. 100), 231 (10), 203 (11), 69 (10).
General Procedure for the Synthesis of Prenylated Xan-
thones 2 - 9
A mixture of 1,3,6,8-tetrahydroxy-9H-xanthen-9-one (1)
(1.02 g; 3.92 mmol) and prenyl bromide (2.09 g; 14.03 mmol)
in 50 mL of 10% aqueous KOH was stirred for 16 hours at

526 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 6
Lim et al.
room temperature. The mixture was then acidified with
10% HCl and extracted with AcOEt (3 ꢀ 40 mL). The
combined organic layers were dried over Na2SO4. After
removal of the solvent under reduced pressure, purification
of the crude product using silica gel column eluted with a
mixture of hexane-ethyl acetate in increasing polarity, fol-
lowed by Sephadex LH-20 column eluted with methanol
yielded successively 2-9.
1,6,8-Trihydroxy-2,2,4,5-tetrakis(3-methylbut-2-enyl)-2H-
xanthene-3,9-dione (4)
2% (35.5 mg) as yellow crystals, mp 182-185 oC; UV ꢃmax
nm (EtOH) (log ꢄ): 204, 218, 266, 351; IR ꢅmax (KBr) cm-1
:
3415, 3035, 2972, 2914, 1672, 1614, 1550, 1455, 1413, 1293,
1
1132, 1194, 1172, 1110, 858, 784, 728; H NMR (500 MHz,
acetone-d6): 12.37 (1H, s, 1-OH), 11.49 (1H, s, 8- OH ), 6.51
(1H, s, H-7), 5.23 (1H, t, J = 6.7 Hz, H-22), 5.12 (1H, t, J =
6.5 Hz, H-17), 4.82 (2H, t, J = 7.6 Hz, H-12 & H-12’), 3.57
(2H, d, J = 6.7 Hz, H-21), 3.06 (2H, d, J = 6.5 Hz, H-16), 2.89
(2H, dd, J = 13.6, 7.6 Hz, H-11 & H-11’), 2.79 (2H, dd, J =
13.6, 7.6 Hz, H-11 & H-11’), 1.86 (3H, s, H-24), 1.74 (3H, s,
H-19), 1.74 (3H, s, H-25), 1.66 (3H, s, H-20), 1.51 (6H, s, H-
14 & H-14’), 1.48 (6H, s, H-15 & H-15’); 13C NMR (125
MHz, acetone-d6): 194.0 (C-3), 181.5 (C-9), 174.4 (C-1),
163.9 (C-6), 163.6 (C-4a), 160.1 (C-8), 154.4 (C-10a), 135.0
(C-13 & C-13’), 131.7 (C-23), 130.3 (C-18), 122.4 (C-17),
122.2 (C-22), 117.4 (C-12 & C-12’), 113.8 (C-4), 108.2 (C-
9a), 107.5 (C-5), 103.0 (C-8a), 100.0 (C-7), 57.1 (C-2), 38.7
(C-11 & C-11’), 25.0 (C-20), 24.9 (C-15, C-15' & C-25), 21.3
(C-21), 20.4 (C-16), 17.3 (C-24), 17.0 (C-14, C-14’ & C-19);
EIMS m/z (rel. int.): 532 ([M]+. 7), 463 (61), 407 (100), 393
(15), 365 (13), 353 (18), 69 (15); EIHRMS: 532.2825 ([M]+.,
C33H40O6+.; calcd 532.2825).
1,8-Dihydroxy-2,2,4,5,7,7-hexakis(3-methylbut-2-enyl)-
2H-xanthene-3,6,9 (7H)-trione (2)
2% (43.2 mg) as a yellow liquid, UV ꢃmax nm (EtOH)
(log ꢄ): 209, 235, 245, 274; IR ꢅmax (KBr) cm-1 : 3436,
2973, 2927, 2856, 1638, 1438, 1381, 1278, 1174, 818, 776;
1H NMR (400 MHz, CDCl3): 11.73 (2H, s, 1- OH & 8- OH
), 5.01 (2H, t, J = 6.7 Hz, H-17 & H-22), 4.72 (4H, t, J =
7.3 Hz, H-12, H-12’, H-27 & H-27’), 3.01 (4H, d, J = 6.7
Hz, H-16 & H-21), 2.94 (4H, dd, J = 14.0, 7.3 Hz, H-11,
H-11’, H-26 & H-26’), 2.68 (4H, dd, J = 14.0, 7.3 Hz, H-
11, H-11’, H-26 & H-26’), 1.69 (6H, s, C-19 & C-24), 1.60
(6H, s, H-20 & H-25), 1.50 (12H, s, H-14, H-14’, H-29 &
H-29’), 1.49 (12H, s, H-15, H-15’, H-30 & H-30’); 13C
NMR (100 MHz, CDCl3): 193.8 (C-3 & C-6), 179.7 (C-9),
173.8 (C-1 & C-8), 161.5 (C-4a & C-10a), 135.7 (C-13, C-
13’, C-28 & C-28’), 130.9 (C-18 & C-23), 121.8 (C-17 &
C-22), 116.9 (C-12, C-12’, C-27 & C-27’), 115.7 (C-4 &
C-5), 114.1 (C-8a & C-9a), 55.6 (C-2 & C-7), 38.5 (C-11,
C11’, C-26 & C-26’), 25.2 (C-20 & C-25), 25.0 (C-15, C-
15’, C-30 & C-30’), 20.6 (C-16 & C-21), 17.3 (C-14, C-
14’, C-29 & C-29’), 17.1 (C-19 & C-24); EIMS m/z (rel.
int.): 668 ([M]+. 5), 599 (31), 531 (85), 475 (88), 419 (82),
363 (33), 149 (35), 69 (100); EIHRMS: 668.4054 ([M]+.,
C43H56O6+.; calcd 668.4077).
1,3,6,8-Tetrahydroxy-2,4,7-tris(3-methylbut-2-enyl)-9H-
xanthen-9-one (5)
3% (50.9 mg) as yellow crystals, mp 179-181 oC; UV ꢃmax
nm (EtOH) (log ꢄ): 216, 237, 261, 297, 341; IR ꢅmax (KBr) cm-
1
: 3474, 3414, 2966, 2913, 1618, 1437, 1317, 1275, 1236,
1176, 1120, 1099, 812, 787; 1H NMR (500 MHz, acetone-d6):
12.34 (1H, s, 1-OH), 12.33 (1H, s, 8- OH ), 6.54 (1H, s, H-5),
5.28 (1H, t, J = 7.2 Hz, H-22), 5.22 (1H, t, J = 7.1 Hz, H-12),
5.21 (1H, t, J = 7.3 Hz, H-17), 3.53 (2H, d, J = 7.2 Hz, H-21),
3.43 (2H, d, J = 7.3 Hz, H-16), 3.36 (2H, d, J = 7.1 Hz, H-11),
1.86 (3H, s, H-24), 1.79 (6H, s, H-14 & H-19), 1.66 (3H, s, H-
20), 1.65 (6H, s, H-15 & H-25); 13C NMR (125 MHz, acetone-
d6 ): 183.5 (C-9), 163.2 (C-6), 160.4 (C-3), 159.8 (C-8), 157.7
(C-1), 155.6 (C-10a), 152.7 (C-4a), 131.7 (C-13), 131.5 (C-
18), 130.8 (C-23), 122.3 (C-22), 122.0 (C-12 & C-17), 110.8
(C-7), 110.5 (C-2), 106.3 (C-4), 101.3 (C-8a), 100.9 (C-9a),
93.5 (C-5), 25.0 (C-15, C-20 & C-25), 21.6 (C-21), 21.2 (C-
11), 21.0 (C-16), 17.2 (C-24), 17.1 (C-14), 17.0 (C-19); EIMS
m/z (rel. int.): 464 ([M]+. 100), 421 (28), 409 (91), 393 (73),
365 (62), 353 (71), 337 (41), 309 (57), 297 (72), 69 (16), 43
(24); EIHRMS: 464.2200 ([M]+., C28H32O6+.; calcd 464.2199).
1,6,8-Trihydroxy-2,2,4,5,7-pentakis(3-methylbut-2-enyl)-
2H-xanthene-3,9-dione (3)
1% (18.8 mg) as a yellow liquid, UV ꢃmax nm (EtOH)
(log ꢄ): 209, 229, 262, 353; IR ꢅmax (KBr) cm-1 : 3608,
3524, 3412, 3004, 2967, 2924, 1722, 1421, 1367, 1220,
1
1092, 902, 785; H NMR (400 MHz, acetone-d6): 12.35
(1H, s, 1-OH), 11.80 (1H, s, 8-OH ), 5.19 (1H, t, J = 7.0
Hz, H-27), 5.11 (1H, t, J = 6.2 Hz, H-22), 5.07 (1H, t, J =
7.0 Hz, H-17), 4.76 (2H, t, J = 7.4 Hz, H-12 & H-12’),
3.61 (2H, d, J = 6.2 Hz, H-21), 3.44 (2H, d, J = 7.0 Hz, H-
26), 3.01 (2H, d, J = 7.0 Hz, H-16), 2.82 (2H, dd, J = 13.6,
7.4 Hz, H-11 & H-11’), 2.72 (2H, dd, J = 13.6, 7.4 Hz, H-
11 & H-11’), 1.82 (3H, s, H-24), 1.76 (3H, s, H-29), 1.70
(3H, s, H-19), 1.64 (6H, s, H-25 & H-30), 1.61 (3H, s, H-
20), 1.45 (6H, s, H-14 & H-14’), 1.42 (6H, s, H-15 & H-
15’); 13C NMR (100 MHz, acetone-d6): 194.2 (C-3), 181.7
(C-9), 174.4 (C-1), 163.7 (C-4a), 161.0 (C-6), 157.0 (C-8),
152.5 (C-10a), 135.1 (C-13 & C-13’), 132.4 (C-23), 132.3
(C-28), 130.5 (C-18), 122.4 (C-17), 122.2 (C-22), 121.3
(C-27), 117.5 (C-12 & C-12’), 113.8 (C-4), 113.3 (C-7),
108.3 (C-9a), 107.3 (C-5), 101.9 (C-8a), 57.1 (C-2), 38.8
(C-11 & C11’), 25.1 (C-25 & 30), 25.0 (C-15 & C-15’),
24.9 (C-20), 21.7 (C-26), 21.5 (C-21), 20.5 (C-16), 15.5
(C-29), 17.2 (C-24), 17.1 (C-19), 17.0 (C-14 & C-14’);
EIMS m/z (rel. int.): 600 ([M]+. 15), 531 (100), 475 (72),
419 (76), 363 (29), 309 (23), 69 (38), 43 (21); EIHRMS:
600.3440 ([M]+., C38H48O6+.; calcd 600.3451).
1,3,6,8-Tetrahydroxy-2,4,5-tris(3-methylbut-2-enyl)-9H-
xanthen-9-one (6)
4% (65.5 mg) as yellow crystals, mp 179-181 oC; UV ꢃmax
nm (EtOH) (log ꢄ): 216, 237, 261, 341; IR ꢅmax (KBr) cm-1
:
3420, 2966, 2921, 1618, 1466, 1313, 1232, 1170, 1085, 815,
1
753; H NMR (500 MHz, acetone-d6): 12.34 (1H, s, 1-OH),
12.01 (1H, s, 8- OH ), 6.32 (1H, s, H-7), 5.28 (1H, t, J = 7.2
Hz, H-22), 5.22 (2H, t, J = 7.1 Hz, H-12 & H-17), 3.59 (2H, d,
J = 6.7 Hz, H-21), 3.49 (2H, d, J = 6.9 Hz, H-16), 3.43 (2H, d,
J = 6.9 Hz, H-11), 1.79 (9H, s, H-14, H-19 & H-24), 1.68 (3H,
s, H-20), 1.66 (6H, s, H-15 & H-25); 13C NMR (125 MHz,
acetone-d6): 183.7 (C-9), 163.0 (C-8), 160.7 (C-6), 160.6 (C-
3), 157.7 (C-1), 154.8 (C-10a), 152.8 (C-4a), 131.9 (C-13),
131.7 (C-18), 131.1 (C-23), 122.6 (C-12 & C-17), 122.0 (C-

Synthesis and Cytotoxic Activity of Prenylated Xanthones
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 6 527
22), 110.6 (C-2), 106.8 (C-4), 106.7 (C-5), 101.0 (C-8a &
C-9a), 97.8 (C-7), 25.0 (C-20), 24.9 (C-15 & C-25), 21.6
(C-21), 21.4 (C-11), 21.2 (C-16), 17.2 (C-24), 17.1 (C-14
& C-19); EIMS m/z (rel. int.): 464 ([M]+. 100), 421 (52),
Stock Solution and Cell Culture
Stock solution of the target compounds was prepared at
concentration of 10 mg/mL in absolute ethanol. The human
cancer cell lines HeLa (cervical carcinoma) and MDA-MB-
231 (human estrogen receptor negative breast cancer) were
obtained from the American Type Culture Collection (ATCC,
USA). Cells were cultured in RPMI-1640 with 5% Fetal Bo-
vine Serum (FBS), 100 IU mL-1 penicillin and 100g/mL strep-
409 (62), 393 (58), 365 (43), 353 (84), 309 (22), 297 (18),
79 (17), 43 (18); EIHRMS: 464.2199 ([M]+., C28H32O6
calcd 464.2199).
;
+.
1,3,6,8-Tetrahydroxy-4,5-bis(3-methylbut-2-en-1-yl)-9H-
o
xanthen-9-one (7)
tomycin by using 25 mL flask in a 37 C incubator with 5%
CO2.
o
4% (57.4 mg) as yellow crystals, mp 250-253 C; UV
ꢁ
max nm (EtOH) (log ꢀ): 234, 260, 338; IR ꢁmax (KBr) cm-1 :
3468, 2966, 2914, 1617, 1508, 1431, 1312, 1273, 1213,
Cytotoxic Assay
1
1173, 1090, 835, 813, 778; H NMR (500 MHz, acetone-
Cytotoxic activities of the test compounds 1-9 against hu-
man HeLa and MDA-MB-231 cancer cell lines were evaluated
using the protocol described in the literature by Rahmat et al.
[17] with some modifications. The exponential growth HeLa
and MDA-MB-231 cells were seeded in 96-well plate at con-
centration of 7500 and 35000 cells per well, respectively in
RPMI 1460 medium. After being incubated for 24 hours at 37
oC under a 5% CO2 atmosphere, cells were treated with six
concentrations of tested compounds at 1.6, 3.1, 6.3, 12.5, 25.0
and 50.0 ꢀg/mL and incubated for another 72 hours. Then, 20
ꢆL of MTT stock solution (5 mg in 1 mL PBS) was added to
each well and the plate was further incubated for 3 hours at 37
oC. 100 ꢆL of ethanol was added to each well and after 30
minutes, the absorbance (OD) of the samples and the reference
were measured by using ELISA spectrophotometer microplate
reader at wavelength 550 nm. The concentration of substance
required for 50% growth inhibition, IC50 was determined from
the absorbance (OD) against concentration curve. Anticancer
drugs, doxorubicin and cisplatin, purchased from Calbiochem
were used as positive controls.
d6): 12.00 (2H, s, 1-OH & 8-OH), 6.31 (2H, s, H-2 & H-7),
5.30 (2H, t, J = 6.8 Hz, H-12 & H-17), 3.53 (4H, d, J = 6.8
Hz, H-11 & H-16), 1.79 (6H, s, H-14 & H-19), 1.67 (6H, s,
H-15 & H-20); 13C NMR (125 MHz, acetone-d6): 183.7
(C-9), 163.1 (C-1 & C-8), 160.7 (C-3 & C-6), 154.9 (C-4a
& C-10a), 131.2 (C-13 & C-18), 122.6 (C-12 & C-17),
107.0 (C-4 & C-5), 100.9 (C-8a & C-9a), 97.9 (C-2 & C-
7), 24.9 (C-15 & C-20), 21.4 (C-11 & C-16), 17.1 (C-14 &
C-19); EIMS m/z (rel. int.): 396 ([M]+. 100), 381 (67), 353
(18), 341 (40), 325 (73), 313 (55), 297 (25), 273 (27), 260
(20), 143 (15), 69 (14), 43 (9); EIHRMS: 396.1555 ([M]+.,
C23H24O6+.; calcd 396.1573).
1,3,6,8-Tetrahydroxy-2-(3-methylbut-2-enyl)-9H-
xanthen-9-one (8)
o
4% (46.9 mg) as yellow crystals, mp 184-186 C; UV
ꢂ
max nm (EtOH) (log ꢀ): 203, 253, 334; IR ꢁmax (KBr) cm-1 :
3436, 2925, 2855, 1630, 1618, 1612, 1467, 1299, 1280,
1188, 1168, 1073, 816, 762; 1H NMR (400 MHz, acetone-
d6): 12.22 (H, s, 1-OH), 12.03 (H, s, 8-OH), 6.43 (1H, s, H-
4), 6.33 (1H, s, H-5), 6.21 (1H, s, H-7), 5.24 (1H, t, J = 7.6
Hz, H-12), 3.31 (2H, d, J = 7.6 Hz, H-11), 1.75 (3H, s, H-
14), 1.62 (3H, s, H-15); 13C NMR (100 MHz, acetone-d6):
183.2 (C-9), 165.6 (C-6), 163.3 (C-3), 163.1 (C-8), 159.8
(C-1), 157.8 (C-10a), 155.6 (C-4a), 130.9 (C-13), 122.2
(C-12), 111.0 (C-2), 101.2 (C-8a), 101.0 (C-9a), 98.4 (C-
7), 94.1 (C-5), 93.6 (C-4), 25.1 (C-15), 21.1 (C-11), 17.1
(C-14); EIMS m/z (rel. int.): 328 ([M]+. 42), 313 (29), 285
(68), 273 (100), 260 (10), 153 (7), 69 (7).
CONCLUSION
In summary, a series of xanthone derivatives with typical
or modified nucleus have been synthesized and most of them
were found to display significant inhibitory activity against
HeLa and MDA-MB-231 cancer cell lines. It is notable that
the presence of chroman-4-one and prenyl moieties is essential
for cytotoxicity of xanthones. In particular, compounds 3 and 4
bearing a novel skeleton were found to show comparable ac-
tivities with the standard drugs used in the assay, and this can
provide useful information for synthesizing new xanthone de-
rivatives with enhanced activity in the future.
1,3,6,8-Tetrahydroxy-4-(3-methylbut-2-enyl)-9H-
xanthen-9-one (9)
o
5% (60.4 mg) as yellow crystals, mp 204-205 C; UV
ꢂ
max nm (EtOH) (log ꢀ): 212, 234, 254, 333; IR ꢁ (KBr)
max
cm-1 : 3370, 2967, 2928, 1654, 1630, 1618, 1511, 1305,
1271, 1163, 1096, 825, 750; 1H NMR (500 MHz, acetone-
d6): 12.13 (H, s, 1-OH), 12.01 (H, s, 8-OH), 6.46 (1H, d, J
= 2.2 Hz, H-5), 6.34 (1H, s, H-2), 6.25 (1H, d, J = 2.2 Hz,
H-7), 5.27 (1H, t, J = 7.2 Hz, H-12), 3.47 (2H, d, J = 7.2
Hz, H-11), 1.86 (3H, s, H-14), 1.66 (3H, s, H-15); 13C
NMR (125 MHz, acetone-d6): 183.4 (C-9), 165.7 (C-6),
163.2 (C-1), 163.1 (C-8), 160.7 (C-3), 157.9 (C-10a),
154.8 (C-4a), 131.0 (C-13), 122.2 (C-12), 106.9 (C-4),
101.2 (C-8a), 100.9 (C-9a), 98.3 (C-7), 98.0 (C-2), 94.2
(C-5), 24.9 (C-15), 21.3 (C-11), 17.1 (C-14); EIMS m/z
(rel. int.): 328 ([M]+. 70), 313 (100), 273 (49), 260 (47),
191 (30), 167 (13), 149 (45), 69 (19), 57 (23), 43 (34).
ACKNOWLEDGEMENTS
This material is based upon work supported by grants from
the Malaysia Toray Science Foundation, and Ministry of Sci-
ence, Technology and Innovation Malaysia (Project No. 02-
02-11-SF0079).
REFERENCES
[1]
Lee, B.W.; Lee, J.H.; Lee, S.T.; Lee, H.S.; Lee, W.S.; Jeong, T.S.;
Park, K.H. Antioxidant and cytotoxic activities of xanthones from
Cudrania tricuspidata. Bioorg. Med. Chem. Lett., 2005, 15, 5548-
5552.

528 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 6
Lim et al.
[2]
[3]
Mahabusarakam, W.; Rattanaburi, S.; Phongpaichit, S.; Kanjana-
Opas, A. Antibacterial and cytotoxic xanthones from Cratoxylum
cochinchinense. Phytochem. Lett., 2008, 1, 211-214.
M.S.J.; Eaton, G. Dihydroxyxanthones prenylated derivatives: Syn-
thesis, 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.
Dua, V.K.; Ojha, V.P.; Roy, R.; Joshi, B.C.; Valecha, N.; Devi,
C.U.; Bhatnagar, M.C.; Sharma, V.P.; Subbarao, S.K. Anti-
malarial activity of some xanthones isolated from the roots of
Andrographis paniculata. J. Ethnopharm., 2004, 95, 247-251.
Woo, S.; Jung, J.; Lee, C.; Kwon, Y.; Na, Y. Synthesis of new
xanthone analogues and their biological activity test—
Cytotoxicity, topoisomerase II inhibition, and DNA cross-linking
study.ꢀBioorg. Med. Chem. Lett. 2007, 17, 1163-1166.
[11]
Li, N.G.; You, Q.D.; Huang, X.F.; Wang, J.X.; Guo, Q.L.; Chen,
X.G.; Li, Y.; Li, H.Y. Synthesis and antitumor activities of structure-
related small molecular compounds of gambogic acid. Chin. Chem.
Lett., 2007, 18, 659-662.
[4]
[5]
[12]
[13]
Li, N.G.; You, Q.D.; Huang, X.F.; Wang, J.X.; Guo, Q.L.; Chen,
X.G.; Li, Y.; Li, H.Y. Structure-activity Relationship of Gambogic
Acid. Chin. J. Nat. Med., 2008, 6, 37- 42.
Boonnak, N.; Karalai, C.; Chantrapromma, S.; Ponglimanont, C.;
Fun, H.K.; Kanjana-Opas, A.; Chantrapromma, K.; Kato, S.
Anti-Pseudomonas aeruginosa xanthones from the resin and
green fruits of Cratoxylum cochinchinense. Tetrahedron, 2009,
65, 3003-3013.
Zhang, Y.; Song, Z.; Hao, J.; Qiu, S.; Xu, Z. Two new prenylated
xanthones and a new prenylated tetrahydroxanthone from the
pericarp of Garcinia mangostana. Fitoterapia, 2010, 81, 595-
599.
Boonnak, N.; Karalai, C.; Chantrapromma, S.; Ponglimanont, C.;
Fun, H.K.; Kanjana-Opas, A.; Laphookhieo, S. Bioactive preny-
lated xanthones and anthraquinones from Cratoxylum formosum
ssp. pruniflorum. Tetrahedron, 2006, 62, 8850-8859.
Han, Q.B.; Yang, N.Y.; Tian, H.L.; Qiao, C.F.; Song, J.Z.;
Chang, D.C.; Chen, S.L.; Luo, K.Q.; Xu, H.X. Xanthones with
growth inhibition against HeLa cells from Garcinia xipshuan-
bannaensis. Phytochemistry, 2008, 69, 2187-2192.
Shadid, K.A.; Shaari, K.; Abas, F.; Israf, D.A.; Hamzah, A.S.;
Syakroni, N.; Saha, K.; Lajis, N.H. Cytotoxic caged-
Liu, Y.; Zou, L.; Ma, L.; Chen, W.H.; Wang, B.; Xu, Z.L. Synthesis
and pharmacological activities of xanthone derivatives as ꢀ-
glucosidase inhibitors. Bioorg. Med. Chem., 2006, 14, 5683-5690.
Varache-Lembege, M.; Moreau, S.; Larrouture, S.; Montaudon, D. ;
Robert, J. ; Nuhrich, A. Synthesis and antiproliferative activity of
aryl- and heteroaryl-hydrazones derived from xanthone carbalde-
hydes. Eur. J. Med. Chem., 2008, 43, 1336-1343.
[14]
[15]
[6]
[7]
[8]
[9]
[10]
Helesbeux, J.-J. ; Duval, O.; Dartiguelongue, C. ; Séraphin, D ; Oger,
J.-M.; Richomme, P. Synthesis of 2-hydroxy-3-methylbut-3-enyl sub-
stituted coumarins and xanthones as natural products. Application of
the Schenck ene reaction of singlet oxygen with ortho-prenylphenol
precursors. Tetrahedron, 2004, 60, 2293-2300.
George, J.H.; Hesse, M.D.; Baldwin, J.E.; Adlington, R.M.
Biomimetic synthesis of polycyclic polyprenylated acylphlorogluci-
nol natural products isolated from Hypericum papuanum. Org. Lett.,
2010, 12 (15), 3532-3535.
Rahmat, A.; Rosli, R.; Zain, W.N.I.W.M.; Endrini, S.; Sani, H.A.
Antiproliferative activity of pure lycopene compared to both ex-
tracted lycopene and juices from watermelon (Citrullus vulgaris) and
papaya (Carica papaya) on human breast and liver cancer cell lines.
J. Med. Sci., 2002, 2, 55-58.
[16]
[17]
polyprenylated xanthonoids and
a xanthone from Garcinia
cantleyana. Phytochemistry, 2007, 68, 2537-2544.
Castanheiro, R.A.P.; Pinto, M.M.M.; Silva, A.M.S.; Cravo,
S.M.M.; , Gales, L.; Damas, A.M.; Nazareth, N.; Nascimento,