Synthesis and evaluation of caged Garcinia xanthones

Article abstract of DOI:10.1039/b612903j

Inspired by the combination of unique structure and potent bioactivities exhibited by several family members of the caged Garcinia xanthones, we developed a synthesis of simplified analogues that maintain the overall caged motif. The caged structure of th

Full text of DOI:10.1039/b612903j

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and evaluation of caged Garcinia xanthones†
Ayse Batova,b Thanh Lam,a Veit Wascholowski,c Alice L. Yu,b Athanassios Giannis*c and
Emmanuel A. Theodorakis*a
Received 6th September 2006, Accepted 5th December 2006
First published as an Advance Article on the web 20th December 2006
DOI: 10.1039/b612903j
Inspired by the combination of unique structure and potent bioactivities exhibited by several family
members of the caged Garcinia xanthones, we developed a synthesis of simplified analogues that
maintain the overall caged motif. The caged structure of these compounds was constructed via a
site-selective Claisen/Diels–Alder reaction cascade. We found that the fully substituted caged structure,
in which are included the C18 and C23 geminal methyl groups, is necessary to maintain bioactivity.
Analogue 17 had comparable activity to the natural products of this family, such as gambogic acid.
These compounds exhibit cytotoxicity in a variety of tumor cell lines at low micromolar concentrations
and were found to induce apoptosis in HUVE cells. In addition, studies with HL-60 and HL-60/ADR
cells indicate that these compounds are not affected by the mechanisms of multidrug resistance,
conferred by P glycoprotein expression, typical of relapsed cancers and thus represent a new and potent
pharmacophore.
Introduction
The Garcinia genus of tropical plants has yielded a structurally
intriguing family of caged xanthone-derived natural products
that have interesting bioactivities and a documented value in
traditional Eastern medicine.1 The structure of forbesione (1,
Fig. 1) typifies this unusual architecture in which a 4-oxa-
tricyclo[4.3.1.03,7] dec-8-en-2-one scaffold has been built onto the
C-ring of a xanthone backbone.2 This motif is further customized
via substitutions at the A-ring and peripheral oxidations to pro-
duce a variety of structural subfamilies such as the morellins (2,3),3
the gaudichaudiones (4,5)4 and the gambogins (6,7).5 Recent
biological reports attest to the biological and pharmacological
potential of these compounds. For instance, desoxymorellin (3)
and gaudichaudione A (4) were found to exhibit potent cytotoxi-
city against several cancer cell lines.5b,6 In addition, gambogic acid
(6) was shown to induce apoptosis in T47D (breast cancer) via
a mechanism that is independent of cell cycle and may involve
binding to the transferring receptor.7 Related studies in MGC-803
Fig. 1 Chemical structures of selected caged Garcinia xanthones.
(gastric carcinoma) cells indicated that 6 regulates expression of
Bax and Bcl-2 proteins that are known to play a crucial role in
apoptosis.8 In addition to its anticancer activity, gambogic acid
was shown to inhibit the growth of Gram positive bacteria.9
Inspired by the therapeutic potential of the caged Garcinia
xanthones, we sought to develop a chemical strategy that would
allow synthetic access to these metabolites, and thus facilitate their
thorough biological and pharmacological evaluation. Along these
lines, we have recently reported a unified strategy for the synthesis
of selected caged Garcinia xanthones.10,11 This strategy relies
on a biomimetic Claisen/Diels–Alder/Claisen reaction cascade12
that produces the pentacyclic motif of forbesione (1) from a
tricyclic prenylated xanthone. Subsequent functionalizations at
the periphery of the A ring of 1 produced desoxymorellin (3),
desoxygaudichaudione A (5) and gambogin (7).10,13 Herein we
present an application of this strategy to the synthesis of simplified
analogues of the parent structures and their biological evaluation.
aDepartment of Chemistry and Biochemistry, University of California, San
Diego, 9500 Gilman Drive Mail Box: 0358, La Jolla, CA, 92093-0358, USA.
E-mail: etheodor@ucsd.edu; Fax: +1-858-822-0386; Tel: +1-858-822-0456
bDepartment of Pediatrics/Hematology-Oncology, University of California,
San Diego, 200 West Arbor Drive, San Diego, CA, 92103-8447, USA.
E-mail: abatova@ucsd.edu; Tel: +1-619-471-6955
Results and discussion
cInstitute of Organic Chemistry, University of Leipzig, Johannisallee 29,
D-04103, Leipzig, Germany. E-mail: giannis@uni-leipzig.de; Fax: +49-341-
9736-599; Tel: +49-341-9736-581
Synthesis of caged Garcinia xanthones
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra for compounds 10, 11, 14, 15, 17, 18, 21 and 22. See DOI:
10.1039/b612903j
The synthesis of simplified analogues of the caged Garcinia motif
is shown in Scheme 1. ZnCl2-induced condensation14 of o-anisic
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fruitless. Nonetheless, this compound was easily separable from
desired product 15 via a simple chromatography on silica gel.
Heating of 15 in DMF (120 ◦C, 1 h) gave rise to the caged motif
17 via a Claisen-rearranged intermediate 16. The excellent site-
selectivity of this Claisen/Diels–Alder reaction cascade (C5 versus
C6 allylation) can be rationalized by considering that the electron-
ically deficient C9 carbonyl group polarizes selectively the O–C18
bond facilitating its rupture.10,17 In addition, recent computational
studies by Houk and co-workers on similar substrates have shown
that the Claisen rearrangement is reversible and that the rate of
the Diels–Alder reaction controls the product selection.18
In a similar manner compound 11 was allylated with allyl
bromide to afford adduct 18 in quantitative yield. We found that
this compound could undergo the Claisen/Diels–Alder reaction
only at elevated temperatures and prolonged heating (DMF, reflux,
16 h). In this case we isolated the regular caged structure 22
together with the neo isomer 21 in a ratio of 1.2 : 1 and a combined
yield of 59%.
It is interesting to note the difference in the Claisen/Diels–
Alder reaction between substrates 15 and 18. Compound 18,
lacking the geminal methyl groups at the C18 and C23 centers,
requires forcing conditions for the dearomatization thus producing
both constitutional isomers of the Claisen/Diels–Alder reaction
cascade. In contrast, substrate 15 undergoes a faster and smoother
dearomatization leading selectively to caged structure 17. This
finding may be attributed to the presence of the geminal methyl
group at C18 of 15 that stabilizes the partial positive charge,
formed at this carbon, during the transition state of the Claisen
rearrangement. Such polar transition states have been supported
by both computational studies and kinetic isotope effects in related
structures.19
Encouraged by recent reports on the solvent-induced accelera-
tion of the Claisen/Diels–Alder reaction in similar substrates,13d,20
we studied the effect of solvent and temperature for the conversion
of 18 to 21 and 22. Table 1 summarizes our findings. No reaction
was observed upon heating of 18 in deuterated toluene at 100 ◦C
for 4 hours (entry 1) and either longer reaction times (16 hours,
entry 2) or higher temperatures (120 ◦C, entry 3) led to only a
small improvement in product formation. Switching the solvent
from toluene to DMF led to an acceleration of the Claisen/Diels–
Alder reaction that started to proceed even at 100 ◦C after 4 hours
(compare entries 1 and 4). Increase of both temperature and
reaction time led to a substantial incre◦ase in product conversion
that reached 79% after heating at 150 C for 16 hours (entry 7).
However, increase of the reaction temperature from 100 to 150 ◦C
led to a decrease in the selectivity of product formation (compare
ratios of 22 : 21 in entries 5 and 7). In deuterated methanol, this
reaction cascade started proceeding even at 60 ◦C, but the overall
product conversion was only 14% even after 16 hours (entry 8).
At 100 ◦C after only 4 hours of heating, this reaction proceeded at
65% conversion (entry 9) and was completed after 16 hours (entry
10). The rate acceleration of this reaction was even more dramatic
when a mixture of CD3OD : D2O 1 : 1 was used as the solvent.
In this case the cyclization proceeded in 73% yield after heating at
60 ◦C for 4 hours (entry 11), while it was completed after heating
at 100 ◦C for 4 hours (entry 12).
Scheme 1 Reagents and conditions: (a) 8 (1.0 equiv.), 9 (1.1 equiv.), ZnCl2
(5.0 equiv.), POCl3, 65 ◦C, 8 h, 78%; (b) aq. NaOH (30%), MeOH, 100 ◦C,
3 d, 71%; (c) K2CO3 (2.2 equiv.), KI (2.2 equiv.), 12 (2.2 equiv.), CuI (0.2
equiv.), acetone, 45 ◦C, 3 h, 13 (35%) and 14 (50%); (d) Pd/BaSO4 (10%),
quinoline, EtOAc, 25 ◦C, 6 h, 89%; (e) DMF, 120 ◦C, 1 h, 78%; (f) K2CO3
(2.2 equiv.), H2C CHCH2Br (2.4 equiv.), acetone, 45 ◦C, 2 h, 100%; (g)
=
DMF, reflux (153 ◦C), 16 h, 22 (32%) and 21 (27%); (h) MeOH : H2O 1/1,
100 ◦C, 0.5 h, 94%; (i) MeOH : H2O 1/1, 100 ◦C, 4 h, 22 (67%) and 21
(22%).
acid (8) with pyrogallol (9) in POCl3 produced benzophenone
adduct 10 that underwent a base-induced cyclization to form
xanthone 11 (55% yield over two steps). Conversion of 11 to
bis(dimethylallyloxy) xanthone 15 was accomplished by a two
steps procedure that involved propargylation of the C5 and C6
phenols with 2-chloro-2-methyl butyne (12) to form 1415 followed
by Lindlar reduction of the pendant alkynes16 (45% combined
yield). The main side-product of this sequence was alkene 13
formed by concomitant reaction of the C5 phenol at the vinyl
organometallic intermediate. Attempts to decrease the amount
of 13 by adding more equivalents of chloride 12 proved to be
In short, the findings presented in Table 1 demonstrate clearly
that polar solvents can accelerate significantly the Claisen/Diels–
Alder reaction. In turn, this leads to an efficient product
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Table 1 Effect of solvent and temperature on the conversion of 18 to 21 and 22
Entry
Solventa
T/◦C
t/h
Conversion 21 + 22 (%)
Ratio 22 : 21
1
2
3
4
5
6
7
8
9
Toluene-d8
Toluene-d8
Toluene-d8
DMF-d7
DMF-d7
DMF-d7
DMF-d7
CD3OD
CD3OD
CD3OD
100
100
120
100
100
150
150
60
4
16
16
4
16
4
16
16
4
16
4
<5
12
23
18
25
26
79
14
65
ND
2.1 : 1
1.8 : 1
2.4 : 1
2.3 : 1
1.3 : 1
1.2 : 1
2.9 : 1
2.5 : 1
2.3 : 1
3.0 : 1
2.8 : 1
100
100
60
10
11
12
100
73
100
CD3OD/D2O
CD3OD/D2O
100
4
a The reactions were carried out in sealed NMR tubes and monitored by 1H NMR spectrometry. In all cases the concentration of starting material 18 was
between 0.04–0.05 mmol.
conversion at relatively low temperatures (between 60–100 ◦C) and
an enhanced product selectivity. Similar observations have also
been made by the Nicolaou group13d and an explanation of this
effect, based upon previous theoretical and experimental work, has
been proposed.20 On a preparative scale, we found that heating of
18 in a mixture of MeOH : H2O (1 : 1) at 100 ◦C for 4 h led to the
isolation of a 3.1 : 1 mixture of 22 and 21 in a combined yield of
89%. Under these optimized conditions the conversion of 15 to 17
proceeded in 94% isolated yield after heating for 0.5 h (Scheme 1).
showed any growth inhibition at the concentrations tested (data
not shown). This supports the notion that the caged motif of this
family is essential for bioactivity.
Importantly, the compounds shown in Table 2 were also
evaluated in HL-60/ADR cells, a multidrug resistant clone
obtained by transfection of HL-60 cells with mdr-1.21 The results
of parallel experiments indicated that HL-60/ADR had similar
sensitivity to the anti-proliferative effects of the caged Garcinia
xanthones as the parental HL-60 cell line. This significant finding
suggests that the caged Garcinia xanthones are not subject to the
mechanism of chemo-resistance resulting from the expression of
mdr characteristic of many relapsed cancers.22
Cell proliferation studies
The ability of the synthesized caged Garcinia xanthones to inhibit
cancer cell growth was evaluated in promyelocytic leukemia cell
line, HL-60, using a 3H-thymidine incorporation assay. Cells were
incubated with increasing concentrations of the compounds for
48 h, and then pulsed with 3H-thymidine for 6 h. Gambogic acid
(6) and gambogin (7) were the most active among all compounds
tested and exhibited an IC50 value of 0.3 and 0.8 lM respectively
(Table 2). Their comparable activity suggests that the carboxylic
acid functionality of 6 does not contribute significantly to its
bioactivity. Interestingly, analogue 17 showed similar activity to
that of the natural products (IC50 = 1.5 lM), while the related
structures 21 and 22 were found to be relatively inactive, inducing
less than 10% growth inhibition at the highest concentrations
tested (2.0 lM). These findings suggest that the geminal methyl
groups at the C18 and C23 centers play an important role in the
bioactivity of the caged Garcinia xanthone system. In addition, we
found that none of the acyclic molecules tested (11, and 13–16)
Active compounds having IC50 values less than 2 lM (com-
pounds 3,6,7 and 17) were selected for further evaluation in a panel
of solid and non-solid tumor cell lines (Table 3). The T-cell acute
lymphoblastic leukemia cell line (CEM) was the most sensitive
among all the cell lines tested. The IC50 values recorded in these
cells were in the submicromolar range (0.15–0.35 lM). The solid
tumor cell lines were slightly less sensitive than CEM cells with
IC50 values of the compounds ranging from 0.4 to 3.1 lM, with the
exception of compound 17 in A549 cells (IC50 > 4 lM). Although,
gambogic acid (6) was the most active among the compounds
evaluated, the differences in activities among them were small
(≤3-fold). This supports the notion that the caged structure of
these compounds is the major contributor for their activity.
Cell viability and apoptosis studies
In order to distinguish between cytostatic and cytotoxic effects,
two independent cell viability studies were performed. In the first,
Table 2 Inhibition of cell proliferation by caged Garcinia xanthones in
adriamycin sensitive and resistant promyelocytic leukemia cells
Table 3 Inhibition of cell proliferation by caged Garcinia xanthones in
solid and non-solid tumor cells
IC50/lM
Compound
Cell line
3
6
7
17
Compound
HL-60
HL-60/ADR
Tissue type
IC50/lM
Forbesione (1)
2.2
2.0
Desoxymorellin (3)
1.0
0.3
0.8
1.1
0.5
1.1
A549
HT29
MCF-7
M21
PC3
CEM
Lung
Colon
Breast
Melanoma
Prostate
Leukemia
2.1
1.2
0.9
1.6
1.2
NDa
1.8
0.7
0.4
1.2
0.4
0.15
1.8
1.0
1.1
1.0
1.1
0.35
>4
Gambogic acid (6)
Gambogin (7)
17
21
22
3.1
NDa
2.2
1.5
1.4
Inactivea
Inactivea
Inactivea
Inactivea
NDa
0.3
a Less than 10% inhibition at 2.0 lM.
a ND: not determined.
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Fig. 2 Effect of gambogin (7) on CEM cell viability. CEM cells were treated with increasing concentrations of 7 for 4 days. Cell viability was determined
by counting cells excluding trypan blue dye. Error bars represent standard deviation.
viability was determined in CEM cells treated with compound 7
for 4 days using the trypan blue exclusion assay. The results, shown
Conclusions
In conclusion, we present herein a study toward the design of
in Fig. 2, demonstrated that CEM cells were very sensitive to 7
simplified structural analogues of the caged Garcinia natural
with an IC50 of 0.3 lM in agreement with that found in the cell
products and preliminary evaluation of their biologic activity in
proliferation studies (Table 3).
a variety of tumor cell lines and HUVE cells. We found that
The second viability study was performed in HUVE (human
analogue 17, which maintains the basic structural motif of the
umbilical vein endothelial) cells treated with compound 17 for
24 h. Cell viability was determined using the WST assay that
measures metabolic activity of cells alive. Compound 17 was found
to be cytotoxic with an IC50 value of 1.38 lM. A proliferation
caged Garcinia xanthones but is devoid of any functionalization
of the xanthone A-ring, maintains the activity exhibited by
the more structurally complex natural products of this family.
In contrast, compounds 21 and 22 that lack the C18 and
assay confirmed the WST assay (data not shown). This finding
C23 geminal methyl groups have substantially reduced activities,
suggests that compound 17 and related caged xanthones may have
a therapeutic potential as inhibitors of angiogenesis.23
suggesting that maintaining intact the caged C-ring is essential
for bioactivity. The active compounds had cytotoxicity at low
to sub-micromolar concentrations in solid and non-solid tumor
cell lines respectively and induced apoptosis in HUVE cells.
Remarkably, similar IC50 values were obtained for the compounds
tested in HL-60 and HL-60/ADR cell lines, suggesting that these
compounds are not subject to the mechanism of drug resistance
resulting from expression of mdr. Therefore, members of this
family of compounds may have therapeutic potential in relapsed
cancers typically resistant to standard chemotherapeutic agents.
In addition, the cytotoxicity observed in HUVE cells suggests that
these compounds are interesting leads for the development of new
inhibitors of angiogenesis. Future work is aimed at determining
tumor cell selectivity in vitro and in vivo with the possibility of
identifying more potent and more selective analogues.
The mechanism of cytotoxicity in HUVE cells was investigated
using an ELISA-based assay that distinguishes between apoptosis
and necrosis. This photometric assay allows the determination of
histone-associated DNA fragments that are released during cell
death. We found that the majority (>90%) of HUVE cells treated
with 17 underwent rapid apoptosis after 10 h in a dose-dependent
manner (Fig. 3). Cell necrosis was not detected at concentrations
lower than 1.5 lM and was only observed in a small subset of cells
(ca 10%) at much higher concentrations (>3 lM), indicating that
apoptosis is the predominant mechanism of cell death.
Experimental
General notes
o-Anisic acid (8), pyrogallol (9) and 2-chloro-2-methyl butyne (12)
were purchased from Aldrich. Gambogic acid (7) was purchased
from Gaia Chemical Corporation (CT, USA). The synthesis
and spectroscopic characterization of compounds 1, 3 and 7
have been reported in reference 10. All reagents were obtained
(Aldrich, Acros) at highest commercial quality and used without
further purification except where noted. Air- and moisture-
sensitive liquids and solutions were transferred via syringe or
stainless steel cannula. Organic solutions were concentrated by
Fig. 3 Induction of HUVE cell apoptosis (black line) and necrosis (gray
line) by 17 at different concentrations after 10 h incubation time; A =
A405 nm − A490 nm; c = concentration of 17.
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rotary evaporation below 45 ◦C at approximately 20 mmHg.
All non-aqueous reactions were carried out under anhydrous
conditions, i.e. using flame-dried glassware, under an argon
atmosphere and in dry, freshly distilled solvents, unless otherwise
noted. Dimethylformamide (DMF) and quinoline were distilled
from calcium hydride under reduced pressure (20 mmHg) and
(90% Et2O–hexane); 1H NMR (400 MHz, DMSO) d 8.14 (dd, J =
8.0, 1.6 Hz, 1H), 7.82–7.78 (m, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.54
(d, J = 8.8 Hz, 1H), 7.43–7.39 (m, 1H), 7.0 (d, J = 8.8 Hz, 1 H);
13C NMR (100 MHz, DMSO) d 175.1, 155.3, 151.4, 146.2, 134.6,
132.5, 125.7, 123.8, 120.7, 117.9, 116.4, 114.6, 113.2; HRMS calc.
for C13H8O4 (M + H+) 229.0501, found 229.0509.
˚
stored over 4 A molecular sieves until needed. Yields refer to
3,4-Bis(2-methylbut-3-yn-2-yloxy)-9H-xanthen-9-one (14). To
a round-bottomed flask containing xanthone 11 (500 mg,
2.19 mmol), KI (800 mg, 4.82 mmol), K2CO3 (666.2 mg,
4.82 mmol), and CuI (42 mg, 0.22 mmol) was added dry acetone
(20 mL). The reaction vessel was then equipped with a reflux
condenser, and the reaction was heated at 45 ◦C under argon. After
20 minutes, 2-chloro-2-methylbut-3-yne (12) (0.55 ml, 4.82 mmol)
was added, and the reaction was heated for two more hours.
The reaction was then cooled to 25 ◦C and acidified with 10%
HCl solution. The reaction mixture was partitioned between ethyl
ether (30 mL) and water. The aqueous layer was back-extracted
(2 × 30 mL), and the combined ethyl ether layers were dried
over MgSO4, filtered, and concentrated. The crude material was
purified through column chromatography (5–20% Et2O in hexane)
to yield compound 14 (394.8 mg, 50%); yellow solid; Rf = 0.45
(50% Et2O–hexane); 1H NMR (400 MHz, CDCl3) d 8.32 (dd, J =
8.0, 1.2 Hz, 1H), 8.06 (d, J = 9.2 Hz, 1H), 7.71–7.67 (m, 1H), 7.65
(d, J = 9.2 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.36 (dd, J = 8.0,
7.2 Hz, 1H), 2.66 (s, 1H), 2.29 (s, 1H), 1.83 (s, 6H), 1.76 (s, 6H);
13C NMR (100 MHz, CDCl3) d 176.5, 155.8, 155.7, 152.2, 134.2,
126.4, 123.7, 121.7, 117.9, 115.8, 74.8, 73.6, 30.6, 29.7; HRMS
calc. for C23H20O4 (M + H+) 361.1440, found 361.1464.
chromatographically and spectroscopically (1H NMR, 13C NMR)
homogeneous materials, unless otherwise stated. Reactions were
monitored by thin-layer chromatography (TLC) carried out on
0.25 mm E. Merck silica gel plates (60F-254) and visualized
under UV light and/or developed by dipping in solutions of
10% ethanolic phosphomolybdic acid (PMA) or p-anisaldehyde
and applying heat. E. Merck silica gel (60, particle size 0.040–
0.063 mm) was used for flash chromatography. Preparative thin-
layer chromatography separations were carried out on 0.25 or
0.50 mm E. Merck silica gel plates (60F-254). NMR spectra
were recorded on Varian Mercury 400 and/or Unity 500 MHz
instruments and calibrated using the residual undeuterated solvent
as an internal reference. The following abbreviations were used
to explain the multiplicities: s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet, b = broad. IR spectra
were recorded on a Nicolet 320 Avatar FT-IR spectrometer and
values are reported in cm−1 units. High resolution mass spectra
(HRMS) were recorded on a VG 7070 HS mass spectrometer under
chemical ionization (CI) conditions or on a VG ZAB-ZSE mass
spectrometer under fast atom bombardment (FAB) conditions.
(2-Methoxyphenyl)(2,3,4-trihydroxyphenyl)methanone
(10).
To a 250 ml round-bottomed flask containing flame-dried under
vacuum ZnCl2 (10.2 g, 75 mmol) was added o-anisic acid (8)
(2.49 g, 15 mmol) and pyrogallol (9) (2.07 g, 16.5 mmol) followed
by POCl3 (20 mL). The reaction vessel was then equipped with a
reflux condenser and stirred under argon at 65 ◦C for 8 h. The red
colored reaction mixture was then cooled to 25 ◦C and poured
into a beaker of about 500 g of ice. The mixture was extracted
with ethyl ether (3 × 100 mL), and the combined organic layers
were dried over MgSO4, filtered, and concentrated. The crude
material was purified through column chromatography (30%
Et2O–hexane) to yield benzophenone 10 (3.04 g, 78%); yellow
solid; Rf = 0.65 (90% Et2O–hexane); 1H NMR (400 MHz, CDCl3)
d 7.45 (ddd, J = 8.4, 7.2, 1.6 Hz, 1H), 7.26 (dd, J = 7.2, 2 Hz,
1H), 7.04–6.98 (m, 2H), 6.87 (d, J = 9.2 Hz, 1H), 6.42 (d, J =
9.2 Hz, 1H), 3.77 (s, 3H); 13C NMR (100 MHz, CDCl3) d 200.5,
156.2, 151, 150.1, 131.5, 130.8, 128.7, 127.4, 126.5, 120.2, 114.2,
111.3, 107.1, 55.7; HRMS calc. for C14H12O5 (M + H+) 261.0763,
found 261.0741.
3,4-Bis(2-methylbut-3-en-2-yloxy)-9H-xanthen-9-one (15). To
a solution of xanthone 14 (230 mg, 0.64 mmol) in EtOAc
(10 mL) was added 10% Pd/BaSO4 (23 mg) and quinoline
(0.66 mL, 0.56 mmol). The reaction mixture was degassed using
argon and stirred under an atmosphere of hydrogen for 3 hours.
Spectroscopic analysis (1H NMR) of the crude mixture revealed
that the reaction had proceeded by ca. 35%. During that time,
an additional amount of 10% Pd/BaSO4 (10 mg) was added to
accelerate the reaction. After stirring for an additional 3 hours
under a hydrogen atmosphere, the reaction was stopped and
1
the reduction, estimated by H NMR of the crude, was found
to be quantitative. The reaction mixture was filtered through
a plug of silica gel, and the residue concentrated and purified
through a column chromatography (3–10% Et2O–hexane) to yield
15 (207.3 mg, 89%); yellow solid; Rf = 0.66 (70% Et2O–hexane);
1H NMR (400 MHz, CDCl3) d 8.26 (dd, J = 6.0, 1.6 Hz, 1H), 7.90
(d, J = 8.8, 0.8 Hz, 1H), 7.65–7.61 (m, 1H), 7.46–7.43 (m, 1H),
7.31–7.27 (m, 1H), 7.09 (dd, J = 9.2, 2.0 Hz, 1H), 6.29–6.10 (m,
2H), 5.18–5.11 (m, 3H), 4.99–4.96 (m, 1H), 1.54 (d, J = 2 Hz, 6H),
1.52 (d, J = 2.4 Hz, 6H); 13C NMR (100 MHz, CDCl3) d 176.2,
156.5, 155.5, 152.1, 143.0, 135.4, 134.0, 126.2, 123.8, 123.5, 121.2,
120.8, 120.7, 117.7, 117.6, 116.9, 116.4, 113.8, 112.8, 83.3, 81.9,
27.1, 26.9; HRMS calc. for C23H24O4 (M + H+) 365.1753, found
365.1740.
3,4-Dihydroxy-9H-xanthen-9-one (11). To a solution of ben-
zophenone 10 (1.6 g, 6.15 mmol) in methanol (20 mL) was added
a solution of aqueous NaOH (30% w/w, 20 mL) and water
(10 mL). The green colored reaction mixture was then refluxed
at 100 ◦C for 3 days. The red colored reaction mixture was
cooled to 25 ◦C and acidified with aqueous HCl (10%, 600 mL).
The reaction mixture was partitioned between water and ethyl
ether (50 mL). The aqueous layer was then back extracted with
ethyl ether (2 × 50 mL). The combined organic layers were
dried over MgSO4, filtered, and concentrated. The crude material
was purified through column chromatography (20–40% Et2O in
hexanes) to yield xanthone 11 (0.99 g, 71%); yellow solid; Rf = 0.4
Caged xanthone 17. A solution of 15 (55 mg, 0.15 mmol) in
DMF (2.0 mL) was heated at 120 ◦C for 1 hour. The yellow
reaction mixture was cooled 25 ◦C and the mixture purified by
column chromatography (15–20% Et2O–hexane) to yield the caged
xanthone 17 (42.5 mg, 78%). Alternatively, a solution of 15 (39 mg,
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0.11 mmol) in MeOH–H2O 1 : 1 (2.0 mL) was heated at 100 ◦C for
0.5 hours. Purification as indicated above yielded caged xanthone
17 (36.6 mg, 94%); white solid; Rf = 0.55 (70% Et2O–hexane);
1H NMR (400 MHz, CDCl3) d 7.93 (dd, J = 8.0, 1.6 Hz, 1H),
7.53–7.49 (m, 1H), 7.42 (d, J = 6.8 Hz, 1H), 7.07–7.03 (m, 2H),
4.42–4.38 (m, 1H), 3.49 (dd, J = 6.8, 4.8 Hz, 1H), 2.67–2.61 (m,
2H), 2.45 (d, J = 9.6 Hz, 1H), 2.33 (dd, J = 13.6, 4.8 Hz, 1H), 1.72
(s, 3H), 1.30 (s, 6H), 0.89 (s, 3H); 13C NMR (100 MHz, CDCl3) d
202.7, 176.2, 159.4, 136.0, 134.7, 134.6, 133.5, 126.8, 121.7, 118.9,
118.8, 117.9, 90.2, 84.5, 83.5, 48.8, 46.8, 30.4, 29.8, 29.2, 25.4,
25.2, 16.8; HRMS calc. for C23H24O4 (M + H+) 365.1753, found
365.1765.
119,1, 118.5, 118.2, 83.7, 76.2, 75.9, 45.7, 45.4; HRMS calc. for
C19H16O4 (M + H+) 309.1127, found 309.1133.
3H-Thymidine incorporation assay. Cells were plated in a 96-
well plate at 10–20 × 103 cells/well in RPMI supplemented
with 10% fetal bovine serum, 2 mM glutamine, 1% peni-
cillin/streptomycin (complete medium). The caged Garcinia xan-
thones were added to the cells at increasing concentrations and
0.1% DMSO was added to control cells. Cells were incubated for
48 h and then pulsed with 3H-thymidine for 6 h. Incorporation of
3H-thymidine was determined in a scintillation counter (Beckman
Coulter Inc., Fullerton, CA) after cells were washed and deposited
onto glass microfiber filters using a cell harvester M-24 (Brandel,
Gaithersbur, MD).
3,4-Bis(allyloxy)-9H-xanthen-9-one (18). To a flask contain-
ing xanthone 11 (500 mg, 2.19 mmol) and K2CO3 (666.2 mg,
4.82 mmol) was added dry acetone (20 mL), followed by allyl-
bromide (0.42 mL, 4.82 mmol). The reaction vessel was then
equipped with a reflux condenser, and the reaction was heated
at 45 ◦C under argon for two hours. The mixture was then
cooled to room temperature and acidified with 10% aqueous HCl
solution. The reaction mixture was partitioned between ethyl ether
(20 mL) and water (20 mL). The aqueous layer was back-extracted
(2 × 20 mL), and the combined ethyl ether layers were dried
over MgSO4, filtered, and concentrated. The crude material was
purified through a column chromatography (5–20% Et2O–hexane)
to yield allylated xanthone 18 (675.2 mg, 100%); white solid; Rf =
0.45 (50% Et2O–hexane); 1H NMR (400MHz, CDCl3) d 8.3 (dd,
J = 8.0, 1.6 Hz, 1H), 8.04 (d, J = 8.8 Hz, 1H), 7.69 (dt, J =
8.4, 1.2 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.35 (dd, J = 8.0,
7.2 Hz), 6.97 (d, J = 8.8 Hz), 6.23–6.03 (m, 2H), 5.49–5.32 (m,
3H), 5.38 (dd, J = 10.4, 0.8 Hz, 1H), 4.71 (dd, J = 6.4, 5.2 Hz);
13C NMR (100 MHz, CDCl3) d 176.2, 156.5, 155.9, 150.6, 134.3,
133.6, 132.1, 126.4, 123.7, 122.1, 121.4, 118.3, 118.1, 117.9, 116.6,
109.8, 74.7, 69.8; HRMS calc. for C19H16O4 (M + H+) 309.1127,
found 309.1134.
Trypan-blue exclusion assay. CEM cells were plated in a 24-
well plate in complete media at 50 × 103 cells/well. Cells were
treated with 7 at concentrations of 0.25, 0.5, 1.0 lM or with 0.1%
DMSO (control cells). Cells were incubated for 4 days and then
the number of viable cells was determined after the addition of
trypan-blue dye by counting the cells which exclude trypan-blue
in a hemocytometer.
WST assay. Compound 17 was dissolved in DMSO and fur-
ther diluted with Endothelial Cell Growth Medium (PromoCell,
Heidelberg, Germany) to obtain a final concentration as indicated.
HUVE cells (PromoCell, Heidelberg, Germany) were seeded into
each well of a 96-well cell culture plate at 7000 cells per well and
◦
incubated at 37 C for 24 h with the indicated concentrations of
each compound. The final volume was 100 lL per well. Control
samples were incubated with the solvent alone. Each experiment
was repeated in triplicate. Afterwards the WST-1 reagent was
added to the cells at 10 lL per well and the cells further incubated
at 37 ◦C for additional 3 h. Then the cell culture plate was agitated
thoroughly for 1 minute on a shaker at 200 U/min. The absorbance
of each sample was measured using a microplate reader at 440 nm.
The reference wavelength was 690 nm.
Caged xanthones 21 and 22. A solution of 18 (46.2 mg,
0.15 mmol) in DMF (2.0 mL) was refluxed (153 ◦C) for 16 h.
The yellow reaction mixture was then cooled to 25 ◦C and the
residue was column chromatographed (10–30% Et2O–hexane)
to yield a mixture of caged xanthones 21 (12.4 mg, 27%) and
22 (14.7 mg, 32%). Alternatively, a solution of 18 (25.1 mg,
0.08 mmol) in MeOH–H2O 1 : 1 (2.0 mL) was heated at 100 ◦C for
4 hours. Purification as indicated above yielded caged xanthones
21 (5.2 mg, 21%) and 22 (16.7 mg, 67%); 21: white solid; Rf =
0.55 (70% Et2O–hexane); 1H NMR (300 MHz, CDCl3) d 7.95 (d,
J = 7.8 Hz, 1H), 7.59–7.54 (m, 1H), 7.34 (d, J = 7.2 Hz, 1H),
7.11–6.98 (m, 2H), 5.29–5.15 (m, 1H), 4.68 (d, J = 9.9 Hz, 1H),
4.56–4.50 (m, 2H), 3.91 (d, J = 7.5 Hz, 1H), 3.54–3.44 (m, 1H),
2.61 (m, 2H), 2.23 (m, 3H); HRMS calc. for C19H16O4 (M + H+)
309.1127, found 309.1132. 22: white solid; Rf = 0.54 (70%, Et2O–
hexane); 1H NMR (400 MHz, CDCl3) d 7.92 (dd, J = 8.0, 2.0 Hz,
1H), 7.58–7.53 (m, 1H), 7.30 (d, J = 6.8 Hz, 1H), 7.18 (d, J =
8.4 Hz, 1H), 7.09–7.05 (m, 1H), 5.65–5.54 (m, 1H), 5.14–5.09 (m,
2H), 4.08 (dd, J = 8.4, 3.6 Hz, 1H), 3.94 (m, 1H), 3.47 (dd, J =
6.4, 4.4 Hz, 1H), 2.63 (dd, J = 14, 6 Hz, 1H), 2.57–2,48 (m, 1H),
2.28 (dd, J = 14, 7.6 Hz, 1H), 2.21 (d, J = 12.8, 5.6 Hz, 2H);
13C NMR (100 MHz, CDCl3) d 198.1, 175.4, 160.2, 136.6, 136.5,
134.5, 134.3, 134.2, 131.6, 131.4, 127.2, 126.9, 122.3, 121.9, 119.6,
Apoptosis assay. The compounds were dissolved in DMSO
and further diluted with Endothelial Cell Growth Medium
(PromoCell, Heidelberg, Germany) to obtain final concentrations
as indicated. HUVE cells were seeded into each well of a 96-well
cell culture plate at 10000 cells per well and incubated at 37 ◦C
for 10 h with the indicated concentrations of each compound. The
final volume was 100 lL per well. Control samples were incubated
with the solvent alone. Each sample was repeated three times.
The proapoptotic effect was detected by using the Cell Death
Detection ELISAPLUS kit (Roche Diagnostics GmbH, Mannheim,
Germany) according to the manufactor’s instructions. The kit con-
stitutes a photometric enzyme-immunoassay for the qualitative
and quantitative in vitro determination of cytoplasmic histone-
associated-DNA-fragments (mono- and oligo-nucleosomes) after
induced cell death. Due to the working procedure the kind of cell
death (apoptosis or necrosis) can be determined. The absorption
values A (A405 nm − A490 nm) measured give a quantitative indication
of the induced amount of apoptosis/necrosis. The higher the
absorption A, the higher the induction of apoptosis/necrosis at
the corresponding concentrations of the compounds.
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Tisdale, I. Slobodov and E. A. Theodorakis, Org. Biomol. Chem., 2003,
1, 4418–4422.
12 (a) A. J. Quillinan and F. Scheinmann, J. Chem. Soc. D, 1971, 966–967;
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13 For related synthetic studies by the Nicolaou group see: (a) K. C.
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