Synthesis of caged 2,3,3a,7a-tetrahydro-3,6-methanobenzofuran-7(6H)-ones: Evaluating the minimum structure for apoptosis induction by gambogic acid

Article abstract of DOI:10.1016/j.bmc.2008.02.084

We have reported the discovery of gambogic acid (GA) as a potent apoptosis inducer and the identification of transferrin receptor as its molecular target. In order to understand the basic pharmacophore of GA for inducing apoptosis and to discover novel and simplified derivatives as potential anti-cancer agents, we explored the synthesis of caged 2,3,3a,7a-tetrahydro-3,6-methanobenzofuran-7(6H)-ones (4-oxatricyclo[4.3.1.0]decan-2-ones). Three types of 2,3,3a,7a-tetrahydro-3,6-methanobenzofuran-7(6H)-ones based on xanthone, 2-phenylchromene-4-one and benzophenone, were synthesized using a Claisen/Diels-Alder reaction cascade. All the reactions produced the targeted caged compound as well as its neo-isomer. The caged compounds based on xanthone and 2-phenylchromene-4-one were found to maintain the apoptosis inducing and cell growth inhibiting activity of GA, although with less potency. The caged compounds based on benzophenone were found to be inactive. Our study determined the minimum structure of GA for its apoptosis inducing activity, which could lead to the development of simple derivatives as potential anti-cancer drugs.

Full text of DOI:10.1016/j.bmc.2008.02.084

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4233–4241
Synthesis of caged 2,3,3a,7a-tetrahydro-3,6-methanobenzofuran-
7(6H)-ones: Evaluating the minimum structure
for apoptosis induction by gambogic acid
Jared Kuemmerle, Songchun Jiang, Ben Tseng, Shailaja Kasibhatla,
John Drewe and Sui Xiong Cai*
Epicept Corporation, 6650 Nancy Ridge Drive, San Diego, CA 92121, USA
Received 22 January 2008; accepted 26 February 2008
Available online 29 February 2008
Abstract—We have reported the discovery of gambogic acid (GA) as a potent apoptosis inducer and the identification of transferrin
receptor as its molecular target. In order to understand the basic pharmacophore of GA for inducing apoptosis and to discover
novel and simplified derivatives as potential anti-cancer agents, we explored the synthesis of caged 2,3,3a,7a-tetrahydro-3,6-meth-
anobenzofuran-7(6H)-ones (4-oxatricyclo[4.3.1.0]decan-2-ones). Three types of 2,3,3a,7a-tetrahydro-3,6-methanobenzofuran-
7(6H)-ones based on xanthone, 2-phenylchromene-4-one and benzophenone, were synthesized using a Claisen/Diels–Alder reaction
cascade. All the reactions produced the targeted caged compound as well as its neo-isomer. The caged compounds based on xan-
thone and 2-phenylchromene-4-one were found to maintain the apoptosis inducing and cell growth inhibiting activity of GA,
although with less potency. The caged compounds based on benzophenone were found to be inactive. Our study determined the
minimum structure of GA for its apoptosis inducing activity, which could lead to the development of simple derivatives as potential
anti-cancer drugs.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
promote apoptosis via targeting key proteins in the
apoptosis pathway, including Bcl-2 inhibitors,6 inhibi-
tors of MDM2-p53 interaction,7 inhibitors of XIAP,8
and Smac mimetics,9 are currently being explored.
Apoptosis, also called programmed cell death, plays
critical roles in normal cell development as well as elim-
ination of excessive or dysfunctional cells.1 Caspases are
known to be crucial for the execution of apoptosis.2
Among the caspases, caspase-3 has been called an execu-
tioner caspase and it cleaves multi-protein substrates in
cells, leading to irreversible cell death.3 Inadequate
apoptosis, such as due to defects in the molecular
machinery to activate the caspase cascade, is one of
the hallmarks of many cancer cells.4
Toward our goal of discovering and developing novel
apoptosis inducers as potential anti-cancer drugs, we
have developed a cell-based high-throughput screening
(HTS) assay for the identification of apoptosis inducers
using a proprietary fluorescent caspase-3 substrate.10,11
Using this technology, we have discovered several series
of potent apoptosis inducers, including 4-aryl-chrom-
enes12 and 3,5-diaryl-oxadiazoles,13 as well as identified
their molecular targets.14,15
It is known that many clinically used anti-cancer drugs
kill tumor cells through the induction of apoptosis.
Therefore, promoting apoptosis is a promising strategy
that could lead to the discovery and development of
new anti-cancer agents.5 Several novel approaches to
We have reported the discovery and SAR studies of
gambogic acid (1, GA) (Chart 1) as a novel apoptosis in-
ducer.16 The 9,10 carbon–carbon double bond of the
a,b-unsaturated ketone in 1 was found to be critical
for its apoptosis inducing activity, and the 9,10 satu-
rated derivative 2 was found to be devoid of apoptosis
inducing and anti-proliferative activity. The 6-hydroxy
and 30-carboxy groups were found to tolerate a
variety of modifications.16 Applying the SAR data, the
Keywords: Gambogic acid; Apoptosis inducer; Cell growth inhibition;
2,3,3a,7a-Tetrahydro-3,6-methanobenzofuran-7(6H)-ones; Claisen/Di-
els–Alder reaction cascade; Potential anti-cancer drugs.
*
Corresponding author. Tel.: +1 858 202 4006; fax: +1 858 202
4000; e-mail: scai@epicept.com
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.084

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J. Kuemmerle et al. / Bioorg. Med. Chem. 16 (2008) 4233–4241
natural products in the genus Garcinia.26 Several natural
CO2H
30
products containing this caged system, includ-
ing 1-O-methylforbesione (3a)27 and forbesione (3b)28
(Chart 1) have been prepared recently via total synthe-
sis. More recently, the synthesis and biological evalua-
tion of simple caged Garcinia xanthones 4 and its
neo-isomer 5 have been reported.29 All these compounds
were prepared using a Claisen/Diels–Alder reaction cas-
cade.30 The reaction in general produced the regular
scaffold that is presented in the natural products from
Garcinia (1–4), as well as a neo-scaffold as in 5.31
O
O
O
O
O
HO
O
O
O
O
9
6
10
O
OH
OR
1,
2,
3a, R = Me,
3b, R = H,
=
=
O
O
O
O
O
O
Since our SAR studies showed that the C@C of the a,b-
unsaturated ketone in 1 is critical for its apoptosis
inducing activity, while the 6-hydroxy and 30-carboxy
group could tolerate a variety of modifications, we have
been interested in the synthesis of simple derivatives of
GA such as 4.32 These molecules would be useful to
determine the role of the side groups and the tetracyclic
pyran–xanthone structure for the apoptosis inducing
activity of GA. In addition, they also could lead to the
development of simple and novel derivatives of GA as
potential anti-cancer drugs. Herein we report the synthe-
sis of three groups of simple derivatives of GA based on
xanthone, 2-phenylchromene-4-one and benzophenone,
and the characterization of these compounds as apopto-
sis inducers.
O
4
5
Chart 1.
30-carboxy group was used to prepare a series of re-
agents, including biotin labeled and fluorescent labeled
molecules, leading to the identification of transferrin
receptor as the molecular target of GA.17 More recently,
GA has been reported to inhibit NF-kappaB signaling
pathway and potentiating apoptosis through its interac-
tion with the transferrin receptor.18 GA also has been re-
ported to selectively induce apoptosis of human
hepatoma SMMC-7721 cells, while having relatively less
effect on human normal embryonic hepatic L02 cells,
suggesting that GA might be a highly effective anti-can-
cer drug candidate with low toxicity to normal tissue.19
2. Results and discussion
2.1. Chemistry
In addition, GA has been reported recently to inhibit the
catalytic activity of human topoisomerase II-a by bind-
ing to its ATPase domain,20 as well as to inhibit angio-
genesis through suppressing vascular endothelial growth
factor-induced tyrosine phosphorylation of KDR/Flk-
1.21 A derivative of GA, acetyl isogambogic acid, has
been reported to efficiently elicit cell death in melanoma
cells and to inhibit ATF2 transcriptional activities, acti-
vate JNK, and increase c-Jun transcriptional activities.22
N-(2-Ethoxyethyl)gambogamide (NG-18), a derivative
of GA, also has been reported to efficiently induce apop-
tosis in cultured human cancer cells associated with up-
regulation of the pro-apoptotic Bcl-2 family member
Bax, and down-regulation of anti-apoptotic protein
Bcl-2.23 Interestingly, gambogic amide, the amide deriv-
ative of GA, has been reported recently to selectively
bind to TrkA and robustly induce its tyrosine phosphor-
ylation and downstream signaling activation, including
Akt and MAPKs, and to strongly prevent glutamate-in-
duced neuronal cell death and provoke prominent neu-
rite outgrowth in PC12 cells, suggesting that gambogic
amide might provide an effective treatment for neurode-
generative diseases and stroke.24
The xanthone-based caged compounds 4 and 5 were
synthesized using procedures similar to Nicolaou and
Li27 and Batova et al.29 as shown in Scheme 1. 3,4-Dihy-
droxy-9H-xanthen-9-one (8) was prepared from reaction
of 2-fluorobenzoyl chloride (7) with pyrogallol and alu-
minum chloride, followed by cyclization of the interme-
diate benzophenone in the presence of sodium
carbonate. Reaction of 8 with allyl bromide in the pres-
ence of potassium carbonate produced 3,4-bis-allyloxy-
9H-xanthen-9-one (9). Compound 9 was heated in di-
phenyl ether at 190 ꢁC to produce targeted compound
1
4 as well as its neo-isomer 5. The H NMR spectra of
4 and 5 have distinct patterns both in the aromatic re-
gion and non-aromatic region, in agreement with what
has been reported by Batova et al.29 Treatment of com-
pound 4 with L-Selectride selectively reduced the C@C
double bond in the a,b-unsaturated ketone to give com-
pound 10, similar to what has been observed for GA.16
The xanthone-based caged compounds 13 and 14 were
prepared using procedures similar to Batova et al.29 as
shown in Scheme 2. Reaction of xanthone 8 with 3-
chloro-3-methyl-1-butyne in the presence of cupric
chloride and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
produced 3,4-bis-(1,1-dimethyl-prop-2-ynyloxy)-9H-
xanthen-9-one (11). Reduction of 11 by hydrogen using
Lindlar catalyst produced 3,4-bis-(1,1-dimethyl-allyl-
oxy)-9H-xanthen-9-one (12). When compound 12 was
refluxed in toluene, it produced the targeted compound
13 as well as its neo-isomer 14. This is different from
GA is a natural product isolated from the gamboge resin
of Garcinia hanburyi tree in Southeast Asia. The struc-
ture of GA was confirmed by X-ray crystallographic
analysis.25 It contains a unique caged 2,3,3a,7a-tetrahy-
dro-3,6-methanobenzofuran-7(6H)-one (4-oxatricyclo
[4.3.1.0]decan-2-one) ring system which is found in the

J. Kuemmerle et al. / Bioorg. Med. Chem. 16 (2008) 4233–4241
4235
OH
OH
O
F
O
F
OH
Br
O
O
OH
ClCOCOCl
CH2Cl2
OH
OH
Cl
1) AlCl3 / CH2Cl2
2) Na2CO3 / DMF, reflux
K2CO3 / acetone
6
7
8
O
O
O
O
O
Ph2O
190 oC
O
O
O
O
O
O
O
O L-Selectride
+
O
9
O
10
O
5
4
Scheme 1.
Cl
OH
O
H2
O
OH
O
O
Lindlar catalyst
CuCl2/DBU
O
O
8
11
O
O
O
O
Toluene
Reflux
O
O
O
O
O
O
O
+
O
14
12
13
Scheme 2.
Batova et al.29 which reported that heating of 12 in
DMF at 120 ꢁC or MeOH/H2O at 100 ꢁC produced only
compound 13. This is probably due to the different sol-
vents and conditions used in the reaction. The 1H NMR
signals of the aromatic region of 14 give a distinct pat-
tern that is similar to that of the corresponding neo-iso-
mer 5. Correspondingly, the pattern of 1H NMR signals
of the aromatic region of isomer 13 have a distinct pat-
tern that is similar to that of the corresponding isomer 4.
to 1.9 ppm are similar to those of the corresponding
neo-isomer 14 from 2.6 to 1.8 ppm.
The benzophenone-based caged compounds 24 and 25
were prepared similar to 4 and 5 as shown in Scheme
4. Reaction of 3,4-dihydroxybenzophenone (20) with
allylbromide in the presence of cesium carbonate pro-
duced 3,4-bis-allyloxy-benzophenone (21), which was
heated in diphenyl ether at 200 ꢁC to produce (2,5-dial-
lyl-3,4-dihydroxy)-benzophenone (22). Reaction of 22
with allylbromide in the presence of cesium carbonate
The 2-phenylchromene-4-one-based caged compounds
18 and 19 were prepared by the method similar to 13
and 14 as shown in Scheme 3. Reaction of 7,8-dihy-
droxy-2-phenyl-4H-chromen-4-one (15) with 3-chloro-
3-methyl-1-butyne in the presence of cupric chloride
and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) produced
7,8-bis-(1,1-dimethyl-prop-2-ynyloxy)-2-phenyl-4H-chro-
men-4-one (16), which was reduced by hydrogenation
using Lindlar catalyst to produce 7,8-bis-(1,1-dimethyl-
allyloxy)-2-phenyl-4H-chromen-4-one (17). Compound
17 was heated in diphenyl ether at 120 ꢁC to produce
the target compound 18 as well as its neo-isomer 19. It
produced
(2,5-diallyl-3,4-bis-allyloxy)-benzophenone
(23). Compound 23 was heated in diphenyl ether at
200 ꢁC to produce the target compound 24 as well as
its neo-isomer 25.
2.2. Induction of apoptosis
The apoptosis inducing activity of the caged molecules
was measured by our cell- and caspase-based HTS as-
say33 in human breast cancer cells T47D, human colon
cancer cells HCT116 and hepatocellular carcinoma can-
cer cells SNU398, and the results are summarized in
Table 1. The xanthone-based compound 4 is about 10
times less active than GA (1) as an apoptosis inducer
in HCT116 and SNU398 cells, two of three cell lines
1
was observed that the H NMR signals of isomer 18
from 2.7 to 2.3 ppm have a pattern that is similar to
those of the corresponding isomer 13 in the same region,
while the 1H NMR signals of the neo-isomer 19 from 2.7

4236
J. Kuemmerle et al. / Bioorg. Med. Chem. 16 (2008) 4233–4241
Cl
OH
O
H2
O
OH
O
O
O
Lindlar catalyst
CuCl2/DBU
O
15
16
O
O
O
Ph2O
O
O
O
O
O
O
O
+
120 °C
O
O
19
17
18
Scheme 3.
OH
Br
O
OH
Ph2O
OH
O
OH
200 oC
Cs2CO3 / acetone
O
O
O
22
20
21
O
Br
Cs2CO3 / acetone
O
O
O
O
Ph2O
O
+
200 oC
O
O
O
25
24
23
Scheme 4.
important for apoptosis inducing activity, and the posi-
tions of the caged structure fused with the bicyclic chro-
man-4-one are not critical for activity.
Table 1. Caspase activation activity of caged 2,3,3a,7a-tetrahydro-3,6-
methanobenzofuran-7(6H)-ones
Compound
EC50 (lM)a
T47D
HCT116
SNU398
When the carbon–carbon double bond in the a,b-unsat-
urated ketone of 4 was reduced, compound 10 was
found to be inactive up to 20 lM in all three cell lines.
This is similar to the reported SAR of GA (10 vs. 2),16
showing that the carbon–carbon double bond is essen-
tial for apoptosis inducing activity of GA and its simple
caged derivatives.
1
2
0.70 0.08
>20
>20
0.70 0.02
>20
0.70 0.07
>20
4
7.1 0.4
>20
>20
5.7 0.1
5.1 0.2
>20
5
5.9 0.4
>20
10
13
14
18
19
24
25
3.0 0.07
2.7 0.05
5.1 0.04
4.8 0.1
>20
2.3 0.2
1.9 0.1
5.4 0.05
3.6 0.4
>20
0.90 0.06
1.5 0.04
2.6 0.04
2.5 0.01
>20
The xanthone-based tetramethyl compound 13 was
found to be about 3- to 7-fold more active than 4, con-
firming that the side group of GA contributes to its
activity. Compound 13 was about three to four times
less active than GA (1) in T47D and HCT116 cells,
and about as active as GA in SNU cells, indicating
that compound 13 has good apoptosis inducing activ-
ity. The neo-isomer 14 was found to be about as active
as 13, confirming that how the caged structure fused
with the bicyclic chroman-4-one is not critical for its
activity. Interestingly, the 4-phenylchromene-2-one-
based compound 18 was found to be only about 2-fold
less active than 13, indicating that the 6-phenylpyran-
4-one ring in 18 can be used to replace the chroman-4-
>20
>20
>20
a Data are means of three or more experiments and are reported as
means standard error of the mean (SEM).
tested, indicating that the simple structure of 4 still
maintains some of the apoptosis inducing activity of
GA, and that the side groups contribute to the apoptosis
activity of GA. The neo-isomer 5 is about as active as 4
in inducing apoptosis and also is active in two of three
cell lines tested, suggesting that the caged structure is

J. Kuemmerle et al. / Bioorg. Med. Chem. 16 (2008) 4233–4241
4237
one ring in 13. Thus the tetracyclic pyran–xanthone
structure of GA can be reduced to a bicyclic chro-
men-4-one structure and still maintain some of its
apoptosis inducing activity. Similarly as observed
above, the neo-isomer 19 was found to be about as ac-
tive as that of 18.
up to 20 lM in all the three cell lines, confirming the
importance of the carbon–carbon double bond in the
a,b-unsaturated ketone for the apoptosis inducing and
growth inhibiting activities of GA and its derivatives.
Interestingly, compounds 13 and 14 were found to have
GI50 values of around 0.2 lM in the three cell lines, and
were about as active as GA (1), indicating these simple
GA derivatives have good cell growth inhibiting activity
but less apoptosis inducing activity. Compounds 18 and
19 were found to be about 5-fold less active than 13 and
14. Compound 24, which was not active in the apoptosis
induction assay, also was found to be inactive in the
growth inhibition assay up to 20 lM in all the three cell
lines.
The benzophenone-based compound 24 was found to
be inactive up to 20 lM in all the three cell lines, sug-
gesting that a bicyclic structure such as that in 18 might
be the minimum to maintain the apoptosis inducing
activity of GA. Similarly, the neo-isomer 25 also was
found to be inactive up to 20 lM in all the three cell
lines. It is also possible that the two extra 2-propenyl
groups in the bridge heads of 24 and 25 might have
negative effects on their biological activity. Another
possible explanation is that because the carbonyl in
the benzoyl group is not part of a ring structure fused
into the caged structure, the carbonyl group is not in
the same plane as the C@C bond in the caged structure
due to some unfavorable steric interaction. Therefore
the a,b-unsaturated ketone in 24 and 25 might not be
able to function similarly as the one in GA as well as
compounds 4 and 14.
3. Conclusion
In conclusion, we have synthesized three groups
of caged 2,3,3a,7a-tetrahydro-3,6-methanobenzofuran-
7(6H)-ones based on tricyclic xanthone, bicyclic 2-phe-
nylchromene-4-one and mono-cyclic benzophenone as
simple derivatives of GA. The xanthone and 2-phenyl-
chromene-4-one-based caged molecules 4, 13 and 18,
as well as the corresponding neo-isomers 5, 14 and 19,
were found to induce apoptosis in our cell-based assay.
These molecules are 5–10 times less potent than GA.
The benzophenone-based caged molecule 24 and its
neo-isomer 25 were found to be inactive in the apoptosis
induction assay. Similar to GA, the carbon–carbon dou-
ble bond in the a,b-unsaturated ketone of compound 4
was found to be essential for its apoptosis inducing
activity and reduction of the carbon–carbon double
bond in 4 produced compound 10 which was found to
be inactive in the apoptosis assay. The compounds that
are active in the caspase activation assay also were
found to be active in the growth inhibition assay. Our
synthesis and biological studies determined the mini-
mum structure of GA for its apoptosis inducing and cell
growth inhibiting activities, which could lead to the de-
sign and synthesis of simple derivatives of GA as poten-
tial anti-cancer drugs.
2.3. Growth inhibition
The caged compounds were also tested by the tradi-
tional growth inhibition assay to confirm that the active
compounds can inhibit cancer cell growth. The growth
inhibition assays in T47D, HCT116 and SNU398 cells
were run in a 96-well microtiter plate as described previ-
ously.34 The 50% growth inhibition (GI50) is defined as
the concentration of drug causing 50% inhibition in
absorbance compared with control untreated cells.
GI50 values are summarized in Table 2.
Compounds 4 and 5 were found to have GI50 values of
1–2 lM in the three cell lines tested and to be about 10-
to 15-fold less active than GA (1). Interestingly, com-
pounds 4 and 5 were reported to be inactive in an inhi-
bition of cell proliferation assay.29 This is probably due
to the difference in the concentration of compounds used
in the assays as well as sensitivity of the assays. Similarly
to compound 2, compound 10 was found to be inactive
4. Experimental
4.1. General methods and materials
Table 2. Inhibition of cell proliferation activity of caged 2,3,3a,7a-
tetrahydro-3,6-methanobenzofuran-7(6H)-ones
The 1H NMR spectra were recorded at Varian 300 MHz.
Chemical shifts are reported in ppm (d) and J coupling
constants are reported in Hz. The 13C NMR spectra were
recorded at Varian 75 MHz and chemical shifts are re-
ported in ppm (d). MS spectra analysis was performed
by HT Laboratory, Inc. (San Diego, CA). Elemental
analyses were performed by Numega Resonance Labs,
Inc. (San Diego, CA). Reagent grade solvents were used
without further purification unless otherwise specified.
Flash chromatography was performed on Merck silica
gel 60 (230–400 mesh) using reagent grade solvents. Hu-
man breast cancer cells T47D, human colon cancer cells
HCT116 and hepatocellular carcinoma cancer cells
SNU398 were obtained from the American Type Culture
Collection (Manasas, VA).
Compound
GI50 (lM)a
T47D
HCT116
SNU398
1
2
0.20 0.01
>20
0.10 0.01
>20
0.20 0.01
>20
4
1.6 0.06
1.4 0.07
>20
1.6 0.03
1.5 0.08
>20
1.9 0.2
1.5 0.08
>20
5
10
13
14
18
19
24
0.20 0.01
0.30 0.08
1.4 0.03
1.0 0.03
>20
0.20 0.01
0.20 0.02
1.1 0.03
0.9 0.01
>20
0.20 0.02
0.30 0.03
1.2 0.02
1.2 0.06
>20
a Data are means of three experiments and are reported as
means standard error of the mean (SEM).

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J. Kuemmerle et al. / Bioorg. Med. Chem. 16 (2008) 4233–4241
4.1.1. 3,4-Dihydroxy-9H-xanthen-9-one (8). To a stirring
solution of 2-fluorobenzoic acid (5.09 g, 36.3 mmol) and
dichloromethane (110 mL) in an ice bath under argon
was added dropwise a solution of oxalyl chloride
(2.0 M in dichloromethane, 21 mL, 42 mmol), followed
by dimethylformamide (6 drops). The ice bath was re-
moved and the solution was stirred at room temperature
for 1.5 h. The solution was then concentrated by rotary
evaporation. The product was dissolved in hexane (3·
50 mL) and the mixture was filtered. The filtrate was ro-
tary evaporated to yield 5.42 g of colorless oil. The oil
was added dropwise to a mixture of pyrogallol (6.48 g,
51.3 mmol), aluminum chloride (14.6 g, 110 mmol),
chloroform (250 mL) and dichloromethane (700 mL),
and the solution was stirred for 17 h at room tempera-
ture. The solution was then refluxed for 3 h and cooled
to room temperature. The solution was washed with 1 N
HCl (3· 500 mL). The organic layer was filtered, dried
over sodium sulfate, and evaporated to yield an oil.
The oil was added to dimethylformamide (120 mL) with
sodium carbonate (8.11 g, 76.5 mmol) and it was re-
fluxed for 3.5 h. The solution was concentrated by ro-
tary evaporation with heating, and the residue was
purified by column chromatography (95:5 chloroform/
methanol) to give a solid. The solid was washed with
hexane (2· 35 mL), filtered and dried to yield compound
7.31 (d, J = 6.9, 1H), 7.19 (dd, J = 8.4, 1.0, 1H), 7.08
(td, J = 7.6, 1.1, 1H), 5.60 (m, 1H), 5.15 (s, 1H), 5.11
(m, 1H), 4.09 (dd, J = 8.3, 3.6, 1H), 3.97 (d, J = 8.2,
1H), 3.48 (dd, J = 6.9, 4.4, 1H), 2.63 (dd, J = 14.3, 6.6,
1H), 2.55 (m, 1H), 2.29 (dd, J = 12.5, 5.9, 1H), 2.23
(d, J = 5.8, 2H). Anal. Calcd. For C19H16O4: C, 74.01;
H, 5.23. Found: C, 73.62; H, 5.55.
4.1.4. 3,3a,4,5,6,6a-Hexahydro-1-(2-propenyl)-1,5-meth-
ano-1H,7H-furo[3,4-d]xanthene-7,13-dione (10). To a
stirring solution of 4 (29 mg, 0.094 mmol) in tetrahydro-
furan (15.0 mL) in a dry ice bath under argon was added
dropwise 1.0 M L-Selectride in THF (150 lL,
0.15 mmol) and the solution was stirred for 25 min.
The dry ice bath was removed and the solution was stir-
red for 20 min. The solution was concentrated by rotary
evaporation and the residue was purified by column
chromatography (2:1 hexanes/ethyl acetate) to yield
1
compound 10 (11 mg, 39%) as white solids. H NMR
(CDCl3): 7.91 (dd, J = 8.1, 1.8, 1H), 7.54 (ddd, J = 8.2,
7.0, 1.5, 1H), 7.08 (m, 2H), 5.86 (m, 1H), 5.27
(m, 1H), 5.12 (dt, J = 10.2, 1.5, 1H), 4.16 (dd, J = 8.0,
4.1, 1H), 3.53 (d, J = 8.0, 1H), 3.40 (dd, J = 11.5, 3.3,
1H), 3.06 (dd, J = 13.5, 6.0, 1H), 2.87 (dd, J = 13.3,
8.9, 1H), 2.82 (dt, J = 14.3, 3.6, 1H), 2.74 (dd, J = 9.9,
4.4, 1H), 2.44 (m, 1H), 2.01 (dd, J = 13.5, 10.4, 1H),
1.78 (ddt, J = 14.2, 11.7, 2.7, 1H), 1.67 (dt, J = 14.1,
3.5, 1H). MS: (M+H+), 311.
1
8 (2.10 g, 25%) as off-white solids. H NMR (DMSO-
d6): 8.16 (d, J = 7.4, 1H), 7.84 (t, J = 7.6, 1H), 7.64 (d,
J = 8.5, 1H), 7.57 (d, J = 8.8, 1H), 7.44 (t, J = 7.4,
1H), 6.94 (d, J = 8.5, 1H).
4.1.5. 3,4-Bis-(1,1-dimethyl-prop-2-ynyloxy)-9H-xanthen-
9-one (11). A solution of compound 8 (1.25 g, 5.47 mmol),
cupric chloride (30.2 mg, 0.225 mmol), 3-chloro-3-
methyl-1-butyne (3.36 mL, 29.9 mmol) and 1,8-diazabi-
cyclo[5.4.0]undec-7-ene (2.00 mL, 13.3 mmol) in acetoni-
trile (100 mL) was stirred at room temperature for 11 h
under argon. The solution was then heated at 75 ꢁC for
2 h and cooled to room temperature. The solution was
partitioned between ethyl acetate (100 mL) and water
(75 mL). The ethyl acetate layer was dried over sodium
sulfate and was concentrated by rotary evaporation.
The product was purified by flash column chromatogra-
phy (10:1 hexanes/ethyl acetate) to yield compound 11
(496 mg, 25%) as light yellow solids. 1H NMR (CDCl3):
8.33 (dd, J = 8.0, 1.7, 1H), 8.07 (d, J = 9.1, 1H), 7.71
(ddd, J = 8.2, 7.1, 1.6, 1H), 7.65 (d, J = 9.1, 1H), 7.52
(dd, J = 8.5, 0.6, 1H), 7.38 (ddd, J = 8.0, 7.2, 0.6, 1H),
2.66 (s, 1H), 2.30 (s, 1H), 1.84 (s, 6H), 1.77 (s, 6H).
4.1.2. 3,4-Bis-allyloxy-9H-xanthen-9-one (9). A stirred
solution of compound 8 (290 mg, 1.27 mmol), allyl bro-
mide (800 lL, 9.20 mmol) and potassium carbonate hy-
drate (1.28 g, 7.77 mmol) in acetone (15.0 mL) was
refluxed for 2.5 h. The solution was cooled to room tem-
perature and dichloromethane was added. The mixture
was filtered and the filtrate was rotary evaporated to
yield compound 9 (372 mg, 95%) as white solids. 1H
NMR (DMSO-d6): 8.33 (dd, J = 7.8, 1.8, 1H), 8.07 (d,
J = 8.8, 1H), 7.72 (ddd, J = 8.1, 7.0, 1.7, 1H), 7.56 (dd,
J = 8.5, 0.6, 1H), 7.38 (ddd, J = 7.8, 7.1, 1.0, 1H), 7.00
(d, J = 9.1, 1H), 6.15 (m, 2H), 5.48 (m, 1H), 5.38 (m,
2H), 5.25 (m, 1H), 4.74 (m, 4H).
4.1.3. 3,3a,4,5-Tetrahydro-1-(2-propenyl)-1,5-methano-
1H,7H-furo[3,4-d]xanthene-7,13-dione (4), and 1,3a,4,
11a-tetrahydro-1-(2-propenyl)-3H-1,4a-methano-10H-furo
[3,4-b]xanthene-10,12-dione (5). A stirred solution of
compound 9 (236 mg, 0.767 mmol) and diphenyl ether
(3.0 mL) was refluxed in an oil bath at 190 ꢁC for 11 h.
The solution was cooled to room temperature and the
product was purified twice by flash column chromatogra-
phy (dichloromethane) to give compound 4 (28 mg, 12%)
as white solids. 1H NMR (CDCl3): 7.95 (dd, J = 7.7, 1.7,
1H), 7.56 (ddd, J = 8.2, 7.1, 1.6, 1H), 7.34 (d, J = 7.1, 1H),
7.08 (m, 2H), 5.22 (m, 1H), 4.69 (m, 1H), 4.53 (m, 2H),
3.91 (d, J = 8.0, 1H), 3.53 (ddd, J = 6.1, 3.4, 2.1, 1H),
2.82 (dd, J = 13.3, 5.4, 1H), 2.63 (m, 1H), 2.51 (dd,
J = 13.6, 9.5, 1H), 1.90 (m, 1H), 1.78 (ddd, J = 12.2,
10.4, 2.4, 1H); MS: (M+H+), 309; and compound 5
4.1.6. 3,4-Bis-(1,1-dimethyl-allyloxy)-9H-xanthen-9-one
(12). To
a solution of compound 11 (318 mg,
0.882 mmol) in methanol (25 mL) was added Lindlar’s
catalyst (Pd, 5 wt% on calcium carbonate, 75 mg) under
hydrogen (1 atm). The mixture was stirred at room tem-
perature for 1 h, then the mixture was filtered through a
syringe filter and the solvent was evaporated. The resi-
due was purified by column chromatography (SiO2,
EtOAc:hexanes/10–25%) to give compound 12
1
(249 mg, 77%) as white solids. H NMR (CDCl3): 8.31
(dd, J = 8.1, 1.8, 1H), 7.93 (d, J = 9.0, 1H), 7.70 (m,
1H), 7.50 (dd, J = 8.7, 0.9, 1H), 7.37 (m, 1H), 7.13 (d,
J = 9.0, 1H), 6.30 (dd, J = 17.7, 10.8, 1H), 6.20 (dd,
J = 17.1, 11.1, 1H), 5.25–5.16 (m, 3H), 5.03 (dd,
J = 10.5, 0.9, 1H), 1.59 (s, 6H), 1.58 (s, 6H).
1
(44 mg, 19%) as white solids. H NMR (CDCl3): 7.93
(dd, J = 8.0, 1.9, 1H), 7.56 (ddd, J = 8.2, 7.0, 1.8, 1H),

J. Kuemmerle et al. / Bioorg. Med. Chem. 16 (2008) 4233–4241
4239
4.1.7. 3,3a,4,5-Tetrahydro-3,3-dimethyl-1-(3-methyl-2-
butenyl)-1,5-methano-1H,7H-furo[3,4-d]xanthene-7,13-
dione (13), and 1,3a,4,11a-tetrahydro-3,3-dimethyl-1-(3-
methyl-2-butenyl)-3H-1,4a-methano-10H-furo[3,4-b]xan-
thene-10,12-dione (14). A solution of compound 12
(229 mg, 0.587 mmol) in toluene (10 mL) was refluxed
under argon for 2 h. The solvent was evaporated and
the residue was purified by column chromatography
(SiO2, EtOAc:hexanes/10–30%) to give compound 13
(145 mg, 63%) as white solids. 1H NMR (CDCl3):
7.95 (dd, J = 7.8, 1.5, 1H), 7.53 (ddd, J = 8.1, 7.2,
1.5, 1H), 7.44 (dd, J = 7.2, 0.6, 1H), 7.07 (m, 2H),
4.42 (m, 1H), 3.50 (dd, J = 6.9, 4.5, 1H), 2.65–2.61
(m, 2H), 2.46 (d, J = 9.3, 1H), 2.35 (dd, J = 12.6, 4.2,
1H), 1.73 (s, 3H), 1.31 (m, 1H), 1.31 (s, 6H), 0.92 (s,
3H); 13C NMR (CDCl3): 202.8, 176.3, 159.5, 136.1,
134.8, 134.7, 133.6, 126.8, 121.8, 119.0, 118.9, 118.0,
90.3, 84.6, 83.5, 48.8, 46.8, 30.4, 29.2, 25.4, 25.2,
16.8; MS: (M+H+), 365; and compound 14 (18 mg,
8%) as white solids. 1H NMR (CDCl3): 7.92 (dd,
J = 8.1, 1.8, 1H), 7.55 (ddd, J = 8.7, 7.2, 1.8, 1H),
7.26 (d, J = 6.9, 1H), 7.20 (dd, J = 8.4, 0.6, 1H), 7.07
(td, J = 7.2, 1.2, 1H), 5.03 (m, 1H), 3.77 (dd, J = 6.9,
4.5, 1H), 2.56 (d, J = 13.2, 1H), 2.50 (dd, J = 15.3,
6.9, 1H), 2.17 (dd, J = 9.6, 4.5, 1H), 2.09 (dd,
J = 14.7, 8.4, 1H), 1.88 (dd, J = 13.2, 9.9, 1H), 1.72
(s, 3H), 1.60 (s, 3H), 1.39 (s, 3H), 1.35 (s, 3H); 13C
NMR (CDCl3): 199.7, 175.4, 160.2, 136.5, 136.1,
135.9, 134.9, 127.0, 122.0, 119.2, 118.3, 117.3, 84.1,
83.7, 78.8, 44.8, 42.1, 33.1, 30.2, 29.7, 26.8, 26.0,
18.2. MS: (M+H+), 365.
4.1.10. 6,7,7a,8-Tetrahydro-8,8-dimethyl-10-(3-methyl-2-
butenyl)-2-phenyl-6,10-methano-4H,10H-furo[3,4-i]-1-benz-
opyran-4,11-dione (18), and 5a,6,8a,9-Tetrahydro-8,8-
dimethyl-6-(3-methyl-2-butenyl)-2-phenyl-8H-6,9a-Meth-
ano-4H-furo[3,4-g]-1-benzopyran-4,10-dione (19). A solu-
tion of compound 17 in diphenyl ether (2 mL) was stirred
at 120 ꢁC under argon for 3 h. The reaction mixture was
cooled to room temperature and the product was purified
by column chromatography (SiO2, EtOAc:hexanes/10–
30%) to give compound 18 (45 mg, 52%) as white solids.
1H NMR (CDCl3): 7.85–7.82 (m, 2H), 7.57–7.46 (m,
3H), 7.30 (d, J = 6.9, 1H), 6.13 (s, 1H), 4.69 (m, 1H),
3.48 (dd, J = 6.9, 4.5, 1H), 2.67–2.56 (m, 2H), 2.52 (d,
J = 9.3, 1H), 2.34 (dd, J = 13.5, 4.5, 1H), 1.69 (s, 3H),
1.40 (s, 3H), 1.36 (m, 1H), 1.31 (s, 6H); 13C NMR
(CDCl3): 203.0, 177.1, 168.4, 134.5, 134.1, 132.9, 131.7,
131.2, 128.8, 126.5, 117.9, 100.8, 92.7, 84.3, 83.2, 49.2,
46.5, 30.4, 29.1, 29.0, 25.7, 25.0, 17.8; MS: (M+H+),
391; and compound 19 (21 mg, 24%) as white solids. 1H
NMR (CDCl3): 7.91–7.87 (m, 2H), 7.55–7.43 (m, 3H),
7.06 (d, J = 6.6, 1H), 6.09 (s, 1H), 5.07 (m, 1H), 3.77
(dd, J = 6.9, 4.5, 1H), 2.69 (d, J = 13.2, 1H), 2.53 (dd,
J = 15.3, 6.6, 1H), 2.16 (dd, J = 9.3, 4.2, 1H), 2.11 (dd,
J = 15.0, 8.7, 1H), 1.91 (dd, J = 13.2, 9.9, 1H), 1.74 (s,
3H), 1.61 (s, 3H), 1.40 (s, 3H), 1.36 (s, 3H); 13C NMR
(CDCl3): 198.7, 175.9, 169.6, 136.1, 135.3, 132.8, 131.8,
131.1, 128.6, 126.8, 117.2, 101.3, 86.0, 83.7, 78.7, 44.5,
41.6, 32.8, 30.2, 29.6, 26.8, 26.0, 18.3; MS: (M+H+), 391.
4.1.11. 3,4-Bis-allyloxy-benzophenone (21). To a suspen-
sion of 3,4-dihydroxybenzophenone (2.14 g, 10 mmol)
and cesium carbonate (7.01 g, 21.6 mmol) in dry acetone
(50 mL) was added allylbromide (5.3 mL, 60 mmol).
The mixture was stirred at 60 ꢁC for 6 h and additional
allybromide (3.0 mL, 34.7 mmol) and cesium carbonate
(3.00 g, 9.2 mmol) were added. The mixture was stirred
for 3 h, cooled to room temperature, filtered and washed
with EtOAc. The filtrate was evaporated under reduced
pressure. The crude product was partitioned between
EtOAc (50 mL) and H2O (15 mL). The EtOAc phase
was separated and evaporated to give compound 21
4.1.8. 7,8-Bis-(1,1-dimethyl-prop-2-ynyloxy)-2-phenyl-
4H-chromen-4-one (16). To a suspension of 7,8-dihy-
droxy-2-phenyl-4H-chromen-4-one (510 mg, 2 mmol)
and CuCl2 (16 mg, 0.12 mmol) in acetonitrile (3 mL)
were added 3-chloro-3-methyl-1-butyne (0.5 mL,
4.56 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene
(0.78 mL, 5.2 mmol) slowly at 0 ꢁC. The dark mixture
was stirred at 0 ꢁC for 5 h and then at room temperature
overnight. The solvent was evaporated and the residue
was purified by column chromatography (SiO2,
EtOAc:hexanes/10–50%) to give compound 16
1
(2.9 g, 99%) as a light yellow oil. H NMR (CDCl3):
7.77 (m, 1H), 7.74 (m, 1H), 7.57 (m, 1H), 7.50–7.44
(m, 3H), 7.38 (ddd, J = 8.7, 2.4, 0.9, 1H), 6.91 (d,
J = 8.4, 1H), 6.08 (m, 2H), 5.47 (m, 1H), 5.41 (m, 1H),
5.32 (m, 2H), 4.68 (m, 4H).
1
(540 mg, 69%) as white solids. H NMR (CDCl3): 8.02
(m, 2H), 7.96 (d, J = 9.0, 1H), 7.69 (d, J = 9.0, 1H),
7.53 (m, 3H), 6.78 (s, 1H), 2.66 (s, 1H), 2.26 (s, 1H),
1.81 (s, 6H), 1.76 (s, 6H).
4.1.12. (2,5-Diallyl-3,4-dihydroxy)-benzophenone (22). A
solution of compound 21 (1.51 g, 5.1 mmol) in diphenyl
ether (3 mL) was stirred at 200 ꢁC for 2 h. The reaction
mixture was cooled and it was purified by column chro-
matography (SiO2, 30% EtOAc in hexanes) to give com-
4.1.9. 7,8-Bis-(1,1-dimethyl-allyloxy)-2-phenyl-4H-chro-
men-4-one (17). To a solution of compound 16 (98 mg,
0.254 mmol) in methanol (20 mL) was added Lindlar’s
catalyst (Pd, 5 wt% on calcium carbonate, 25 mg) under
hydrogen (1 atm). The mixture was stirred at room tem-
perature for 50 min, then it was filtered through a syr-
inge filter and the solvent was evaporated. The residue
was purified by column chromatography (SiO2,
EtOAc:hexanes/10–25%) to give compound 17 (72 mg,
1
pound 22 (0.84 g, 56%) as a light yellow oil. H NMR
(CDCl3): 7.79 (m, 2H), 7.57 (m, 1H), 7.44 (t, J = 7.8,
2H), 6.78 (s, 1H), 6.16–5.86 (m, 3H), 5.60 (br s, 1H),
5.12 (m, 4H), 3.51 (d, J = 6.0, 2H), 3.39 (d, J = 6.6, 2H).
4.1.13. (2,5-Diallyl-3,4-bis-allyloxy)-benzophenone (23).
A suspension of compound 22 (840 mg, 2.85 mmol), ce-
sium carbonate (2.78 g, 8.55 mmol) and allybromide
(1.5 mL, 17.3 mmol) was stirred at 60 ꢁC for 20 h. The
mixture was filtered and washed with EtOAc. The filtrate
was evaporated and the crude product was purified by col-
1
73%) as white solids. H NMR (CDCl3): 7.98 (m, 2H),
7.81 (dd, J = 9.0, 0.9, 1H), 7.52 (m, 3H), 7.16 (dd,
J = 9.0, 0.6, 1H), 6.75 (d, J = 0.6, 1H), 6.22 (m, 2H),
5.24–5.12 (m, 3H), 4.98 (dd, J = 10.2, 1.2, 1H), 1.56 (s,
6H), 1.55 (s, 6H).

4240
J. Kuemmerle et al. / Bioorg. Med. Chem. 16 (2008) 4233–4241
umn chromatography (SiO2, 10–30% EtOAc:hexanes) to
give compound 23 (880 mg, 83%) as a light yellow oil. 1H
NMR (CDCl3): 7.81–7.77 (m, 2H), 7.57 (tt, J = 7.2, 1.5,
1H), 7.47–7.41 (m, 2H), 6.90 (s, 1H), 6.10 (m, 2H), 5.89
(m, 2H), 5.43 (m, 1H), 5.38 (m, 1H), 5.27 (m, 1H), 5.24
(m, 1H), 5.04 (m, 1H), 5.00 (m, 1H), 4.88 (m, 1H), 4.83
(dq, J = 10.2, 1.5, 1H), 4.57–4.51 (m, 4H), 3.50 (dt,
J = 6.3, 1.5, 2H), 3.89 (dt, J = 6.6, 1.2, 2H).
exposed continuously to test compound for 48 h. CellTit-
er-Glo reagent (Promega) was added. The samples were
mixed by agitation, incubated at room temperature for
10–15 min and then read using a luminescent plate reader
(Model Spectrafluor Plus Tecan Instrument). The GI50 is
defined as 50% inhibition of cell proliferation.
References and notes
4.1.14. 4-Benzoyl-2,3,3a,7a-tetrahydro-3a,6,7-tri-(2-pro-
penyl)-3,6-methanobenzofuran-7(6H)-one (24), and 5-ben-
zoyl-2,3,3a,7a-tetrahydro-3a,6,7a-tri-(2-propenyl)-3,6-
methanobenzofuran-7(6H)-one (25). A solution of com-
pound 23 (474 mg, 1.27 mmol) in diphenyl ether (3 mL)
was stirred at 200 ꢁC for 2 h. The reaction mixture was
cooled to room temperature and the product was purified
by column chromatography (SiO2, CH2Cl2) to give com-
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1
pound 24 (190 mg, 35%) as solids. H NMR (CDCl3):
7.80–7.75 (m, 2H), 7.59 (m, 1H), 7.48–7.43 (m, 2H), 6.23
(s, 1H), 5.85–5.58 (m, 2H), 5.36 (m, 1H), 5.21–5.06 (m,
3H), 5.02–4.91 (m, 3H), 4.14 (dd, J = 8.7, 4.5, 1H), 3.72
(d, J = 8.4, 1H), 3.01 (dd, J = 13.8, 5.7, 1H), 2.93 (dd,
J = 14.4, 7.2, 1H), 2.66–2.51 (m, 3H), 2.41–2.29 (m,
2H), 2.08 (dd, J = 13.2, 10.2, 1H), 1.70 (d, J = 13.5, 1H);
13C NMR (CDCl3): 204.4, 192.8, 141.6, 139.8, 136.5,
133.8, 133.2, 132.5, 132.0, 129.7, 128.3, 119.3, 118.8,
118.5, 83.5, 53.4, 51.1, 50.9, 39.6, 39.3, 34.3, 33.5, 32.7;
MS: (M+H+), 375; and compound 25 (79 mg, 14%) as sol-
ids. 1H NMR (CDCl3): 7.79–7.76 (m, 2H), 7.60 (tt,
J = 6.6, 1.5, 1H), 7.50–7.44 (m, 2H), 6.33 (s, 1H), 5.98–
5.73 (m, 2H), 5.60 (m, 1H), 5.16–4.95 (m, 6H), 4.18 (dd,
J = 8.4, 4.5, 1H), 3.70 (d, J = 8.7, 1H), 3.49 (m, 1H),
2.81 (dd, J = 14.4, 8.7, 1H), 2.67 (dd, J = 9.9, 4.5, 1H),
2.56–2.50 (m, 2H), 2.45–2.37 (m, 1H), 2.24 (dd,
J = 14.7, 9.3, 1H), 2.02 (dd, J = 12.9, 10.2, 1H), 1.69 (d,
J = 13.8, 1H); 13C NMR (CDCl3) 205.5, 193.8, 141.2,
141.1, 137.3, 133.7, 133.3, 132.9, 132.0, 129.9, 128.3,
118.9, 118.7, 118.3, 82.7, 74.3, 53.2, 49.5, 39.7, 38.5,
35.3, 34.0, 30.6; MS: (M+H+), 375.
4.2. Caspase activation assay (EC50)
The caspase assay was run as previously reported.33 In
brief, T47D, HCT116 and SNU398 cells were exposed
continuously to test compound for 24 h. The caspase-3
fluorogenic
bonyl-R110 in a caspase buffer was then added, followed
substrate
N-(Ac-DEVD)-N-ethoxycar-
by incubation at 37 ꢁC for 2 h.
Calculation. The relative fluorescence unit (RFU) values
measured on a Model Spectrafluor Plus Tecan Instru-
ment were used to calculate the sample readings as fol-
lows: the activity of caspase activation was determined
by the ratio of the net RFU value for the test compound
to that of control samples. The EC50 (lM) was deter-
mined by
a sigmoidal dose–response calculation
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