A-ring oxygenation modulates the chemistry and bioactivity of caged Garcinia xanthones

Article abstract of DOI:10.1039/c3ob40395e

Natural products of the caged Garcinia xanthones (CGX) family are characterized by a unique chemical structure, potent bioactivities and promising pharmacological profiles. We have developed a Claisen/Diels-Alder reaction cascade that, in combination with a Pd(0)-catalyzed reverse prenylation, provides rapid and efficient access to the CGX pharmacophore, represented by the structure of cluvenone. To further explore this pharmacophore, we have synthesized various A-ring oxygenated analogues of cluvenone and have evaluated their bioactivities in terms of growth inhibition, mitochondrial fragmentation, induction of mitochondrial-dependent cell death and Hsp90 client inhibition. We found that installation of an oxygen functionality at various positions of the A-ring influences significantly both the site-selectivity of the Claisen/Diels-Alder reaction and the bioactivity of these compounds, due to remote electronic effects.

Full text of DOI:10.1039/c3ob40395e

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A-ring oxygenation modulates the chemistry and
bioactivity of caged Garcinia xanthones†
Cite this: DOI: 10.1039/c3ob40395e
Kristyna M. Elbel,‡a Gianni Guizzunti,‡b Maria A. Theodoraki,c Jing Xu,a
Ayse Batova,a Marianna Dakanalia and Emmanuel A. Theodorakis*a
Natural products of the caged Garcinia xanthones (CGX) family are characterized by a unique chemical
structure, potent bioactivities and promising pharmacological profiles. We have developed a Claisen/Diels–
Alder reaction cascade that, in combination with a Pd(0)-catalyzed reverse prenylation, provides rapid and
efficient access to the CGX pharmacophore, represented by the structure of cluvenone. To further explore
this pharmacophore, we have synthesized various A-ring oxygenated analogues of cluvenone and have
evaluated their bioactivities in terms of growth inhibition, mitochondrial fragmentation, induction of mito-
chondrial-dependent cell death and Hsp90 client inhibition. We found that installation of an oxygen func-
tionality at various positions of the A-ring influences significantly both the site-selectivity of the Claisen/
Diels–Alder reaction and the bioactivity of these compounds, due to remote electronic effects.
Received 24th February 2013,
Accepted 22nd March 2013
DOI: 10.1039/c3ob40395e
www.rsc.org/obc
low micromolar kd to the heat shock protein Hsp90,6 thereby
reducing the expression levels of Hsp90-dependent clients that
Introduction
Tropical plants of the genus Garcinia have led to the identifi- are involved in cell growth, apoptosis, angiogenesis and
cation of an intriguing family of xanthone-derived natural pro- metastasis.7
ducts that have interesting bioactivities and a documented
Intrigued by the unusual chemical motif and therapeutic
value in traditional Eastern medicine.1 Collectively referred to promise of CGXs, we sought to design a general synthetic strat-
as caged Garcinia xanthones (CGXs),2 these compounds are egy that would allow us to further explore and optimize its bio-
structurally defined by an unusual architecture in which a logical properties.8,9 These studies led to the identification of
4-oxa-tricyclo[4.3.1.03,7]dec-8-en-2-one scaffold has been built cluvenone (2, CLV), a structurally simplified CGX that retains
onto the C-ring of a xanthone backbone. This motif is further the biological activities of 1.10 Along these lines, we have
decorated via substitutions at the A-ring and peripheral oxi- shown that CLV parallels the activity of GA in inducing cell
dations to produce a variety of related subfamilies. Gambogic stress and apoptosis in various cancer lines at low micromolar
acid (1, GA),3 the archetype of this family, has been studied as concentration. Fluorescent labeling studies have indicated that
a potent antitumor and anti-inflammatory agent and has both 1 and 2 localize in mitochondria and induce significant
entered phase I clinical trials in China.4 Various biological
studies have suggested that 1 can induce cell apoptosis by acti-
vating the mitochondrial pathway via mechanisms that are
partly dependent on the Bcl-2 family of proteins.5 Although
the primary direct molecular target of 1 is still under investi-
gation, recent studies have suggested that it can bind with a
aDepartment of Chemistry & Biochemistry, University of California, San Diego, 9500
Gilman Drive MC: 0358, La Jolla, CA 92093-0358, USA. E-mail: etheodor@ucsd.edu;
Fax: +1-858-822-0386; Tel: +1-858-822-0456
bDepartment of Cell Biology and Infection, Membrane Traffic and Pathogenesis Unit,
Pasteur Institute, Paris, France
cThe City College of New York, Department of Biology, 160 Convent Avenue,
NY 10031, USA
†Electronic supplementary information (ESI) available: Experimental procedures
for all reactions and biological assays and copies of 1H and 13C NMR spectra for
compounds 3, 4 and 9–21. See DOI: 10.1039/c3ob40395e
‡These authors contributed equally.
Fig. 1 Structures of selected caged Garcinia xanthones (CGXs).
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changes in the morphology and structure of this organelle,
thereby leading to cell apoptosis.11 It has been proposed that
the C9–C10 enone functionality of these compounds contrib-
utes to their bioactivity, presumably by acting as a conjugate
electrophile.9a,b To further explore and optimize the CGX
pharmacophore, we synthesized A-ring oxygenated xanthones
3 and 4 and compared their activities with those of 1, 2 and
5.10 Here we show that installation of an oxygen group at C6 or
C18 positions of the CGX motif (gambogic acid numbering)
affects significantly both the synthesis and the bioactivity of
these compounds (Fig. 1).
Results and discussion
Synthesis of A-ring hydroxylated caged Garcinia xanthones
The synthesis of caged Garcinia xanthones containing a pro-
tected phenolic group at the C6 and C18 positions of the A-ring
is highlighted in Schemes 1 and 2. Acyl chloride 7a, prepared
via oxalyl chloride/DMF-mediated chlorination of commer-
cially available 2,6-dimethoxybenzoic acid (6a), was subjected
to an AlCl3-catalyzed Friedel–Crafts acylation with pyrogallol
trimethyl ether (8) (Scheme 1). The resulting benzophenone
intermediate was treated with NaOH to produce xanthone 9a
in 90% combined yield. Demethylation of 9a with 48% HBr in
AcOH gave rise to hydroxylated xanthone 10a (95% yield). Pro-
tection of the C-ring catechol of 9a as the corresponding
diphenylketal, followed by alkylation of the C6 phenol with
MOMCl/NaH and deprotection of the ketal functionality
yielded xanthone 11a (3 steps, 56% overall yield).9d Pd(0)-cata-
lyzed reverse prenylation of 11a with carbonate 12 produced
Scheme 2 Reagents and conditions: (a) 1.0 equiv. ZnCl2 (0.5 M in THF), THF,
2–24 h, 40 °C, 93% for 14, 0% for 18, 75% for 4 and 75% for 21; (b) DMF,
120 °C, 2 h, 100%.
diallyl ether 13a in 63% yield.10 In a similar manner, 13b was
produced from 2,4-dimethoxybenzoic acid (6b) in 7 steps and
17% overall yield.
Conversion of 13a and 13b to the corresponding caged
structures was achieved via the tandem Claisen/Diels–Alder
reaction cascade (Scheme 2).12 To this end, heating of 13a
(DMF, 120 °C, 2 h) produced two isomeric caged xanthones:
the “regular” caged structure 15 (80% yield) that is formed
upon initial Claisen migration of the C12 allyl ether at the C13
position, and the “neo” caged structure 16 (20% yield) that is
formed upon initial Claisen migration of the C13 allyl ether at
the C12 position (gambogic acid numbering). On the other
hand, ZnCl2-induced cleavage of the MOM group (40 °C, 2 h)
of 13a formed 14 (93% yield) that, upon heating, underwent
the Claisen/Diels–Alder reaction to form regular caged
xanthone 3 (92% yield) together with the neo caged compound
17 (8% yield). In a similar manner, heating of 13b (DMF, 2 h,
120 °C) produced the regular caged structure 19 (80% yield)
together with the neo structure 20 (20% yield). To our surprise,
removal of the MOM group from 13b under ZnCl2 conditions
proved to be problematic and led to partial or full loss of the
dimethylallyl ethers. Thus, this deprotection was accom-
plished after conversion of 13b to the corresponding caged
compounds 19 and 20. To this end, treatment of 19 and 20
with ZnCl2 (40 °C, 24 h) afforded caged xanthones 4 and 21
respectively in ca. 75% yield.13
The products distribution of the Claisen/Diels–Alder reac-
tion deserves some comments. We have previously postulated
that the site selectivity of this reaction can be rationalized by
considering the electronic deficiency of the B-ring carbonyl
group that, being para to the C12 allyl ether, facilitates its
rupture during the inaugural Claisen rearrangement.13 In
turn, Claisen migration of this group at the C13 position leads
to a site selective Diels–Alder reaction ultimately forming the
regular caged motif as the major product. In contrast, the
Scheme 1 Reagents and conditions: (a) 5.0 equiv. oxalyl chloride, DMF (cat.),
DCM, 16 h, 25 °C; (b) 3.0 equiv. AlCl3, Et2O, 18 h, 25 °C then 9.0 equiv. NaOH,
MeOH–H2O (1.5 : 1), 48 h, 110 °C, 90% for 9a, 58% for 9b; (c) 48% HBr–HOAc
(1 : 2), 12 h, 120 °C, 95% for 10a, 81% for 10b; (d) 1.6 equiv. Ph2CCl2, Ph2O,
4 h, 175 °C; (e) 2.5 equiv. MOMCl, 2.5 equiv. NaH, acetone, 12 h, 25 °C; (f) H2,
10% Pd/C, THF–MeOH (3 : 1), 18 h, 25 °C, 56% for 11a; 43% for 11b (over 3
steps); (g) 10 equiv. 12, 10 mol% Pd(PPh3)4, THF, 2 h, 5 °C, 63% for 13a, 84%
for 13b.
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competing Claisen migration of the C13 dimethylallyl ether at
It is interesting to relate the activities of these CGXs to their
the C12 position is not electronically favored. Thus, the corres- structure, in particular as they relate to the reactivity of the C9–
ponding neo caged motif is formed only as minor C10 enone as a conjugate electrophile (Table 1 and Fig. 2).
a
product.14,15 The results of our study with compounds 13a, Comparison of 3 with 15 suggests that the presence of a free
13b and 14 further support this proposal. Specifically, the phenolic group at the C6 position of the A ring (i.e. compound
Claisen/Diels–Alder reaction of 14 produces the regular and 3) increases the potency of this compound, while its protection
the neo caged compounds 3 and 17 in a 92 : 8 ratio. This as a MOM ether (i.e. compound 15) decreases its potency. This
enhanced site selectivity can be explained by considering that modulation of potency parallels the effect of the C6 oxygen on
the C6 hydroxyl group increases the electron deficiency of the the activation of the C9–C10 enone. Specifically, in compound
B-ring carbonyl group via hydrogen bonding and thus facili- 3, the C6 free phenolic functionality can create a hydrogen
tates the rupture of the C12 allyl ether, leading predominantly bond with the adjacent carbonyl group, thereby enhancing the
to the regular caged motif of 3. In contrast, the presence of a electrophilicity of the C10 center. In contrast, the MOM ether
MOM ether at C6 or C18 (i.e. 13a or 13b) donates electron functionality at C6 of 15 enriches the electron density of the B-
density at the B-ring carbonyl group, thus attenuating the site ring carbonyl group (vinylogous carbonyl system), thereby
selectivity of the Claisen/Diels–Alder reaction (regular/neo reducing the electrophilicity of the C10 center. In a similar
motifs: 80/20).
manner, compound 19 (containing a MOM ether at C18) is
twice as potent as 4 (that contains a free phenolic group at
C18). This is consistent with the prediction that a free phenolic
group para to the B ring carbonyl would act as a strong elec-
tron donor, thereby reducing the electrophilicity of the C10
center. Along these lines, compound 5 (containing a methyl
group at C10) is about twenty times less potent than 2 in which
the C10 center is not substituted and thus more accessible for
a conjugate addition reaction with a bionucleophile. Overall,
these comparative studies support the notion that increasing
the electrophilicity of the C10 enone enhances the potency of
the CGX motif.
Cell proliferation studies of A-ring hydroxylated caged Garcinia
xanthones
The ability of the synthesized CGXs to inhibit cancer cell
growth was evaluated in the T-cell acute lymphoblastic leuke-
mia cell line (CEM). Based on our previous studies, this cell
line was found to be one of the most sensitive.8 The inhibitory
concentrations at 50% inhibition (IC50) of the active CGXs
ranged from 0.12 to 1.0 μM. Compound 510 was found to be
relatively inactive with an IC50 greater than 5 μM (Table 1 and
Fig. 2).
Effect of GA and CLV on mitochondria
We have previously reported that CLV (2) and GA (1) localize in
mitochondria, and induce mitochondrial fragmentation
leading to activation of caspase-3 (C3) ultimately resulting in
cell apoptosis.11 Despite having similar phenotypes, the cellu-
lar effects of 1 and 2 have not been compared. To this end, we
began by analyzing the timing of induction of the apoptotic
pathway, by western blot (WB) analysis of active caspase-9, a
known initiator caspase,16 and caspase-3, the known down-
stream executioner of apoptosis.17 Caspase-9 (C9) was exam-
ined due to its well established connection to the
mitochondrial apoptotic pathway and its subsequent acti-
vation of C3. HeLa cells were treated with 2 μM GA (Fig. 3a) or
CLV (Fig. 3b) from 0 min (control cells, incubated with equal
volume of DMSO) to 180 min. Both compounds induced a
similar activation of C9 and C3 after about 120 min of incu-
bation. In fact, C9 appears to be activated right before C3 since
it is visible after about 90 min of incubation.
Table 1 Inhibition of cell proliferation by CGXs in CEM cells
Compound
IC50 (μM)
1
2
3
4
5
15
19
0.12
0.18
0.3
1.0
5.1
0.5
0.5
We then evaluated the status of the mitochondrial network
by immunofluorescence (IF). As shown in Fig. 3(g and h), both
GA and CLV induced a similar degree of mitochondrial disrup-
tion. Both compounds were found to induce a first degree of
fragmentation after 30 min incubation, at which time the
mitochondrial network appeared fragmented in larger discrete
elements (Fig. 3d for GA and Fig. 3g for CLV). At longer incu-
bation times (1 h treatment for both compounds) the mito-
chondria appeared collapsed in the perinuclear area and the
mitochondrial network was no longer visible (Fig. 3e and 3h).
Fig. 2 Inhibition of 3H-thymidine incorporation by selected CGXs in CEM cells.
Cells were treated with increasing concentrations of various CGXs for 48 h and
pulsed with 3H-thymidine prior to harvesting.
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Fig. 4 Effect of selected CGXs on apoptosis. Cells were treated for 60 min,
90 min, 120 min, 180 min with 2 μM CLV (lanes 2–5), 2 μM 3 (lanes 6–9), 2 μM
4 (lanes 10–13) and 80 μM 5 (lanes 14–17). Control cells treated with an equal
volume of DMSO are in lane 1. The cells were then collected and analyzed by
WB to measure the induction of apoptosis by caspase-3 (C3) activation. Both
CLV and 3 show active caspase-3 starting at 90 min (lanes 3 and 7 respectively);
4 requires 120 min incubation to achieve comparable levels of activation; 5
induced caspase-3 activation only after 180 min incubation at 80 μM (lane 17).
Tubulin (bottom line) was used as a loading control.
and provide additional arguments in favor of the role of A ring
hydroxylation in modulating the reactivity of the C9–C10 enone
as an electrophile.
We also tested compounds 3, 4 and 5 for their ability to
induce mitochondrial fragmentation. Control untreated cells
are shown in Fig. 5a. Compound 3 induced mitochondrial dis-
ruption after 30 min of incubation (Fig. 5b), while extensive
levels of fragmentation were reached after 60 minutes of incu-
bation (Fig. 5c). The action of compound 3 was therefore
similar to that of CLV at identical time points (compare with
Fig. 3g and 3h respectively). To achieve similar levels of frag-
mentation, compound 4 had to be incubated for 60 min
Fig. 3 Comparison between GA and CLV with respect to mitochondrial frag-
mentation and induction of apoptosis. Cells treated with GA (a) or CLV (b) for
the indicated amounts of time (from 0 min to 180 min) were processed for WB
analysis to show activation of the mitochondria-dependent apoptosis pathway,
represented by caspase-9 (C9) and by the downstream executioner caspase-3
(C3). Cells treated with GA for 0 min (c), 30 min (d) and 60 min (e) or with CLV
for 0 min (f), 30 min (g) and 60 min (h) were analyzed by IF to reveal the status
of the mitochondria. Mitochondria are in red; nuclei are in blue. Tubulin
(bottom line) was used as a loading control.
Comparison of the IF results with the WB analysis supports
the notion that mitochondrial fragmentation (visible after
30 min incubation) appears before the activation of the initia-
tory C9 (visible after 90 min incubation). This observation con-
firms that both GA and CLV induce cell death via the intrinsic
pathway, in which mitochondrial damage leads initially to C9
activation that subsequently activates C3. These results also
suggest that GA and CLV act with a similar timing to activate
the mitochondrial-dependent (intrinsic) apoptotic pathway.
Effect of A-ring hydroxylated caged Garcinia xanthones on
apoptosis and mitochondria
We then evaluated the ability of compounds 3, 4 and 5 to
induce mitochondrial fragmentation and cell death in HeLa
cells. Cells incubated for 60 to 180 min with CLV and deriva-
tives 3, 4 and 5 were analyzed to assess induction of apoptosis
by WB analysis of active caspase-3 (C3). As shown in Fig. 4, 2
and 3 were able to induce apoptosis after 90 min incubation
(compare lanes 3 and 7). On the other hand, compound 4,
which carried the phenolic group at C18, was less effective,
Fig. 5 Effect of 3, 4 and 5 on mitochondria. Cells treated with compounds 3, 4
inducing C3 activation only after 120 min treatment (lane 12). and 5 were imaged to visualize the mitochondrial structure by IF. DMSO-treated
control cells are in (a). Top panel: treatment with 3 for 30 min (b) and 60 min
Moreover, compound 5 proved to be inactive at the concen-
trations tested (2 μM). Even at much higher concentrations (up
to 80 μM), 5 proved to be the least effective of the compounds,
(c). Middle panel: treatment with 4 for 30 min (d), 60 min (e) and 90 min (f).
Bottom panel: treatment with 5 for 60 min (g), 90 min (h), 120 min (i). Mito-
chondria are shown in red; nuclei are in blue. Intact mitochondria are shown in
requiring 180 min to induce comparable levels of apoptosis.
a, d, g and h. Initial level of fragmentation is observed in b, e, and i. More exten-
The results parallel the potency profile of these compounds sive fragmentation is observed in c and f.
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(Fig. 5e) and 90 min (Fig. 5f). Moreover, compound 5 showed recover for 90 min in fresh medium. The cells were then ana-
the lowest levels of fragmentation, being able to induce partial lyzed by western blot in order to visualize caspase-3 activation.
mitochondrial disruption only after 120 min incubation at As shown in Fig. 6(g and h), no signs of apoptosis (measured
80 μM concentration (Fig. 5i). Overall, the findings suggest by caspase-3 activation) were visible prior to 90 min incubation
that the extent of mitochondrial fragmentation parallels the with either GA (Fig. 6g, lanes 1–5) or CLV (Fig. 4h, lanes
activation of the C9–C10 enone as an electrophile.
10–13). However, when cells were treated for 45 min and then
allowed to recover for 90 min (Fig. 6g lane 7 and Fig. 6h lane
15), caspase-3 was active, indicating that, during 45 min of
incubation, both GA and CLV were inducing irreversible mor-
phological changes to mitochondrial structure, leading to the
irreversible activation of the apoptotic pathway.
We then tested the irreversibility of mitochondrial fragmen-
tation upon exposure to compounds 3, 4 and 5 (Fig. 7). The
cells were treated as in Fig. 6; after incubation with CGXs for
different amounts of time, the cells were subjected to 90 min
recovery in fresh medium and were then processed for IF and
WB analyses to detect mitochondria fragmentation and
caspase-3 activation.
All the compounds were incubated at 2 μM (with the excep-
tion of 5 that was used at 80 μM) for times ranging from 0 min
to the minimum time necessary to induce caspase-3 activation,
at 15 min intervals. This time varies from compound to com-
pound, being equal to 90 min for 3, 120 min for 4 and
180 min for 5. After washing the compounds, the cells were
allowed to recover for 90 min and processed for IF. Fig. 7
The caged Garcinia xanthones induce irreversible
mitochondrial fragmentation
Inspired by the above findings we sought to evaluate whether
the effect of CGXs on mitochondria can be reversed upon
removal of these compounds from the incubation media. We
began by treating HeLa cells with 2 μM of GA or CLV for 0 min,
15 min and 30 min. The cells were then allowed to recover for
90 min in fresh medium and were subsequently fixed and pro-
cessed for IF. As shown in Fig. 6, 30 min incubation with GA
(Fig. 6c) and with CLV (Fig. 6f) were necessary and sufficient to
induce a permanent mitochondrial fragmentation, while a
shorter time point (15 min) proved to be not effective and
mitochondrial integrity was recovered (Fig. 6b for GA and
Fig. 6e for CLV).
To assess whether the phenotype of the irreversible mito-
chondrial fragmentation corresponded to an irreversible effect
on apoptosis we incubated cells with GA and CLV for 0 min,
30 min, 45 min, 60 min and 90 min, and then allowed them to
Fig. 6 Irreversibility of mitochondrial fragmentation by GA and CLV. (a–f) Cells
were assessed by IF for their ability to regain mitochondrial integrity after
90 min of recovery in fresh medium following treatment with GA for 0 min (a),
15 min (b) and 30 min (c) or CLV for 0 min (d), 15 min (e) and 30 min (f ). Recov-
ery after 30 min of treatment for both GA (c) and CLV (f ) could not restore mito-
chondrial integrity. Mitochondria are shown in red; nuclei are in blue. (g–h)
Western blot analysis was used to measure the degree of irreversibility by
caspase-3 (C3) activation. As shown in (g) (GA) and (h) (CLV), while incubation
times shorter than 90 min did not induce C3 activation (lanes 1–4 and lanes
10–12), after recovery in the absence of the drugs, C3 was shown to be active
once the compounds had been present on the cells for more than 30 min (lanes
7–9 and 15–17). Tubulin (bottom line) was used as a loading control.
Fig. 7 Irreversibility of mitochondrial fragmentation by 3, 4 and 5. Cells were
incubated with CGXs for the specified amounts of time, then subjected to
90 min recovery in fresh medium and processed for IF. Mitochondria are in red;
nuclei are in blue. Top panel: Treatment with 3 prior to recovery for 0 min (a),
15 min (b) and 30 min (c). Bottom panel: Treatment with 4 prior to recovery for
15 min (d), 30 min (e) and 45 min (f). Signs of irreversibility (indicated by the
failure to recover the intact mitochondrial network) are shown in (c) and (f). WB
analysis indicating caspase-3 activation is shown in (g). Compound 3 needs a
minimum of 45 min to induce activation of caspase-3 (lane 3). Compound 4
needs at least 60 min (lane 9), while 5 proved to be reversible up to 120 min
incubation (data not shown). Tubulin (bottom line) was used as a loading
control.
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shows the results for compounds 3 and 4. Compound 5 proved
to have a reversible effect at the times and concentrations
tested (data not shown). Compound 3 behaved similarly to
CLV, since 30 min incubation was sufficient to induce an irre-
versible mitochondrial fragmentation (Fig. 7c). On the other
hand, compound 4 proved to be less effective, requiring
45 min (Fig. 7f) to induce the same level of mitochondrial dis-
ruption. We then performed WB analysis to confirm that the
irreversible mitochondria fragmentation corresponded to the
activation of caspase-3. As shown in Fig. 7g, compound 3
induced caspase activation after 45 min of incubation, repro-
ducing the results obtained for both CLV and GA. Analogue 4
was once again less effective, inducing caspase-3 activation fol-
lowing 45 min of treatment. Compound 5 was ineffective for
up to 120 min of incubation.
Effect of A-ring hydroxylated caged Garcinia xanthones on the
modulation of Hsp90
Hsp90 is a nodal protein that plays a critical role in protein
folding and is essential to cell proliferation.7 In cancer cells
this protein is overexpressed and also exists in a high-affinity
conformation that facilitates malignant progression.18 As
such, it represents an important target for the development of
anticancer agents.19 Treatment of different cancer cell lines
with known Hsp90 inhibitors is known to induce degradation
of kinases, transcription factors and other protein clients of
Hsp90.20 Encouraged by recent studies suggesting that Hsp90
is a major target of GA,6 we evaluated the effect of CLV and its
A-ring hydroxylated derivatives 3 and 4 on this protein in
SKBR3 cells, a breast cancer cell line. The cells were treated
with different concentrations of GA, CLV, 3 and 4 or just the
solvent (DMSO) for 24 hours and analyzed for several different
client kinases, as well as Hsp70 and Hsp90. As seen in Fig. 8,
the active form of the survival kinase Akt, pAkt (anti-phospho
Ser473), disappears faster than the total Akt (tAkt). The kinase
Raf-1 that functions in the MAPK/ERK signaling pathway con-
trolling cell growth degrades in a concentration dependent
manner for all the compounds tested. Also, Her2 kinase, a well
established therapeutic target for breast cancer, proves to be
extremely sensitive to treatment with GA, CLV and 3 (starts
degrading at the lowest dose tested) and less sensitive to com-
pound 4 (degradation happens only at the higher concen-
tration used). Additionally, the mass induction of Hsp70 and
the less prominent induction of Hsp90 are indicators of Hsp90
inhibition, upon which the heat shock factor (HSF1) becomes
activated and leads to transcriptional induction of the heat
shock genes (e.g. Hsp70). Comparison of the amounts of client
degradation and induction of Hsp70 using different com-
pounds suggests that both CLV and 3 behave similarly and
they resemble the client degradation rates and Hsp70 induc-
tion observed with GA.
Fig. 8 Gambogic acid (GA), CLV, 3 and 4 induce degradation of Hsp90 client
proteins. SKBR3 cells were treated with different concentrations of the drugs or
DMSO (control) for 24 hours. Cell lysates were fractionated by SDS-PAGE and
analyzed for pAkt (S473), tAkt (total Akt), Raf-1, Her2 (indicated by an arrow-
head; the asterisk depicts a non-specific band), Hsp70 and Hsp90. GAPDH was
used as a loading control.
Fig. 9 GA, CLV,
3 and 4 induce apoptosis in a concentration dependent
manner in SKBR3 cells. SKBR3 cells, after treatment with the indicated concen-
trations of the compounds or DMSO (control) for 24 hours, were lysed and ana-
lyzed for cleavage of PARP by SDS-PAGE. GAPDH was used as a control for equal
loading.
24 hours post treatment, even at the low concentration of
0.5 μM both GA and CLV induce cleavage of PARP (∼MW 89-
lower band), which clearly indicates that the cells are already
undergoing apoptosis. Comparison between CLV and 3 shows
that both compounds are able to induce apoptosis at similar
rates (compare CLV and 3 at 2 μM). As expected, compound 4
did not induce significant apoptosis at the concentrations
used.
Conclusions
In order to test the SKBR3 cells for induction of apoptosis,
after treating the cells with the same concentrations of GA, Inspired by the biological potential of cluvenone (CLV) and
CLV, 3 and 4 that lead to a degradation of Hsp90 clients, we related caged Garcinia xanthones (CGXs), we synthesized
analysed the cell lysates for cleavage of PARP, a DNA polymer- selected A-ring hydroxylated analogues and evaluated their
ase involved in DNA repair and cell death (Fig. 9). Again, effect on cell growth, mitochondrial fragmentation,
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mitochondriotoxicity and Hsp90 client protein degradation.
We found that both the C6 and C18 hydroxylated cluvenones
inhibit the growth of CEM cells at low micromolar concen-
trations and induce cell death via the mitochondrial pathway.
In addition, gambogic acid, CLV and the hydroxylated cluve-
nones induce Hsp90-dependent protein client degradation at
low micromolar concentrations. Interestingly, the site of A ring
hydroxylation significantly influences the site selectivity of the
Claisen/Diels–Alder cascade. For instance, introduction of a
MOM ether group at C6 or C18 (e.g. compound 4) increases via
resonance the electron density of the B ring carbonyl group
leading to the formation of both regular and neo caged motifs
(4 : 1 ratio in favor of the regular caged structure). In contrast,
the presence of a free phenolic group at C6 (i.e. compound 3)
decreases the electron density of the B ring carbonyl, thus
favoring the formation of the regular motif (produced in an
11.5 : 1 ratio over the neo caged motif). Importantly, the site of
A ring hydroxylation also affects the bioactivity of the CGX
motif. Specifically, 3 is approximately 3 times more active than
4 in inhibiting cell growth of CEM cells. A similar trend is
observed in the mitochondrial fragmentation, the caspase acti-
vation and the Hsp90 client inhibition assays. We postulate
that this trend stems from tuning the reactivity of the C9–C10
enone as a conjugate electrophile. In support of this postulate,
methylation at C10 (i.e. compound 5) significantly limits the
reactivity of this enone thereby decreasing the overall bioacti-
vity of the CGX motif. Overall, our studies indicate that the
A-ring of the caged Garcinia xanthones is amenable to
functionalization in a manner that modulates not only the site
selectivity of the Claisen/Diels–Alder reaction cascade but also
their biological potency.
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Financial support from the National Institutes of Health (CA
133002) is gratefully acknowledged. We thank the National
Science Foundation for instrumentation grants CHE9709183
and CHE0741968. We also thank Dr Anthony Mrse (UCSD
NMR Facility) and Dr Yongxuan Su (UCSD MS Facility).
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