17β-Hydroxywithanolides as Sensitizers of Renal Carcinoma Cells to Tumor Necrosis Factor-α Related Apoptosis Inducing Ligand (TRAIL) Mediated Apoptosis: Structure-Activity Relationships

Article abstract of DOI:10.1021/acs.jmedchem.7b00069

Renal cell carcinoma (RCC) is a cancer with poor prognosis, and the 5-year survival rate of patients with metastatic RCC is 5-10%. Consequently, treatment of metastatic RCC represents an unmet clinical need. Screening of a 50 000-member library of natural and synthetic compounds for sensitizers of RCC cells to TRAIL-mediated apoptosis led to identification of the 17β-hydroxywithanolide (17-BHW), withanolide E (1), as a promising lead. To explore structure-activity relationships, we obtained natural and semisynthetic withanolides 1, 2a, 2c, and 3-36 and compared their ability to sensitize TRAIL-mediated apoptosis in a panel of renal carcinoma cells. Our findings revealed that 17-BHWs with a α-oriented side chain are superior to known TRAIL-sensitizing withanolides belonging to withaferin A class with a β-oriented side chain and demonstrated that the 17-BHW scaffold can be modified to enhance sensitization of RCCs to TRAIL-mediated apoptosis, thereby assisting development of natural-product-inspired drugs to treat metastatic RCC.

Full text of DOI:10.1021/acs.jmedchem.7b00069

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Article
17#-Hydroxywithanolides as Sensitizers of Renal Carcinoma Cells
to Tumor Necrosis Factor-# Related Apoptosis Inducing Ligand
(TRAIL)–Mediated Apoptosis: Structure–Activity Relationships
Ya-Ming Xu, Alan D. Brooks, E. M. Kithsiri Wijeratne, Curtis J Henrich,
Poonam Tewary, Thomas J. Sayers, and A. A. Leslie Gunatilaka
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00069 • Publication Date (Web): 03 Mar 2017
Downloaded from http://pubs.acs.org on March 4, 2017
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17
β
ꢀHydroxywithanolides as Sensitizers of Renal Carcinoma Cells to
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Tumor Necrosis Factorꢀα Related Apoptosis Inducing Ligand (TRAIL)–
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Mediated Apoptosis: Structure–Activity Relationships
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YaꢀMing Xu,† Alan D. Brooks,‡,§ E. M. Kithsiri Wijeratne,† Curtis J. Henrich, ‡,¶ Poonam Tewary,‡,§
Thomas J. Sayers*,‡,§ and A. A. Leslie Gunatilaka*,†
†Natural Products Center, School of Natural Resources and the Environment, College of Agriculture
and Life Sciences, University of Arizona, 250 E. Valencia Road, Tucson, Arizona 85706, United
States
‡Basic Research Program, Leidos Biomedical Research Inc., Frederick National Laboratory for
Cancer Research, Maryland 21702, United States
§Cancer and Inflammation Program, National Cancer InstituteꢀFrederick, Maryland 21702, United
States
¶Molecular Targets Laboratory, National Cancer InstituteꢀFrederick, Maryland 21702, United States
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ABSTRACT: Renal cell carcinoma (RCC) is a cancer with poor prognosis and the 5ꢀyear survival
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rate of patients with metastatic RCC is 5–10%. Consequently, treatment of metastatic RCC represents
an unmet clinical need. Screening of a 50,000ꢀmember library of natural and synthetic compounds for
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sensitizers of RCC cells to TRAILꢀmediated apoptosis led to identification of the 17βꢀ
hydroxywithanolide (17ꢀBHW), withanolide E (1), as a promising lead. To explore structure–activity
relationships, we obtained natural and semiꢀsynthetic withanolides 1, 2a, 2c, and 3–36 and compared
their ability to sensitize TRAILꢀmediated apoptosis in a panel of renal carcinoma cells. Our findings
revealed that 17ꢀBHWs with a
αꢀoriented sideꢀchain are superior to known TRAILꢀsensitizing
withanolides belonging to withaferin A class with a
β
ꢀoriented sideꢀchain and demonstrated that the
17ꢀBHW scaffold can be modified to enhance sensitization of RCCs to TRAILꢀmediated apoptosis,
thereby assisting development of natural productꢀinspired drugs to treat metastatic RCC.
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■
INTRODUCTION
Renal carcinoma is among the ten most common cancers in the US and according to the
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American Cancer Society’s most recent estimates, about 63,990 new cases of kidney cancer will
occur and over 14,400 people will die from this disease in 2017.1 There are over 338,000 new renal
cancer cases worldwide each year2 and it is often diagnosed with poor prognosis at the metastatic
stage.3 Approximately 90% of renal cancers arise in the renal parenchyma and are termed renal cell
carcinoma (RCC). The current standard of care, immunotherapy using interferon or interleukinꢀ2, has
had some success with responses in a small fraction of patients with metastatic RCC.4 Although some
antiꢀangiogenic therapies including sunitinib and sorafenib (Supporting Information, Figure S1) have
provided modest responses, it remains unclear which patients would have durable responses.5 Thus,
the treatment of advanced RCC represents an unmet clinical need and the discovery of innovative
approaches for this purpose is urgently required.
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Current strategies for cancer therapy aim to overcome two key hallmarks of cancer
including excessive proliferation and avoidance of apoptosis.6 In contrast to inhibiting
proliferation, which will mostly achieve stable disease by limiting tumor outgrowth, induction of
apoptosis has the potential to eliminate cancer cells, which could provide an opportunity for cure.
Consequently, methods of inducing apoptosis have become an important approach in the design
of effective cancer therapies. TRAIL (tumor necrosis factorꢀrelated apoptosisꢀinducing ligand) is
a protein predominantly derived from immune cells, that functions as a ligand to induce cell
death by apoptosis.7 TRAIL binding to death receptors (DR4 and DR5) assembles the deathꢀ
inducing signaling complex (DISC) through recruitment of FASꢀassociated protein with death
domain (FADD) and caspaseꢀ8, and autocatalytic activation of caspaseꢀ8 causes a cascade of
caspase activation ultimately resulting in cell death.8 TRAIL has a significant advantage in its
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selectivity for targeting cancer cells due to their relatively higher expression of death receptors
than normal cells, which in contrast highly express decoy receptors, DcR1 and DcR2.9 TRAIL
binds to decoy receptors, but these complexes are unable to activate the apoptotic signaling
pathway. Thus, TRAIL has little effect on apoptosis of normal cells. Furthermore in preclinical
models of renal cancer, TRAIL produced by immune cells has been shown to limit cancer
progression.10 However, many cancers acquire resistance to TRAIL therapy by downꢀregulating
DRs and upꢀregulating antiꢀapoptotic proteins including cellular FLICEꢀlike inhibitory protein
(cFLIP).11 Since many cancer cells, particularly those freshly isolated from cancer patients, are
resistant to TRAILꢀmediated apoptosis, the search for novel sensitizers to overcome TRAIL
resistance is considered an effective approach for anticancer drug discovery and the future
improvement of TRAILꢀbased therapies.
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Historically, plants have proven to be a valuable source of clinically useful anticancer
agents and they continue to play a major role in drug discovery as evidenced by the number of
promising new agents in clinical development based on their selective activity against cancerꢀ
related molecular targets.12 Several plantꢀderived natural products are also known to enhance
TRAILꢀinduced apoptosis through modulation of diverse signaling pathways suggesting that
combination therapy involving TRAIL and appropriate natural products might offer an attractive
strategy for treatment of cancer.13 Among the natural products capable of promoting TRAILꢀ
induced apoptosis of renal carcinoma cells are the wellꢀknown withanolide, withaferin A (2a)
derived from Withania somnifera,14 and its new analogue 2b (Figure 1) recently encountered in
Physalis pubescens.15 In our search for sensitizers of renal carcinoma cells to TRAILꢀinduced
apoptosis, we previously used a cellꢀbased highꢀthroughput assay to screen a 50,000ꢀmember
library of natural products and synthetic compounds.16 The most promising compound identified
in this study was withanolide E (1), a constituent of the plant, Physalis peruviana.17 It was also
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found that this 17
β
ꢀhydroxywithanolide (17ꢀBHW) was about 5ꢀfold more potent than 2a in
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sensitizing cancer cells to TRAILꢀmediated apoptosis. In addition, 1 was found to induce
TRAILꢀmediated apoptotic signaling via a rapid decline in the levels of cFLIP proteins, and
animal studies revealed that it sensitized human renal carcinoma cells to apoptosis at
concentrations that did not promote any obvious manifestations of toxicity in mice.17 More
importantly, in contrast to 2a, 1 did not cause growth inhibition, dramatic changes in cell
morphology or trigger a cell stress response at concentrations that sensitized cancer cells to
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undergo apoptosis, suggesting that biological activity of 17ꢀBHWs with a
αꢀoriented side chain
(e.g. 1) may differ considerably from those with a
β
ꢀoriented side chain (e.g. 2a and 2b).18
Based on its unique and promising activity, 1 was considered a lead molecule suitable for
structure–activity relationship (SAR) studies to identify natural productꢀbased compounds that
can selectively promote apoptosis in renal cancer cells and therefore potentially be used to treat
renal carcinoma.
■
RESULTS AND DISCUSSION
Isolation and Semiꢀsynthesis of Withanolides 1–36. Naturally occurring withanolides
1–5, 20, 27–31, and 34–36 for biological evaluation and preparation of semisynthetic 17ꢀBHWs were
obtained from aeroponically cultivated Withania somnifera,19 Physalis crassifolia,20 and P.
peruviana.21 17ꢀBHWs encountered in these plant species belonged to three distinct structural subꢀ
classes based on their oxygenation pattern and were grouped as analogues of withanolide E (WE) (5–
14; Figure 2), physachenolide C (PCC) (15–26; Figure 3), and physachenolide D (PCD) (27–36;
Figure 4). 3βꢀAzidoꢀ4βꢀhydroxywithanolide E (6) was obtained by treating 4βꢀhydroxywithanolide E
(5) with Me3SiN3/AcOH/Et3N.22 Acetyl derivatives 7, 12, 32 and 33 were prepared by acetylation of
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their corresponding hydroxy analogues with Ac2O/pyridine. Treatment of 5 with 1,1′ꢀ
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carbonyldiimidazole gave 9 which on methanolysis afforded 8. Oxidation of 5 with MnO2 yielded
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withaperuvin E (10). Reduction of 10 with NaBH4/CeCl3 afforded 4αꢀhydroxywithanolide E (11) as
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the major product formed as a result of regioꢀ and stereoꢀspecific reduction of its ring A eneꢀdione
moiety.23 When treated with Ph3P/I2, 10 underwent deoxygenation to afford withaperuvin M (13)
which on NaBH4/CeCl3 reduction afforded 4ꢀepiꢀphysapruin A (14). Catalytic hydrogenation of 3
with Pd/C in EtOH containing Et3N afforded 2,3ꢀdihydrophysachenolide C (15) and its desacetyl
analogue 24. Michael adducts of 3 at Cꢀ3, 2,3ꢀdihydroꢀ3 ꢀazidophysachenolide C (16), 2,3ꢀdihydroꢀ
β
3β
ꢀimidazolylphysachenolide C (17), and 2,3ꢀdihydroꢀ3βꢀmethoxyphysachenolide C (18) were
obtained by reacting 3 with Me3SiN3/AcOH/CH2Cl2,22 imidazole/CH2Cl2, and MeOH/Et3N,
respectively. Epoxidation of 4 with mꢀCPBA yielded 3, its epimer, 5,6ꢀepiꢀphysachenolide C (19),
and 24α,25αꢀepoxyphysachenolide C (26) whereas similar treatment of 36 afforded 15αꢀ
hydroxyphysachenolide C (21) and 15αꢀhydroxyꢀ5,6ꢀepiꢀphysachenolide C (23).24 Treatment of 3
with tꢀBuOK/tꢀBuOH/CH2Cl2/–5 oC afforded its18ꢀdesacetyl analogue 25. All new analogues were
characterized using their spectroscopic data (see Experimental Section and Supporting Information,
Figures S2–S60).
TRAILꢀInduced Apoptosis. We have previously demonstrated that a combination of WE
(1) and TRAIL eliminated longꢀterm survival of ACHN renal carcinoma cells while either agent
alone had no effect.17 In this study we first determined the ability of each of the withanolides 1, 2a,
2c, and 3–36 to sensitize ACHN cells to apoptosis in the presence of TRAIL (50.0 ng/mL) (Table 1
and Supporting Information, Figure S61). Of these, the 17ꢀBHW analogues 3–5, 7–9, 11–14, 16 and
17 had activities similar or superior to 1 (Figure 5) suggesting that those belonging to WE and PCC
subꢀclasses of 17ꢀBHWs (Figures 2 and 3, respectively) bearing 5β,6βꢀepoxy moieties exhibited
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potent activity. Among those active, 3 and the novel WE analogue, 4αꢀhydroxywithanolide E (11),
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were selected for further evaluation and 1 and 2a were included for comparison purposes. Analysis of
a panel of human renal carcinoma cells (Cakiꢀ1, SN12C, and UOꢀ31) for sensitization to TRAILꢀ
induced apoptosis demonstrated that the relative activities of these analogues were similar for all
renal cancer cells tested other than ACHN, with 3 and 11 being significantly more active than 1; 2a
displayed no activity at the concentrations tested (Figure 6). Since our previous studies have shown
that treatment of ACHN cells with 1 resulted in a drop in levels of the antiꢀapoptotic cFLIP proteins
thus promoting enhanced caspaseꢀ8 activation upon subsequent exposure of cells to TRAIL,17 we
assessed the effects of these 17ꢀBHWs on levels of cFLIP and caspase activation. As depicted in
Figure 7, 1 caused some reduction of cFLIP, but this was much more pronounced in cells treated with
the same concentration of 3 or 11. Interestingly other active withanolides (7 and 8) also caused a
reduction of cFLIP levels (Supporting Information, Figure S62). On subsequent exposure of cells to
TRAIL, the conversion of procaspaseꢀ8 to the active form the enzyme, and the subsequent proteolytic
activation of the executioner caspaseꢀ3 was also more dramatic in cells treated with 3 and 11
compared to 1 with weak activity and 2a with no activity (Figure 8). This suggested that reduction of
the antiꢀapoptotic cFLIP proteins in renal carcinoma cells by these withanolides, resulted in an
enhanced activation of caspaseꢀ8 on subsequent exposure to TRAIL, and this is likely an important
component of their molecular mechanism of action. Previous studies have reported that reducing
levels of cFLIP with these same RCC cell lines using siRNA was sufficient to sensitize them to
TRAILꢀmediated apoptosis.11b Therefore the efficient reduction of cFLIP levels by 3 and 11 is likely
to be critical for apoptosis sensitization of RCC cells. Thus these decreases in cFLIP proteins result in
an amplification of proximal TRAILꢀinduced extrinsic apoptosis signaling by increasing caspaseꢀ8
activation. Kinetic studies showed that a 6 h treatment of cells with 3 was sufficient to enhance
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caspaseꢀ8 activation following a short (90 min) exposure to TRAIL (Supporting Information, Figure
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S63). For these RCCs a contribution of the intrinsic or mitochondrial signaling pathway might not be
required for apoptosis to occur.
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It is known that 2a14 and its analogue 2b15 could sensitize RCC cells to TRAILꢀsensitized
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apoptosis at high concentrations (1.2
ꢀM and 1.0–2.0 ꢀM, respectively). Data presented here suggests
that 2a and its analogue 2,27ꢀdiacetylꢀ4ꢀepiꢀwithaferin A (2c)23 with a
β
ꢀoriented side chain are much
less potent as TRAILꢀsensitizers and are less effective in reducing cFLIP levels than 17ꢀBHWs with a
ꢀoriented side chain (Supporting Information, Figure S64). In contrast to the effects on TRAIL
α
sensitization, the active 17ꢀBHWs (1, 3 and 11) were less directly cytotoxic to ACHN cells than 2a
and 2c (Supporting Information, Figure S65). In addition, 1 and 3 in contrast to 2a and 2c did not
significantly increase the cellular levels of proteins CHOP, BIP and HSP70 that are elevated in
response to cell stress (Supporting Information, Figure S66). Many compounds that have been
proposed as TRAIL sensitizers, including the chemotherapeutic agent, bortezomib25 (Supporting
Information, Figure S1), and natural products such as cucurbitacin A,13b triptolide13d and 2a14 as well
a novel smallꢀmolecule, 7ꢀbenzylꢀ4ꢀ(2ꢀmethylbenzyl)ꢀ2,4,6,7,8,9ꢀhexahydroimidazo[1,2ꢀ
a]pyrido[3,4ꢀe]pyrimidinꢀ5(1H)ꢀone (ONC201)26 (see Supporting Information, Figure S1), are known
to induce significant levels of cell stress. Furthermore, this cell stress induction has been proposed to
contribute to increased TRAILꢀinduced apoptosis, since increases in CHOP levels are reported to
increase the levels of DR5 on cancer cells resulting in a stronger apoptosis response following
exposure to TRAIL.27 Interestingly, the17ꢀBHWs found to be active in this study did not increase
levels of DR5 on RCC (data not shown). The lack of direct cytotoxicity or induction of cell stress by
active 17ꢀBHWs suggests that in contrast to many other TRAILꢀsensitizers, induction of a cell stress
response is not a part of their mechanism of action, and they may have a novel and unique molecular
mechanism of action that differs from that of many other TRAILꢀsensitizing agents.
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Cytotoxicity and Thiol Reactivity. The ability of 2a to sensitize TRAILꢀinduced
apoptosis of renal carcinoma cells has been attributed to ROSꢀmediated upregulation of DR5 and
downregulation of cFLIP.14 Withanolide 2a has also been reported to exert its cytotoxicity and
cellular stress induction activities by depletion of intracellular glutathione (GSH).28 Thus it was of
interest to determine if sensitization of TRAILꢀinduced apoptosis of ACHN cells by active 17ꢀBHWs
is associated with cytotoxicity and/or cellular stress. The IC50 data (Figure 9) for 1, 3, and those 17ꢀ
BHWs (5, 7, 8, 11 and 12) with TRAILꢀsensitization activity superior or comparable to that of 1
suggested the order of cytotoxic activity to be 1 = 3 < 5 < 11 < 8 < 12 < 7. The 17ꢀBHWs, 7, 8, 11,
and 12 with strong activity as sensitizers of ACHN cells to TRAILꢀinduced apoptosis were found to
be comparatively more cytotoxic than 1 and 3 (Figure 9). Furthermore, testing of a larger panel of
various human cancer cell lines showed no significant differences between 3 and 11 for TRAIL
sensitizing activity (data not shown). However, 11 was found to undergo decomposition on storage
suggesting that it may not be a good candidate for further development. Therefore 3 was selected for
further testing in studies currently underway in mouse preꢀclinical cancer models.
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The chemical structures of 2a and 17ꢀBHWs used in this study contain three potential
electrophilic sites (2,3ꢀenꢀ1ꢀone in ring A, 5,6ꢀoxirane in ring B, and α,βꢀunsaturated lactone in
the side chain), but it is known that among these reactive sites ring A enone moiety is the most
susceptible for nucleophilic attack,28,29 and we have recently shown that cellular stress caused by
2a is due to the formation of Michael adducts of its ring A enone moiety with GSH and that
cytotoxicity of 2a was counteracted by the thiolꢀbased antioxidant, Nꢀacetylcysteine (NAC).28
Thus, it was of interest to compare thiol reactivity (thiophilicity) of some withanolides used in
this study with their ability to affect TRAIL sensitization. Previous work has shown that the
reaction of GSH with enones proceeded rapidly at basic pH (pH = 8), but extremely slowly and
irreversibly at low pH.29 However, after some experimentation with reaction conditions, we
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found that the addition of GSH to 2a took place at a measurable rate at the physiological pH (pH
= 7.4). Thus, the 17ꢀBHWs 1, 3, 5, 7, 8, 11 and 12 in DMSO were treated with aqueous solutions
of GSH and NAC at pH 7.4 and the amount of unreacted withanolide was monitored by HPLC
for 24 h. The cytotoxic withanolide, 2a,23 and comparatively less cytotoxic, 5,6ꢀepiꢀPCC (19),24
were included for comparison purposes. As shown in Figure 10, these withanolides varied
considerably in their relative ability for irreversible thiol capture and suggested that they belong
to 3 distinct classes, those with high (2a, 5, 7, 8, 11, and 12), medium (1 and 3), and low (19)
thiophilicities. Intriguingly, for 17ꢀBHWs the observed thiophilicity was found to parallel their
cytotoxic activity (Figure 9). Withanolides (2a, 5 and 11) with relatively high Michael acceptor
capacity all contain a hydroxy group at the allylic position (Cꢀ4) which is known to accelerate
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thiol addition.30 We have previously shown that the
βꢀhydroxy group at Cꢀ4 in 5 caused
enhancement of its cytotoxic activity compared to 1 lacking this hydroxy group, and had weak
activity for sensitization of ACHN cells to TRAIL17 The least active withanolide 19 displays an
enone moiety in a transꢀdecalin ring system compared to other 17ꢀBHWs which contain this
moiety in a cisꢀdecalin system. Based on reported studies on related molecules,31 it is likely that
the transꢀdecalinꢀtype enone in 19 has a less favorable equilibrium for thiol capture than the cisꢀ
decalinꢀtype enones in 1, 3, 5, 7, 8, 11, and 12.
StructureꢀActivity Relationships. In order to identify structural features of withanolides
responsible for sensitizing renal carcinoma cells to TRAILꢀinduced apoptosis, we examined thirty
four natural and semiꢀsynthetic 17ꢀBHWs (3–36) bearing an
αꢀoriented side chain and compared their
activities with those of 1 and withanolides with a ꢀoriented side chain, 2a and its analogue 2c. It
β
was found that the 17ꢀBHWs had superior desirable activity as TRAILꢀsensitizers and therefore we
focused our attention to structureꢀactivity relationships (SARs) within this group of withanolides.
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Summary of SAR data for 17ꢀBHWs evaluated is depicted in Figure 11. These data suggested that in
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addition to the
oxirane functionality in ring B are essential for activity and that
hydroxylation/ ꢀmethoxycarbonylation at Cꢀ4, and acetoxylation at Cꢀ18 lead to enhancement of
activity, ꢀhydroxylation at Cꢀ4 has no effect on activity, whereas hydroxylation at Cꢀ15, Cꢀ18, Cꢀ26
and Cꢀ27 and ꢀepoxidation of the double bond at Cꢀ24(25), all lead to decrease or loss of activity.
α
ꢀoriented sideꢀchain and/or 17
β
ꢀhydroxy group, enone moiety in ring A and 5
β,6βꢀ
α
ꢀ and ꢀacetoxylation/
β
αꢀ
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β
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■
CONCLUSIONS
Withanolides, a class of steroidal lactones occurring in some plants used in traditional
medicines, continue to attract attention due to their intriguing biological activities.32 Among all
withanolides, the most extensively studied is 2a with a
βꢀoriented side chain at Cꢀ17 that has been
evaluated for its anticancer activity.28,33 Of over 650 withanolides encountered to date, only about 50
contain a
αꢀoriented side chain at Cꢀ17 which is not a part of a cyclic system, and most of these bear
a
β
ꢀhydroxy group at this carbon.32 These 17ꢀBHWs have been reported to have in vitro cytotoxic
activity against breast,34 and prostate35 cancers. Our recent studies demonstrated that 1 was able to
sensitize TRAIL to induce apoptosis of ACHN renal carcinoma cells17 and 3 had potent and selective
cytotoxic activity to prostate cancer cells,24 suggesting the future promise of this class of withanolides
as potential anticancer agents.
Although this study demonstrated the ability of active 17ꢀBHWs to reduce levels of
cFLIP and that increased caspaseꢀ8 activation to be critical for sensitization of human RCC to
TRAILꢀinduced apoptosis, this may not be sufficient for apoptosis sensitization in other cell
types. Interestingly, resistance to TRAIL apoptosis can be due to multiple redundant pathways
for normal cells, whereas many cancer cells may rely on a single nonꢀredundant resistance
mechanism.36 Therefore it is likely for RCC that the overexpression of cFLIP proteins may
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constitute a single dominant apoptosis resistance mechanism. However, genetic and
pharmacological screens have also identified multiple inhibitors of TRAILꢀmediated cell death
in certain cancer cells.37 Thus, for TRAILꢀsensitizing compounds such as the kinase inhibitors
sorafenib38 (Supporting Information, Figure S1) and (Nꢀ(5ꢀ(((5ꢀ(tertꢀbutyl)oxazolꢀ2ꢀ
yl)methyl)thio)thiazolꢀ2ꢀyl)piperidineꢀ4ꢀcarboxamide (SNSꢀ032)39 (see Supporting Information,
Figure S1), their molecular mechanism of action seems to involve the reduction in cellular levels
of a number of other antiꢀapoptotic proteins including various Bclꢀ2 family members and IAPs.
As such, these compounds promote not only increases in extrinsic apoptosis signaling but also
intrinsic apoptosis signaling, both of which may be necessary for optimal TRAIL apoptosis in
many cancer cells. The lack of direct cytotoxicity or induction of cell stress by 17ꢀBHWs 1 and 3
suggests that in contrast to many other TRAILꢀsensitizers, induction of cell stress response is not
a part of their mechanism of action, and these may have a novel and unique molecular
mechanism of action that differs from that of many other TRAILꢀsensitizing agents. Thus, taken
together these findings suggest that 3 represents a promising natural productꢀbased molecule to
be developed as an effective and nonꢀtoxic agent to treat metastatic renal carcinomas. Since
some active 17ꢀBHWs can promote apoptosis in cancer cells other than RCC cells,40 we are
currently investigating in more detail if they can have additional effects on other apoptosis
modifying proteins in addition to reducing levels of cFLIP.
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■
EXPERIMENTAL SECTION
Chemistry. Optical rotations were measured in MeOH or CHCl3 with a Jasco DIPꢀ370
digital polarimeter. 1D and 2D NMR spectra were recorded in CDCl3, unless otherwise stated, using
residual solvents as internal standards on Bruker Avance III 400 spectrometer at 400 MHz for 1H
NMR, and 100 MHz for 13C NMR, respectively. The chemical shift values (δ) are given in parts per
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million (ppm), and the coupling constants (J values) are in Hz. LRꢀMS were recorded using
Shimadzu LCMSꢀQP8000α and HRꢀMS were recorded using JEOL HX110A or Agilent TOF mass
spectrometers. Analytical thinꢀlayer chromatography was carried out on silica gel 60 F254 Aluminumꢀ
backed TLC plates (Merck). Preparative thinꢀlayer chromatography (TLC) was performed on
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Analtech silica gel 500 ꢀm glass plates. Compounds were visualized with shortꢀwavelength UV and
by spraying with anisaldehydeꢀsulfuric acid spray reagent and heating until the spots appeared. Silica
gel column chromatography (CC) was accomplished using 230–400 mesh silica gel. Sephadex LHꢀ20
was obtained from Amersham Biosciences. HPLC was carried out on a Phenomenex Luna 5ꢁ C18 (2)
column (10 × 250 mm) or Alltech Econosil normal phase silica column (250 × 10 mm, 10 ꢁ) with
Waters Delta Prep system consisting of a PDA 996 detector. All yields refer to yields of isolated
compounds. Unless otherwise stated, chemicals and solvents were of reagent grade and used as
obtained from commercial sources without further purification. HPLC analysis was conducted for all
compounds that were subjected to bioassays on a Hitachi Lꢀ6200A LC system equipped with Hitachi
ASꢀ4000 Intelligent Auto Sampler, Hitachi Lꢀ4500 Photodiode array detector and Shimadzu ELSDꢀ
LT detector using a Phenomenex Luna 5ꢀ C18 (2) 100 Å (4.6 mm x 250 mm) column and a gradient
solvent system of MeOH/H2O [from 40:60 to 100:0 (v/v) in 30 min]. All the assayed compounds
displayed a chemical purity of >95% by both ELSD and UV (at 230 nm) detection methods.
Isolation of Naturally Occurring Withanolides 1, 2a, 3–5, 20, 27–31, and 34–36.
Withanolide E (1) and 4β
ꢀhydroxywithanolide E (5) were obtained from Physalis peruviana,21
withaferin A (2a) from Withania somnifera,19 and physachenolide C (3), physachenolide D (4), 15αꢀ
acetoxyphysachenolide C (20), 2,3ꢀdihydrophysachenolide Dꢀ3βꢀOꢀsulfate (27), 15αꢀ
acetoxyphysachenolide D (28), 15αꢀacetoxyꢀ2,3ꢀdihydrophysachenolide Dꢀ3βꢀOꢀsulfate (29), 15αꢀ
acetoxyꢀ28ꢀhydroxyphysachenolide D (30), 15αꢀacetoxyꢀ27ꢀhydroxyphysachenolide D (31), 15αꢀ
acetoxyꢀ28ꢀOꢀβꢀglucopyranosylphysachenolide D (34), 15αꢀacetoxyꢀ27ꢀOꢀβꢀ
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glucopyranosylphysachenolide D (35), and 15αꢀhydroxyphysachenolide D (36) were obtained from
Physalis crassifolia20 as previously described.
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General Procedure for Acetylation of Withanolides. To a solution of the
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withanolide (2.0 mg) in anhydrous pyridine (0.2 mL) was added Ac2O (0.5 mL), and the mixture
was stirred at 25 °C until the reaction was complete (judged by the disappearance of the starting
material by TLC). The reaction mixture was evaporated under reduced pressure and the resulting
crude product was purified by RPꢀHPLC or preparative TLC to yield the corresponding acetyl
derivative.
Preparation of Withanolide Analogues 2c, 21 and 23. 2,27ꢀDiacetylꢀ4ꢀepiꢀwithaferin
A (2c) was obtained from withaferin A (2a) as described previously.23 Epoxidation of 15αꢀ
hydroxyphysachenolide D (36) with mꢀchloroperbenzoic acid (mꢀCPBA) afforded 15αꢀ
hydroxyphysachenolide C (21) and 15αꢀhydroxyꢀ5,6ꢀepiꢀphysachenolide C (23).24
Preparation of 2,3ꢀDihydroꢀ3βꢀazidoꢀ4βꢀhydroxywithanolide E (6). To a
solution of 4βꢀhydroxywithanolide E (5, 7.0 mg) in anhydrous CH2Cl2 (0.8 mL) were added
AcOH (19.7 ꢁL), Et3N (1.93 ꢁL), and Me3SiN3 (45.5 ꢁL) and stirred at 25 oC. After 20 h, the
reaction was quenched with aqueous NH4Cl and extracted with EtOAc (3 x 10 mL), combined
organic layer was washed with brine, dried over Na2SO4, and evaporated under reduced pressure.
The residue thus obtained was purified by silica gel preparative TLC using hexanes/EtOAc (2:8)
to give 6 (6.2 mg, 82%, Rf = 0.47) as a white powder; [α] 25 –159 (c 0.11, CHCl3); 1H NMR (400
D
MHz, CDCl3) δ 4.82 (1H, dd, J = 10.0, 6.1 Hz, Hꢀ22), 4.00 (1H, dt, J = 8.2, 3.9 Hz, Hꢀ3), 3.43
(1H, d, J = 3.9 Hz, Hꢀ4), 3.32 (1H, t, J = 1.7 Hz, Hꢀ6), 2.91 (1H, dd, J = 15.7, 7.3 Hz, Haꢀ2),
2.69 (1H, ddd, J = 14.5, 11.0, 8.5 Hz, Haꢀ16), 2.62 (1H, dd, J = 15.7, 4.2 Hz, Hbꢀ2), 2.51ꢀ2.46
(2H, m, H2ꢀ23), 2.27 (1H, dt, J = 16.9, 4.6 Hz, Haꢀ12), 2.13 (1H, dd, J = 14.5, 10.8 Hz, Haꢀ7),
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2.03 (1H, ddd, J = 14.5, 6.5, 1.7 Hz, Hbꢀ7), 1.91 (3H, s, H3ꢀ28), 1.86 (3H, s, H3ꢀ27), 183 (1H, m,
Hꢀ8), 1.72 (1H, dt, J = 11.5, 3.7 Hz, Hꢀ9), 1.64 (1H, dt, J = 12.0, 3.7 Hz, Haꢀ15), 1.56 (1H, dt, J
= 12.0, 3.4 Hz, Hbꢀ15), 1.42 (2H, dd, J = 14.5, 8.2 Hz, Haꢀ11, Hbꢀ16), 1.40 (3H, s, H3ꢀ21), 1.35
(1H, dd, J = 15.0, 5.5 Hz, Hbꢀ11), 1.29 (1H, m, Hbꢀ12), 1.26 (3H, s, H3ꢀ19), 1.00 (3H, s, H3ꢀ18);
13C NMR (100 MHz, CDCl3) δ 208.1 (C, Cꢀ1), 165.8 (C, Cꢀ26), 150.6 (C, Cꢀ24), 121.5 (C, Cꢀ
25), 87.7 (C, Cꢀ17), 81.5 (C, Cꢀ14), 79.5 (CH, Cꢀ22), 79.1 (C, Cꢀ20), 75.7 (CH, Cꢀ4), 64.0 (C, Cꢀ
5), 60.3, (CH, Cꢀ6), 58.7 (C, Cꢀ13), 54.5 (CH, Cꢀ3), 50.4 (C, Cꢀ10), 38.8 (CH2, Cꢀ2), 37.9 (CH2,
Cꢀ16), 35.6 (CH, Cꢀ8), 34.2 (CH2, Cꢀ23), 33.8 (CH, Cꢀ9), 32.2 (CH2, Cꢀ15), 29.3 (CH2, Cꢀ12),
25.5 (CH2, Cꢀ7), 21.0 (CH2, Cꢀ11), 20.6 (CH3, Cꢀ28), 20.1 (CH3, Cꢀ18), 19.7 (CH3, Cꢀ21), 15.0
(CH3, Cꢀ19), 12.4 (CH3, Cꢀ27); HRESIꢀMS m/z 568.2634 [M+Na]+ (calcd for C28H29N3NaO8
568.2634).
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Preparation of 4βꢀAcetoxywithanolide E (7). Acetylation of withanolide E (1) by
the general procedure afforded 7.
4βꢀAcetoxywithanolide E (7). White powder; [α] 2D5 +136 (c 0.1, CHCl3); 1H and 13C
NMR data were consistent with those reported.41
Preparation of 4βꢀMethoxycarbonyloxywithanolide E (8). A solution of 9 (3.0
mg) in MeOH (0.5 mL) was stirred at 25 oC. After 1 h solvent was evaporated under reduced
pressure and the crude product was purified by preparative TLC (silica gel) using CH2Cl2/MeOH
(94:6) as eluent to give 8 (1.2 mg, 43%).
4βꢀMethoxycarbonyloxywithanolide E (8). White amorphous solid; [α] 2D5 +108 (c 0.5,
CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.99 (1H, dd, J = 10.1, 6.0 Hz, Hꢀ3), 6.27 (1H, d, J =
10.1 Hz, Hꢀ2), 4.83 (1H, dd, J = 11.4, 5.5 Hz, Hꢀ22), 4.54 (1H, d, J = 6.0 Hz, Hꢀ4), 3.74 (3H, s,
OCH3), 3.28 (1H, t, J = 1.7 Hz, Hꢀ6), 2.69 (1H, ddd, J = 15.0, 11.0, 8.5 Hz, Haꢀ16), 2.52ꢀ2.45
(2H, m, H2ꢀ23), 2.21 (1H, m, Haꢀ12), 2.06ꢀ2.01 (2H, m, H2ꢀ7), 1.92 (3H, s, H3ꢀ28), 1.86 (3H, s,
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H3ꢀ27), 1.81 (1H, m, Hꢀ8), 1.67ꢀ1.52 (5H, m, Hꢀ9, H2ꢀ11, H2ꢀ15), 1.41 (dd, J = 15.0, 8.5 Hz, 1H,
Hbꢀ16), 1.39 (3H, s, H3ꢀ21), 1.37 (3H, s, H3ꢀ19), 1.29 (1H, brd, J = 12.5 Hz, Hbꢀ12), 1.04 (3H, s,
H3ꢀ18); 13C NMR (100 MHz, CDCl3) δ 200.6 (C, Cꢀ1), 165.7 (C, Cꢀ26), 155.0 (C, OCOOCH3),
150.6 (C, Cꢀ24), 134. 3 (C, Cꢀ3), 135.0 (C, Cꢀ2), 121.5 (C, Cꢀ25), 87.5 (C, Cꢀ17), 81.6 (C, Cꢀ14),
79.4 (CH, Cꢀ22), 79.1 (C, Cꢀ20), 75.4 (CH, Cꢀ4), 61.2 (CH, Cꢀ6), 61.1 (C, Cꢀ5), 55.1 (CH3,
OCH3), 54.5 (C, Cꢀ13), 48.0 (C, Cꢀ10), 37.9 (CH2, Cꢀ16), 36.7 (CH, Cꢀ8), 34.3 (CH2, Cꢀ23),
34.0 (CH, Cꢀ9), 32.3 (CH2, Cꢀ15), 29.4 (CH2, Cꢀ12), 25.6 (CH2, Cꢀ7), 20.9 (CH2, Cꢀ11), 20.6
(CH3, Cꢀ28), 20.2 (CH3, Cꢀ18), 19.6 (CH3, Cꢀ21), 15.2 (CH3, Cꢀ18), 12.4 (CH3, Cꢀ27); HRESIꢀ
MS (m/z) 583.2514 [M+Na]+ (calcd for C30H40NaO10 583.2514).
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Preparation of 4βꢀImidazolylcarbonyloxywithanolide E (9). To a solution of 4
hydroxywithanolide E (5, 10.0 mg) in anhydrous CH2Cl2 (1.0 mL) was added 1,1'ꢀ
βꢀ
carbonyldiimidazole (8.0 mg) and stirred at 25 oC. After 1 h, reaction mixture was filtered
through a short bed of silica gel (0.5 mg) using EtOAc as eluent to give 9 (8.1 mg, 68%).
4βꢀImidazolylcarbonyloxywithanolide E (9). White amorphous solid; [α] 2D5 +108 (c
0.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.04 (1H, s, NꢀCH=N), 7.31 (1H, t, J = 1.6 Hz, Nꢀ
CH), 7.04 (1H, m, NꢀCH), 7.04 (1H, dd, J = 9.8, 6.1 Hz, Hꢀ3), 6.37 (1H, d, J = 9.8 Hz, Hꢀ2),
4.89 (1H, d, J = 6.1 Hz, Hꢀ4), 4.83 (1H, dd, J = 11.5, 5.5 Hz, Hꢀ22), 3.37 (1H, t, J = 2.0 Hz, Hꢀ
6), 2.69 (1H, ddd, J = 15.0, 11.0, 8.5 Hz, Haꢀ16), 2.54–2.42 (2H, m, H2ꢀ23), 2.24 (1H, dt, J =
11.8, 5.7 Hz, Haꢀ12), 2.13–2.05 (2H, m, H2ꢀ7), 1.92 (3H, s, H3ꢀ28), 1.86 (3H, s, H3ꢀ27), 1.81
(1H, dd, J = 11.0, 6.3 Hz, Hꢀ8), 1.70–1.52 (5H, m, Hꢀ9, H2ꢀ11, H2ꢀ15), 1.43 (1H, m, Hbꢀ16),
1.42 (3H, s, H3ꢀ19), 1.39 (3H, s, H3ꢀ21), 1.29 (1H, brd, J = 11.8 Hz, Hbꢀ12), 1.05 (3H, s, H3ꢀ18);
13C NMR (100 MHz, CDCl3) δ 200.1 (C, Cꢀ1), 165.7 (C, Cꢀ26), 150.6 (C, Cꢀ24), 147.9 (C,
NC=O), 137.1 (CH, NꢀCH=N), 136.9 (CH, Cꢀ3), 136.0 (CH, Cꢀ2), 131.0 (CH, NꢀCH), 121.5 (C,
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Cꢀ25), 117.1 (CH, NꢀCH), 87.6 (C, Cꢀ17), 81.6 (C, Cꢀ14), 79.4 (CH, Cꢀ22), 79.1 (C, Cꢀ20), 76.1
(CH, Cꢀ4), 61.5 (CH, Cꢀ6), 60.9 (C, Cꢀ5), 54.5 (C, Cꢀ13), 48.0 (C, Cꢀ10), 37.8 (CH2, Cꢀ16), 36.8
(CH, Cꢀ9), 34.3 (CH2, Cꢀ23), 33.9 (CH, Cꢀ8), 32.3 (CH2, Cꢀ15), 29.4 (CH2, Cꢀ12), 25.6 (CH2, Cꢀ
7), 20.9 (CH2, Cꢀ11), 20.7 (CH3, Cꢀ28), 20.2 (CH3, Cꢀ18), 19.7 (CH3, Cꢀ21), 15.4 (CH3, Cꢀ19),
12.4 (CH3, Cꢀ27); APCIꢀMS (+) m/z 597 [M+1]+; APCIꢀMS (–) m/z 595 [M–1]–; HRESIꢀMS m/z
619.2624 [M+Na]+ (calcd for C32H40N2NaO9 619.2632).
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Oxidation of 4βꢀHydroxywithanolide E (5) to Withaperuvin E (10). To a
solution 5 (10.0 mg) in CHCl3/EtOAc (1:1, 3.0 mL) was added activated MnO2 (60.0 mg) and
stirred at 25 oC. After 6 h, reaction mixture was passed through a short column of silica gel (1.0
g) using CH2Cl2/MeOH (94:6) as eluent to give 10 (8.1 mg, 81%) as a pale yellow solid; [α] D25
–
74 (c 0.5, CH3CN) [lit.42 –72.7 (c 0.7, CH3CN]; 1H and 13C NMR data were consistent with those
reported.42
Reduction of Withaperuvin E (10) to 4αꢀHydroxywithanolide E (11). To stirred
solution of 10 (4.0 mg) in MeOH (1.0 mL) was added CeCl3.7H2O (30.0 mg). The reaction
mixture was cooled to 0 oC and stirred for 5 min. To this mixture was added NaBH4 (ca 0.5 mg)
and stirred at 0 oC. After 5 min, the reaction mixture was quenched with ice, evaporated under
reduced pressure and extracted with EtOAc (3 x 5 mL). Combined organic layer was washed
with brine, dried over anhydrous Na2SO4 and evaporated under reduced pressure and the
resulting residue was chromatographed over a column of silica gel (500.0 mg) made up in
CH2Cl2 and eluted with CH2Cl2 containing increasing amounts of MeOH. Fractions eluted with
CH2Cl2/MeOH (94:6) were combined and evaporated under reduced pressure and the crude
product was further purified by RPꢀHPLC using MeOH/H2O (70:30) as eluent to give 11 (3.0
mg, 75%, tR = 15 min).
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α
ꢀHydroxywithanolide E (11). White amorphous solid; [α] 2D5 +65 (c 0.2, CHCl3); 1H
5
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8
9
NMR (400 MHz, CDCl3) δ 6.78 (1H, dd, J = 10.1, 1.7 Hz, Hꢀ3), 5.99 (1H, dd, J = 10.1, 2.6 Hz,
Hꢀ2), 4.84 (1H, dd, J = 11.2, 5.6, Hz, Hꢀ22), 4.65 (1H, brs, Hꢀ4), 3.71 (1H, brt, J = 1.9 Hz, Hꢀ6),
2.69 (1H, ddd, J = 15.1, 11.0, 8.0 Hz, Haꢀ16), 2.52–2.42 (2H, m, H2ꢀ23), 2.24 (1H, dt, J = 12.5,
4.9 Hz, Haꢀ12), 2.13 (1H, d, J = 2.8 Hz, D2O exchangeable, OHꢀ4), 1.98 (2H, m, H2ꢀ7), 1.91
(3H, s, H3ꢀ28), 1.85 (3H, s, H3ꢀ27), 1.83 (2H, m, Hꢀ8, Haꢀ11), 1.69–1.53 (4H, m, Hꢀ9, Hbꢀ11,
H2ꢀ15), 1.42 (1H, dd, J = 15.1, 8.3 Hz, Hbꢀ16), 1.39 (3H, s, H3ꢀ21), 1.27 (1H, brd, J = 12.5 Hz,
Hbꢀ12), 1.20 (3H, s, H3ꢀ19), 1.06 (3H, s, H3ꢀ18); 13C NMR (100 MHz, CDCl3) δ 200.6 (C, Cꢀ1),
165.7 (C, Cꢀ26), 150.5 (C, Cꢀ24), 146.9 (CH, Cꢀ3), 128.7 (CH, Cꢀ2), 121.5 (C, Cꢀ25), 87.6 (C,
Cꢀ17), 81.8 (C, Cꢀ14), 79.5 (CH, Cꢀ22), 79.1 (C, Cꢀ20), 65.6 (C, Cꢀ5), 64.7 (CH, Cꢀ4), 56.2 (CH,
Cꢀ6), 54.5 (C, Cꢀ13), 47.5 (C, Cꢀ10), 37.9 (CH2, Cꢀ16), 37.8 (CH, Cꢀ9), 34.2 (CH2, Cꢀ23), 34.1
(CH, Cꢀ8), 32.3 (CH2, Cꢀ15), 29.7 (CH2, Cꢀ12), 25.6 (CH2, Cꢀ7), 21.6 (CH2, Cꢀ11), 20.6 (CH3,
Cꢀ28), 20.4 (CH3, Cꢀ18), 19.7 (CH3, Cꢀ21), 13.2 (CH3, Cꢀ19), 12.4 (CH3, Cꢀ27); HRESIꢀMS m/z
525.2463 [M+Na]+ (calcd for C28H38NaO8 525.2463).
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4αꢀAcetoxywithanolide E (12). Acetylation of 11 by the general procedure afforded 12
as an amorphous white solid; [α] 2D5 +100 (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.62 (1H,
dd, J = 10.3, 1.9 Hz, Hꢀ3), 6.05 (1H, dd, J = 10.3, 2.8 Hz, Hꢀ2), 5.85 (dd, J = 2.8, 1.9 Hz. 1H, Hꢀ
4), 4.85 (1H, dd, J = 11.4, 5.1 Hz, Hꢀ22), 3.58 (1H, brs, Hꢀ6), 2.69 (1H, dt, J = 12.8, 10.6 Hz,
Haꢀ16 ), 2.54–2.42 (2H, m, H2ꢀ23), 2.25 (1H, dt, J = 13.0, 5.0 Hz, Haꢀ12), 2.09 (3H, s, OAc),
2.03–1.93 (2H, m), 1.92 (3H, s, H3ꢀ28), 1.90–1.82 (2H, m), 1.86 (3H, s, H3ꢀ27), 1.73 (1H, m),
1.67–1.53 (3H, m), 1.41 (1H, dd, J = 15.1, 8.3 Hz, Hbꢀ16), 1.39 (3H, s, H3ꢀ21), 1.28 (1H, m, Hbꢀ
12), 1.27 (3H, s, H3ꢀ19), 1.06 (3H, s, H3ꢀ18); 13C NMR (100 MHz, CDCl3) δ 200.3 (C, Cꢀ1),
169.8 (C, OAc), 165.7 (C, Cꢀ26), 150.3 (C, Cꢀ24), 144.4 (CH, Cꢀ3), 129.6 (CH, Cꢀ2), 121.5 (C,
Cꢀ25), 87.6 (C, Cꢀ17), 81.7 (C, Cꢀ14), 79.4 (CH, Cꢀ22), 79.1 (C, Cꢀ20), 65.6 (C, Cꢀ5), 63.2 (CH,
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Cꢀ4), 56.5 (CH, Cꢀ6), 54.5 (C, Cꢀ13), 47.9 (C, Cꢀ10), 37.8 (CH2, Cꢀ16), 37.7 (CH, Cꢀ9), 34.3
(CH2, Cꢀ23), 34.0 (CH, Cꢀ8), 32.4 (CH2, Cꢀ15), 29.8 (CH2, Cꢀ12), 25.5 (CH2, Cꢀ7), 21.9 (CH2,
Cꢀ11), 20.8 (CH3, Cꢀ28), 20.6 (CH3, Cꢀ18), 20.4 (CH3, OAc), 19.7 (CH3, Cꢀ21), 13.9 (CH3, Cꢀ
19), 12.4 (CH3, Cꢀ27); HRESIꢀMS m/z 567.2565 [M+Na]+ (calcd for C30H40NaO9 567.2563).
Conversion of Withaperuvin E (10) to Withaperuvin M (13). To a stirred solution
10 (23.0 mg) in anhydrous CH2Cl2 (2.0 mL) at 0 oC was added a solution of Ph3P (14.6 mg) and
I2 (14.0 mg) in CH2Cl2 (0.3mL). Ice bath was removed and reaction mixture was stirred at 25 oC.
The reaction was monitored by TLC and after disappearance of the starting material, the reaction
mixture was diluted with CH2Cl2 (25.0 mL), washed with aq. Na2S2O3 solution, H2O, dried over
anhydrous Na2SO4 and evaporated under reduced pressure to give crude product mixture (35.0
mg). This was separated by RPꢀHPLC using MeOH/H2O (65:35) to give 13 (9.8 mg, 44%). 1H
and 13C NMR data were consistent with those reported;43 APCIꢀMS (+) (m/z) 485 [M+1]+.
Conversion of Withaperuvin M (13) to 4ꢀEpiꢀphysapruin A (14). To a solution
of 13 (9.5 mg) in MeOH (1.0 mL) and THF (0.2 mL) at 0 oC was added CeCl3.7H2O (100.0 mg)
and stirred at 0 oC for 5 min. To this solution was added NaBH4 (ca 0.5 mg) and continued
stirring at 0 oC. After 5 min, the reaction mixture was quenched with ice, solvents were
evaporated under reduced pressure and extracted with EtOAc (3 x 5.0 mL). Combined organic
layer was washed with brine, dried over anhydrous Na2SO4 and evaporated under reduced
pressure to give the crude product (9.8 mg) which was purified by silica gel preparative TLC
using CH2Cl2/MeOH (93:7, double elution) to give 14 (5.8 mg, 59%, Rf = 0.34).
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4ꢀEpiꢀphysapruin A (14). White amorphous solid; [α] 2D5 +118 (c 0.2, CHCl3); 1H NMR
(400 MHz, CDCl3) δ 6.72 (1H, dd, J = 10.5, 2.1 Hz, Hꢀ3), 6.01 (1H, dt, J = 6.3, 2.1 Hz, Hꢀ6),
5.80 (1H, dd, J = 10.5, 2.5 Hz, Hꢀ2), 4.91 (1H, m, Hꢀ4), 4.87 (1H, dd, J = 11.6, 5.2 Hz, Hꢀ22),
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2.67 (1H, ddd, J = 14.5, 11.0, 8.5 Hz, Haꢀ16), 2.50ꢀ2.45 (2H, m, H2ꢀ23), 2.42 (1H, m, Haꢀ12),
2.21 (1H, m, Haꢀ11), 2.28 (1H, m, Hꢀ8), 2.14 (1H, m, Haꢀ7), 1.92 (1H, m, Hbꢀ7), 1.90 (3H, s,
H3ꢀ28), 1.83 (3H, s, H3ꢀ27), 1.77 (1H, dt, J = 11.2, 5.6 Hz, Hꢀ9), 1.66 (1H, m, Haꢀ15), 1.58 (1H,
m, Hbꢀ11), 1.55 (1H, m, Hbꢀ15), 1.40 (1H, dd, J = 14.5, 7.8 Hz, Hbꢀ16), 1.35 (3H, s, H3ꢀ21),
1.28 (1H, brd, J = 14.5 Hz, Hbꢀ12), 1.21 (3H, s, H3ꢀ19), 1.08 (3H, s, H3ꢀ18); 13C NMR (100
MHz, CDCl3) δ 204.2 (C, Cꢀ1), 151.2 (C, Cꢀ24), 148.6 (CH, Cꢀ3), 139.6 (C, Cꢀ5), 127.6 (CH, Cꢀ
2), 121.8 (CH, Cꢀ6), 121.3 (C, Cꢀ25), 87.8 (C, Cꢀ17), 82.1 (C, Cꢀ14), 80.6 (CH, Cꢀ22), 78.6 (C,
Cꢀ20), 66.9 (CH, Cꢀ4), 54.4 (C, Cꢀ13), 50.8 (C, Cꢀ10), 37.6 (CH2, Cꢀ16), 36.7 (CH, Cꢀ9), 36.2
(CH, Cꢀ8), 34.3 (CH2, Cꢀ23), 32.4 (CH2, Cꢀ15), 30.3 (CH2, Cꢀ12), 25.2 (CH2, Cꢀ7), 22.6 (CH2,
Cꢀ11), 20.6 (CH3, Cꢀ28), 20.5 (CH3, Cꢀ18), 19.5 (CH3, Cꢀ21), 19.2 (CH3, Cꢀ19), 12.2 (CH3, Cꢀ
27); HRESIꢀMS m/z 509.2500 [M+Na]+ (calcd for C28H38NaO7 509.2515).
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Conversion of Physachenolide C (3) to 2,3ꢀDihydrophysachenolide C (15) and
18ꢀDeacetylꢀ2,3ꢀdihydrophysachenolide C (24). To a solution of 3 (5.0 mg) in EtOH (2.0 mL)
and Et3N (20 ꢁL) was added 10% Pd/C (0.5 mg) and the mixture was stirred under an atmosphere of
H2 for 7 h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure
to give the product, which was separated by silica gel preparative TLC CHCl3/MeOH (95:5) to afford
15 (2.6 mg) and 24 (1.5 mg).
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D
2,3ꢀDihydrophysachenolide C (15). Offꢀwhite amorphous powder;
[
α]
+3 (c 0.1, MeOH);
1H NMR (400 MHz, CDCl3) δ 4.83 (1H, dd, J = 8.8, 8.0 Hz, Hꢀ22), 4.34 (1H, d, J = 11.6 Hz, Haꢀ18),
4.27 (1H, d, J = 11.6 Hz, Hbꢀ18), 3.17 (1H, brs, Hꢀ6α), 2.05 (3H, s, 18ꢀOAc), 1.91 (3H, s, CH3ꢀ28),
1.87 (3H, s, CH3ꢀ27), 1.39 (3H, s, CH3ꢀ21), 1.12 (3H, s, CH3ꢀ19); 13C NMR (100 MHz, CDCl3) δ
213.2 (C, Cꢀ1), 169.9 (C, 18ꢀOAc), 165.5 (C, Cꢀ26), 149.8 (C, Cꢀ24), 121.9 (C, Cꢀ25), 87.9 (C, Cꢀ
17), 81.2 (C, Cꢀ14), 79.3 (CH, Cꢀ22), 78.9 (C, Cꢀ20), 64.8 (CH2, Cꢀ18), 64.3 (C, Cꢀ5), 60.9 (CH, Cꢀ
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6), 57.6 (C, Cꢀ13), 52.4 (C, Cꢀ10), 38.2 (CH2, Cꢀ16), 35.6 (CH, Cꢀ9), 34.7 (CH2, Cꢀ2), 33.9 (CH2, Cꢀ
23), 33.7 (CH, Cꢀ8), 32.9 (CH2, Cꢀ15), 30.0 (CH2, Cꢀ4), 26.6 (CH2, Cꢀ7), 25.2 (CH2, Cꢀ12), 21.4
(CH2, Cꢀ11), 21.3 (CH3, 18ꢀOAc), 20.7 (CH3, Cꢀ28), 19.1 (CH3, Cꢀ21), 17.8 (CH2, Cꢀ3), 12.5 (CH3,
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Cꢀ19), 12.4 (CH3, Cꢀ27); APCIꢀMS (+) m/z 529 [MH–
H2O]+.
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18ꢀDeacetylꢀ2,3ꢀdihydrophysachenolide C (24). Offꢀwhite amorphous powder;
[
α]
–20
1
(c 0.04, MeOH); H NMR (400 MHz, CDCl3) δ 4.85 (1H, dd, J = 12.8, 4.0 Hz, Hꢀ22), 3.90 (1H, d, J
= 10.8 Hz, Haꢀ18), 3.81 (1H, d, J = 10.8 Hz, Hbꢀ18), 3.17 (1H, brs, Hꢀ6α), 1.91 (3H, s, CH3ꢀ28), 1.86
(3H, s, CH3ꢀ27), 1.39 (3H, s, CH3ꢀ21), 1.12 (3H, s, CH3ꢀ19); 13C NMR (100 MHz, CDCl3) δ 213.1
(C, Cꢀ1), 165.6 (C, Cꢀ26), 150.2 (C, Cꢀ24), 121.6 (C, Cꢀ25), 89.0 (C, Cꢀ17), 80.9 (C, Cꢀ14), 79.0
(CH, Cꢀ22), 78.8 (C, Cꢀ20), 64.4 (C, Cꢀ5), 63.4 (CH2, Cꢀ18), 60.9 (CH, Cꢀ6), 58.7 (C, Cꢀ13), 52.5 (C,
Cꢀ10), 37.5 (CH2, Cꢀ16), 35.6 (CH, Cꢀ9), 34.6 (CH2, Cꢀ2), 34.1 (CH2, Cꢀ23), 33.4 (CH, Cꢀ8), 33.1
(CH2, Cꢀ15), 30.0 (CH2, Cꢀ4), 26.5 (CH2, Cꢀ7), 24.1 (CH2, Cꢀ12), 21.3 (CH2, Cꢀ11), 20.7 (CH3, Cꢀ
28), 19.0 (CH3, Cꢀ21), 17.7 (CH2, Cꢀ3), 12.5 (CH3, Cꢀ19), 12.4 (CH3, Cꢀ27); APCIꢀMS (+) m/z 527
[M+Na]+.
Preparation of 2,3ꢀDihydroꢀ3βꢀazidophysachenolide C (16). To a solution of 3
(7.0 mg) in anhydrous CH2Cl2 (0.8 mL) were added AcOH (13.35 ꢁL), Et3N (1.82 ꢁL), and
TMSꢀN3 (30.8 ꢁL) and stirred at 25 oC. After 20 h, the reaction mixture was quenched with
aqueous NH4Cl and extracted with EtOAc (3 x 10 mL). Combined organic layer was washed
with brine, dried over Na2SO4, and evaporated under reduced pressure and the resulting residue
was purified by silica gel preparative TLC using hexanes/EtOAc (2:8) as eluent to afford 16 (3.1
mg, 67%, Rf = 0.58) and unreacted 3 (2.7 mg).
2,3ꢀDihydroꢀ3βꢀazidophysachenolide C (16). White powder; [α] 2D5 –145 (c 0.11,
CHCl3); 1H NMR (400 MHz, CDCl3) δ 4.83 (1H, t, J = 9.1 Hz, Hꢀ22), 4.34 (1H, d, J = 11.6 Hz,
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Haꢀ18), 4.23 (1H, d, J = 11.6 Hz, Hbꢀ18), 4.03 (1H, dt, J = 7.5, 6.6 Hz, Hꢀ3), 3.30 (1H, brs, Hꢀ
6), 2.76 (1H, dd, J = 14.1, 6.2 Hz, Haꢀ2), 2.73 (1H, dd, J = 14.1, 7.6 Hz, Hbꢀ2), 2.70 (1H, m, Haꢀ
16), 2.49 (2H, brd, J = 9.2 Hz, H2ꢀ23), 2.28 (1H, dd, J = 14.6, 5.8 Hz, Haꢀ4), 2.20 (1H, dd, J =
10.1, 5.4 Hz, Haꢀ7), 2.13 (1H, d, J = 11.6, Haꢀ12), 2.06 (3H, s, OAc), 2.04 (1H, d, J = 11.6, Hbꢀ
12), 1.93 (1H, m, Hꢀ9), 1.91 (3H, s, H3ꢀ28), 1.87 (3H, s, H3ꢀ27), 1.74 (1H, dd, J = 10.9, 4.1 Hz,
Hꢀ8), 1.68 (2H, m, Hbꢀ7, Haꢀ15), 1.59ꢀ1.50 (2H, m, Hbꢀ15, Hbꢀ16), 1.44 (1H, dd, J = 14.6, 2.2
Hz, Hbꢀ4), 1.39 (3H, s, H3ꢀ21), 1.37 (1H, m, Haꢀ11), 1.26 (1H, m, Hbꢀ11), 1.13 (3H, s, H3ꢀ19);
13C NMR (100 MHz, CDCl3) δ 209.2 (C, Cꢀ1), 170.0 (C, OAc), 165.5 (C, Cꢀ26), 149.7 (C, Cꢀ
24), 121.9 (C, Cꢀ25), 87.9 (C, Cꢀ17), 81.1 (C, Cꢀ14), 79.3 (CH, Cꢀ22), 78.9 (C, Cꢀ20), 64.8
(CH2, Cꢀ18), 61.6 (C, Cꢀ5), 61.4 (CH, Cꢀ6), 57.5 (C, Cꢀ13), 53.8 (CH, Cꢀ3), 52.1 (C, Cꢀ10), 41.4
(CH2, Cꢀ2), 38.2 4 (CH2, Cꢀ16), 36.3 (CH2, Cꢀ4), 35.4 (CH, Cꢀ9), 33.9 (CH2, Cꢀ23), 33.6 (CH,
Cꢀ8), 32.9 (CH2, Cꢀ15), 26.1 (CH2, Cꢀ12), 25.2 (CH2, Cꢀ6), 21.5 (CH2, Cꢀ11), 21.3 (CH3, OAc),
20.7 (CH3, Cꢀ28), 19.1 (CH3, Cꢀ21), 13.1 (CH3, Cꢀ19), 12.4 (CH3, Cꢀ27); HRESIꢀMS m/z
610.2735 [M+Na]+ (calcd for C30H41N3NaO9 610.2740).
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Preparation of 2,3ꢀDihydroꢀ3βꢀimidazolylphysachenolide C (17). To a solution
of 3 (5.0 mg) in anhydrous CH2Cl2 (1.0 mL) was added imidazole (8.0 mg) and stirred at 25 oC.
After 6 h, reaction mixture was diluted with EtOAc (15.0 mL), washed with brine (3 x 10.0 mL),
dried over anhydrous Na2SO4 and evaporated under reduced pressure. The resulting residue was
then passed through a short column of Sephadex LHꢀ20 (0.5 g) made up in hexanes/CH2Cl2 (1:4)
and eluted with hexanes/CH2Cl2 (1:4) followed by CH2Cl2/acetone (3:2) to give 17 (4.3 mg,
76%).
2,3ꢀDihydroꢀ3βꢀimidazolylphysachenolide C (17). While amorphous solid; [α] D25
–128 (c 0.2, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.63 (1H, s, NCHN), 7.08 (1H, s, NCH),
6.93 (1H, s, NCH), 4.82 (1H, dd, J = 10.3, 6.3 Hz, Hꢀ22), 4.68 (1H, m, Hꢀ3), 4.37 (1H, d, J =
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11.4 Hz, Haꢀ18), 4.24 (1H, d, J = 11.4 Hz, Hbꢀ18), 3.16 (1H, dd, J = 14.0, 8.8 Hz, Haꢀ2), 3.10
(1H, brs, Hꢀ6), 2.88 (1H, dd, J = 14.0, 8.1 Hz, Hbꢀ2), 2.71 (1H, dt, J = 17.6, 3.2 Hz, Haꢀ16), 2.55
(1H, dd, J = 15.4, 6.6 Hz, Haꢀ4), 2.52ꢀ2.48 (2H, m, H2ꢀ23), 2.27 (1H, dd, J = 16.4, 3.7 Hz, Haꢀ
7), 2.13 (1H, dd, J = 12.3, 6.3 Hz, Haꢀ12), 2.07 (3H, s, OAc), 2.04 (1H, m, Hbꢀ12), 1.96 (1H, m,
Hꢀ9), 1.91 (3H, s, H3ꢀ28), 1.86 (3H, s, H3ꢀ27), 1.78 (1H, dd, J = 11.2, 4.1 Hz, Hꢀ8), 1.76 (1H, dd,
J = 11.2, 4.1 Hz, Hbꢀ7), 1.72 (1H, m, Haꢀ15), 1.65 (1H, m, Hbꢀ15), 1.58 (1H, dd, J = 17.6, 2.0
Hz, Hbꢀ16), 1.40 (3H, s, H3ꢀ21), 1.34ꢀ1.24 (2H, m, H2ꢀ11), 1.19 (3H, s, H3ꢀ19); 13C NMR (100
MHz, CDCl3) δ 208.8 (C, Cꢀ1), 170.0 (C, OAc), 165.7 (C, Cꢀ26), 149.8 (C, Cꢀ24), 136.1 (CH,
NCN), 130.2 (CH, NCH), 121.9 (C, Cꢀ25), 116.8 (C, NC), 88.0 (C, Cꢀ17), 81.1 (C, Cꢀ14), 79.5
(CH, Cꢀ22), 78.6 (C, Cꢀ20), 64.7 (CH2ꢀ Cꢀ18), 61.5 (C, Cꢀ5), 61.2 (CH, Cꢀ6), 57.5 (C, Cꢀ13),
52.0 (C, Cꢀ10), 49.2 (CH, Cꢀ3), 42.1 (CH2, Cꢀ2), 38.3 (CH2, Cꢀ4), 38.1 (CH2, Cꢀ16), 35.6 (CH,
Cꢀ9), 33.9 (CH2, Cꢀ23), 33.5 (CH, Cꢀ8), 32.9 (CH2, Cꢀ15), 26.2 (CH2, Cꢀ12), 25.2 (CH2, Cꢀ7),
21.5 (CH2, Cꢀ11), 21.4 (CH3, OAc), 20.7 (CH3, Cꢀ28), 19.1 (CH3, Cꢀ21), 13.1 (CH3, Cꢀ19), 12.4
(CH3, Cꢀ27); HRESIꢀMS m/z 635.2940 [M+Na]+ (calcd for C33H44N2NaO9 635.2945).
Preparation of 2,3ꢀDihydroꢀ3βꢀmethoxyphysachenolide C (18). To a solution of
17 (2.8 mg) in MeOH (0.5 mL) was added Et3N (5.0 ꢁL) and stirred at 60 oC. After 1h reaction
mixture was evaporated under reduced pressure and the resulting residue was purified by RPꢀ
HPLC using MeOH/H2O (1:1) as eluent and the product was acetylated by the general procedure
to give 18 (1.1 mg, 42%).
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2,3ꢀDihydroꢀ3βꢀmethoxyphysachenolide C (18). White amorphous solid; [α] 2D5 –142
(c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3) δ 4.82 (1H, dd, J = 10.3, 6.3 Hz, Hꢀ22), 4.34 (1H, d,
J = 11.8 Hz, Haꢀ18), 4.25 (1H, d, J = 11.8 Hz, Hbꢀ18), 3.70 (1H, m, Hꢀ3), 3.27 (1H, brs, Hꢀ6),
3.25 (3H, s, OCH3), 2.79 (1H, dd, J = 13.6, 5.6 Hz, Haꢀ2), 2.71 (1H, dd, J = 13.6, 8.1 Hz, Hbꢀ2),
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2.67 (1H, m, Haꢀ16), 2.49 (2H, brd, J = 8.5 Hz, H2ꢀ23), 2.20 (1H, m, Haꢀ7), 2.15 (1H, ddd, J =
14.4, 5.6, 1.8 Hz, Haꢀ4), 2.10 (1H, m, Haꢀ12), 2.06 (3H, s, OAc), 2.00 (1H, dt, J = 14.8, 3.2 Hz,
Hbꢀ12), 1.92 (1H, m, Hꢀ9), 1.91 (3H, s, H3ꢀ28), 1.87 (3H, s, H3ꢀ27), 1.75 (1H, dt, J = 11.1, 4.2
Hz, Hꢀ8), 1.67 (1H, m, Hbꢀ7), 1.60 (1H, dd, J = 12.2, 3.7 Hz, Haꢀ15), 1.57 (1H, dd, J = 12.2, 8.5
Hz, Hbꢀ15), 1.50 (1H, m, bꢀ16), 1.39 (3H, s, H3ꢀ21), 1.30ꢀ1.27 (2H, m, H2ꢀ11), 1.12 (3H, s, H3ꢀ
19); 13C NMR (100 MHz, CDCl3) δ 210.7 (C, Cꢀ1), 169.9 (C, OAc), 165.2 (C, Cꢀ26), 149.6 (C,
Cꢀ24), 121.9 (C, Cꢀ25), 87.9 (C, Cꢀ17), 81.2 (C, Cꢀ14), 79.2 (CH, Cꢀ22), 78.9 (C, Cꢀ20), 72.5
(CH, Cꢀ3), 64.8 (CH2, Cꢀ18), 62.1 (C, Cꢀ5), 61.5 (CH, Cꢀ6), 57.6 (C, Cꢀ13), 56.0 (CH3, OCH3),
52.0 (C, Cꢀ10), 42.7 (CH2, Cꢀ2), 38.2 (CH2, Cꢀ16), 36.4 (CH2, Cꢀ4), 35.4 (CH, Cꢀ9), 33.9 (CH2,
Cꢀ23), 33.6 (CH, Cꢀ8), 32.9 (CH2, Cꢀ15), 26.3 (CH2, Cꢀ12), 25.2 (CH2, Cꢀ7), 21.5 (CH2, Cꢀ11),
21.3 (CH3, OAc), 20.7 (CH3, Cꢀ28), 19.2 (CH3, Cꢀ21), 13.0 (CH3, Cꢀ19), 12.4 (CH3, Cꢀ27);
HRESIꢀMS m/z 599.2829 [M+Na]+ (calcd for C31H44NaO10 599.2832).
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Epoxidation of 15αꢀHydroxyphysachenolide D. To a solution of 15αꢀ
hydroxyphysachenolide D (36)20 (5.5 mg) in anhydrous CH2Cl2 (0.5 mL) at 0 oC was added mꢀ
CPBA (4.8 mg) and stirred for 30 min. Ice bath was then removed and reaction mixture was
stirred at 25 oC for additional 2 h, after which CH2Cl2 was evaporated under reduced pressure
and the resulting residue was separated by silica gel preparative TLC using CH2Cl2/MeOH (94:6,
double elution) as eluent to give 21 (2.3 mg, 41%, Rf = 0.62) and 23 (1.3 mg, 23%, Rf = 0.54).
15αꢀHydroxyphysachenolide C (21). White amorphous solid; [α] 2D5 +68 (c 0.2,
CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.66 (1H, ddd, J = 10.1, 4.9, 2.3 Hz, Hꢀ3), 5.86 (1H, dd,
J = 10.1, 2.8 Hz, Hꢀ2), 4.80 (1H, brd, J = 12.0 Hz, Hꢀ22), 4.43 (1H, d, J = 11.3 Hz, Haꢀ18), 4.10
(1H, d, J = 11.3 Hz, Hbꢀ18), 3.92 (1H, t, J = 8.5 Hz, Hꢀ15), 3.03 (1H, dt, J = 14.5, 2.9 Hz, Haꢀ
4), 3.02 (1H, t, J = 2.8 Hz, Hꢀ6), 2.54 (1H, brd, J = 18.0 Hz, Haꢀ23), 2.39 (1H, m, Hbꢀ23), 2.38
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(1H, m, Haꢀ11), 2.29 (1H, m, Haꢀ12), 2.27 (1H, m, Haꢀ16), 2.25 (1H, m, Hꢀ9), 2.08 (2H, m, H2ꢀ
7), 2.03 (3H, s, OAc), 1.91 (1H, dd, J = 15.5, 8.5 Hz, Hbꢀ16), 1.87 (3H, s, H3ꢀ28), 1.81 (3H, s,
H3ꢀ27), 1.77 (1H, dd, J = 14.5, 4.9 Hz, Hbꢀ4), 1.75 (1H, m, Hꢀ8), 1.61 (1H, brd, J = 11.7 Hz, Hbꢀ
12), 1.29 (3H, s, H3ꢀ19), 1.28 (3H, s, H3ꢀ21), 1.17 (1H, ddd, J = 18.7, 13.2, 5.5 Hz, Hbꢀ11); 13C
NMR (100 MHz, CDCl3) 203.0 (C, Cꢀ1), 170 (C, OCOCH3), 167.2 (C, Cꢀ26), 150.9 (C, Cꢀ24),
142.6 (CH, Cꢀ3), 128.6 (CH, Cꢀ2), 121.4 (C, Cꢀ25), 84.6 (C, Cꢀ20), 80.7 (C, Cꢀ14), 79.1 (CH, Cꢀ
22), 78.4 (C, Cꢀ17), 74.0 (CH, Cꢀ15), 64.7 (C, Cꢀ5), 64.5 (CH2, Cꢀ18), 58.9 (C, Cꢀ13), 56.8 (CH,
Cꢀ6), 48.6 (C, Cꢀ10), 47.3 (C, Cꢀ16), 35.5 (CH, Cꢀ8), 33.9 (CH2, Cꢀ23), 33.8 (CH2, Cꢀ4), 31.8
(CH, Cꢀ9), 25.5 (CH2, Cꢀ12), 23.9 (CH2, Cꢀ7), 21.9 (CH2, Cꢀ11), 21.2 (CH3, OAc), 20.6 (CH3,
Cꢀ28), 15.2 (CH3, Cꢀ19), 12.1 (CH3, Cꢀ27); APCIꢀMS (+) m/z 561 [M+H]+; APCIꢀMS (–): m/z
559 [M–H]ꢀ; HRESIMS m/z 583.2541 [M+Na]+ (calcd for C30H40NaO10 583.2519).
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15αꢀHydroxyꢀ5,6ꢀepiꢀphysachenolide C (23). White amorphous solid; [α] 2D5 +73 (c
0.1, CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.81 (1H, ddd, J = 10.0, 6.1, 2.4 Hz, Hꢀ3), 6.00 (1H,
dd, J = 10.0, 2.7 Hz, Hꢀ2), 4.86 (1H, t, J = 8.5 Hz, Hꢀ22), 4.47 (1H, d, J = 11.1 Hz, Haꢀ18), 4.25
(1H, d, J = 11.1 Hz, Hbꢀ18), 4.09 (1H, m, Hꢀ15), 3.14 (1H, d, J = 2.8 Hz, Hꢀ6), 2.93 (1H, dt, J =
18.5, 3.2 Hz, Haꢀ4), 2.49 (2H, m, H2ꢀ23), 2.47 (1H, m, Haꢀ12), 2.36 (1H, m, Haꢀ16), 2.30 (1H,
m, Haꢀ7), 2.12 (1H, m, Haꢀ11), 2.10 (3H, s, OAc), 2.00 (1H, m, Hbꢀ16), 1.96 (1H, m, Hꢀ9), 1.93
(1H, m, Hꢀ8), 1.92 (3H, s, H3ꢀ28), 1.87 (3H, s, H3ꢀ27), 1.86 (1H, m, Hbꢀ4), 1.85 (1H, m, Hbꢀ7),
1.65 (1H, m, Hbꢀ12), 1.45 (1H, m, Hbꢀ11), 1.36 (3H, s, H3ꢀ21), 1.22 (3H, s, H3ꢀ19); 13C NMR
(100 MHz, CDCl3) 203.1 (C, Cꢀ1), 170 (C, OAc), 167.2 (C, Cꢀ26), 150.9 (C, Cꢀ24), 144.2 (CH,
Cꢀ3), 129.4 (CH, Cꢀ2), 121.8 (C, Cꢀ25), 84.8 (C, Cꢀ20), 80.1 (C, Cꢀ14), 79.2 (CH, Cꢀ22), 77.7
(C, Cꢀ17), 73.7 (CH, Cꢀ15), 65.3 (CH2, Cꢀ18), 61.6 (C, Cꢀ5), 63.9 (CH, Cꢀ6), 57.2 (C, Cꢀ13),
48.6 (C, Cꢀ10), 48.3 (C, Cꢀ16), 36.8 (CH, Cꢀ8), 34.6 (CH, Cꢀ9), 34.0 (CH2, Cꢀ23), 32.8 (CH2, Cꢀ
4), 26.3 (CH2, Cꢀ12), 26.0 (CH2, Cꢀ7), 22.9 (CH2, Cꢀ11), 21.3 (CH3, OAc), 20.7 (CH3, Cꢀ28),
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14.9 (CH3, Cꢀ19), 12.3 (CH3, Cꢀ27); APCIꢀMS (+) m/z 561 [M+H]+; APCIꢀMS (–) m/z 559 [M–
H]ꢀ; HRESIꢀMS m/z 583.2525 [M+Na]+ (calcd for C30H40NaO10 583.2519).
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Epoxidation of 15αꢀAcetoxyphysachenolide C (28). To a stirred solution of 28 (10.0
mg) in CHCl3 (1.0 mL) was added mꢀCPBA (6.0 mg), and the mixture was stirred at 25 oC for 4 h
(TLC control). The reaction mixture was concentrated under reduced pressure and the crude product
was purified by silica gel preparative TLC using CHCl3/MeOH (95:5) as eluent to afford epoxides 20
(3.8 mg) and 22 (2.6 mg) of which 20 was identified as 15αꢀacetoxyphysachenolide C by comparison
of its NMR and MS data with those reported.20
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15αꢀAcetoxyꢀ5,6ꢀepiꢀphysachenolide C (22). Offꢀwhite amorphous powder; [α]
25 +83 (c
D
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0.11, MeOH); H NMR (400 MHz, CDCl3) δ 6.68 (1H, ddd, J = 10.0, 4.8, 2.0 Hz, Hꢀ3), 5.91 (1H,
dd, J = 10.0, 2.0 Hz, Hꢀ2), 5.15 (1H, dd, J = 9.2, 8.8 Hz, Hꢀ15β), 4.84 (1H, m, Hꢀ22), 4.66 (1H, d, J =
11.6 Hz, Hꢀ18), 4.18 (1H, d, J = 11.6 Hz, Hꢀ18), 3.05 (1H, dt, J = 19.6, 2.4 Hz,, Hꢀ4β), 3.01 (1H, d, J
= 4.8 Hz,, Hꢀ6β), 2.13 (3H, s, 18ꢀOAc), 2.07 (3H, s, 15ꢀOAc), 1.92 (3H, s, CH3ꢀ28), 1.87 (3H, s,
CH3ꢀ27), 1.38 (3H, s, CH3ꢀ21), 1.33 (3H, s, CH3ꢀ19); 13C NMR (100 MHz, CDCl3) δ 202.4 (C, Cꢀ1),
171.3 (C, 15ꢀOAc), 170.1 (C, 18ꢀOAc), 165.5 (C, Cꢀ26), 149.9 (C, Cꢀ24), 142.2 (CH, Cꢀ3), 128.9
(CH, Cꢀ2), 121.8 (C, Cꢀ25), 85.0 (C, Cꢀ17), 79.5 (C, Cꢀ22), 79.3 (C, Cꢀ14), 79.0 (CH, Cꢀ20), 65.0
(CH2, Cꢀ18), 64.2 (C, Cꢀ5), 58.5 (CH, Cꢀ6), 57.5(C, Cꢀ13), 48.5 (C, Cꢀ10), 43.6 (CH2, Cꢀ16), 35.6
(CH, Cꢀ9), 33.9 (2xCH2, Cꢀ4 and Cꢀ23), 31.7 (CH, Cꢀ8), 25.9 (CH2, Cꢀ12), 23.2 (CH2, Cꢀ7), 21.8
(CH2, Cꢀ11), 21.5 (CH3, 15ꢀOAc), 21.3 (CH3, 18ꢀOAc), 20.6 (CH3, Cꢀ28), 19.6 (CH3, Cꢀ21), 15.2
(CH3, Cꢀ19), 12.4 (CH3, Cꢀ27); APCIꢀMS (+) m/z 603 [MH]+.
Deacetylation of Physachenolide C (3). To a solution of 3 (10.2 mg) in tꢀBuOH
(1.0 mL) and CH2Cl2 (0.4 mL) at ꢀ5 oC was added a 1.0 M solution of tꢀBuOK in tꢀBuOH (60
µL) and stirred at ꢀ5 oC. After 45 min, reaction mixture was diluted with EtOAc (15 mL) and
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washed with H2O (3 x 10 mL). Organic layer was evaporated under reduced pressure and the
residue was separated by silica gel preparative TLC using CH2Cl2/MeOH (90:10) as eluent to
give 25 (2.6 mg, 35%, Rf = 0.66) and unreacted 3 (2.2 mg, 22%, Rf = 0.72).
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18ꢀDesacetylphysachenolide C (25). White amorphous solid; [α]D25 +87 (c 0.1,
CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.80 (1H, ddd, J = 10.0, 6.4, 2.4 Hz, Hꢀ3), 6.00 (1H, dd,
J = 10.0, 2.7 Hz, Hꢀ2), 4.88 (1H, dd, J = 12.8, 4.0 Hz, Hꢀ22), 3.99 (1H, d, J = 10.8 Hz, Haꢀ18),
3.83 (1H, d, J = 10.8 Hz, Hbꢀ18), 3.16 (1H, brs, Hꢀ6), 2.92 (1H, dt, J = 18.8, 2.8 Hz, Haꢀ4), 2.63
(1H, m, Haꢀ16), 2.51 (1H, m, Haꢀ23), 2.43 (1H, m, Hbꢀ23), 2.17 (1H, dt, J = 12.5, 2.8 Hz, Haꢀ
12), 2.08 (1H, m, Haꢀ11), 1.98ꢀ1.92 (2H, m, H2ꢀ7), 1.91 (3H, s, H3ꢀ28), 1.90 (1H, m, Hꢀ9), 1.87
(3H, s, H3ꢀ27), 1.84 (2H, m, Hbꢀ4 and Hbꢀ12), 1.75 (1H, dt, J = 11.6, 2.4 Hz, Hꢀ8), 1.62ꢀ1.58
(3H, m, H2ꢀ15, Hbꢀ16), 1.53 (1H, dt, J = 14.5, 2.1 Hz, Hbꢀ11), 1.38 (3H, s, H3ꢀ21), 1.22 (3H, s,
H3ꢀ19); 13C NMR (100 MHz, CDCl3) 202.9 (C, Cꢀ1), 165.8 (C, Cꢀ26), 150.3 (C, Cꢀ24), 143,8
(CH, Cꢀ3), 129.8 (CH, Cꢀ2), 122.6 (C, Cꢀ25), 89.2 (C, Cꢀ20), 81.2 (C, Cꢀ14), 79.3 (CH, Cꢀ22),
78.8 (C, Cꢀ14), 64.1 (CH, Cꢀ6), 63.6 (CH2, Cꢀ18), 62.2 (C, Cꢀ5), 58.7 (C, Cꢀ13), 48.5 (C, Cꢀ10),
37.5 (CH2, Cꢀ16), 36.8 (CH, Cꢀ9), 34.2 (CH2, Cꢀ23), 34.0 (CH, Cꢀ8), 33.3 (CH2, Cꢀ15), 32.6
(CH2, Cꢀ4), 26.3 (CH2, Cꢀ7), 24.6 (CH2, Cꢀ12), 22.7 (CH2, Cꢀ11), 20.7 (CH3, Cꢀ28), 19.0 (CH3,
Cꢀ21), 14.7 CH3, Cꢀ19), 12.4 CH3, Cꢀ27); HRESIꢀMS m/z 525.2461 [M+Na]+ (calcd for
C28H38NaO8 525.2464).
Epoxidation of Physachenolide D (4). To a stirred solution of 4 (20.0 mg) in CHCl3 (2.0
mL) was added mꢀCPBA (12.0 mg) and the mixture was stirred at 25 oC for 4 h (TLC control). The
reaction mixture was concentrated under reduced pressure and the product mixture was separated by
C18 RP HPLC (60% aq. MeOH, 3 mL/min, UV detection at 230 nm) to afford physachenolide C (3)
(8.5 mg, tR = 23.4 min) and a mixture of two compounds (tR = 17.7 min). This mixture (tR = 17.7
min) was further separated by silica gel preparative TLC using CHCl3/MeOH (92:8) as eluent to
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afford 19 (5.0 mg) and 26 (1.6 mg). Compounds 3 and 19 were identified by comparison of their
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24α,25αꢀEpoxyphysachenolide C (26). Offꢀwhite amorphous powder; [α]
25 +54 (c 0.85,
D
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MeOH); H NMR (400 MHz, CDCl3) δ 6.80 (1H, ddd, J = 2.4, 6.4, 10.0 Hz, Hꢀ3), 6.01 (1H, dd, J =
2.8, 10 Hz, Hꢀ2), 5.07 (1H, dd, J = 2.8, 12.0 Hz, Hꢀ22), 4.39 (1H, d, J = 11.6 Hz, Hꢀ18), 4.32 (1H, d,
J = 11.6 Hz, Hꢀ18), 3.15 (1H, brs, Hꢀ6α), 2.93 (1H, dt, J = 18.8, 2.4 Hz, Hꢀ4), 2.64 (1H, m, Hꢀ23),
2.58 (1H, dd, J = 15.2, 2.8 Hz, Hꢀ23), 2.10 (3H, s, 18ꢀOAc), 1.56 (3H, s, CH3ꢀ28), 1.45 (3H, s, CH3ꢀ
27), 1.29 (3H, s, CH3ꢀ21), 1.22 (3H, s, CH3ꢀ19); 13C NMR (100 MHz, CDCl3) δ 202.8 (C, Cꢀ1), 170.1
(C, OAc), 168.9 (C, Cꢀ26), 143.7 (CH, Cꢀ3), 129.7 (CH, Cꢀ2), 87.9 (C, Cꢀ17), 81.5 (C, Cꢀ14), 78.7
(CH, Cꢀ22), 77.4 (C, Cꢀ20), 64.9 (CH2, Cꢀ18), 63.8 (CH, Cꢀ6), 62.8 (C, Cꢀ25), 62.1 (C, Cꢀ24), 59.4
(C, Cꢀ5), 57.5 (C, Cꢀ13), 48.5 (C, Cꢀ10), 38.1 (CH2, Cꢀ16), 36.9 (CH, Cꢀ9), 34.2 (CH, Cꢀ8), 32.9
(CH2, Cꢀ4), 32.8 (CH2, Cꢀ15), 29.7 (CH2, Cꢀ23), 26.4 (CH2, Cꢀ7), 25.4 (CH2, Cꢀ12), 22.8 (CH2, Cꢀ
11), 21.3 (CH3, OAc), 18.8 (CH3, Cꢀ21), 17.9 (CH3, Cꢀ28), 14.6 (CH3, Cꢀ19), 13.5 (CH3, Cꢀ27);
HRESIꢀMS m/z 583.2511 [M+Na]+ (calcd for C30H40O10Na, 583.2519).
Preparation of 15α,28ꢀDiacetoxyphysachenolide D (32). Acetylation of 15αꢀacetoxyꢀ
28ꢀhydroxyphysachenolide D (30) by the general procedure afforded 32 as an offꢀwhite amorphous
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powder;
[
α
]
25 +61 (c 0.02, MeOH); H NMR (400 MHz, CDCl3) δ 6.74 (1H, ddd, J = 10.0, 5.2, 2.8
D
Hz, Hꢀ3), 5.84 (1H, dd, J = 10.0, 2.0 Hz, Hꢀ2), 5.52 (1H, d, J = 6.0 Hz, Hꢀ6), 5.20 (1H, dd, J = 9.2,
8.4 Hz, Hꢀ15β), 4.89 (1H, brd, J = 12.8 Hz, Hꢀ22), 4.77 (1H, d, J = 13.6 Hz, Hꢀ28), 4.73 (1H, d, J =
13.6 Hz, Hꢀ28), 4.69 (1H, d, J = 11.6 Hz, Hꢀ18), 4.21 (1H, d, J = 11.6 Hz, Hꢀ18), 3.24 (1H, brd, J =
21.6 Hz,, Hꢀ4β), 2.80 (1H, dd, J = 21.2, 4.8 Hz,, Hꢀ4α), 2.14 (3H, s, 18ꢀOAc), 2.08 (3H, s, 27ꢀOAc),
2.07 (3H, s, 15ꢀOAc), 1.95 (3H, s, CH3ꢀ27), 1.40 (3H, s, CH3ꢀ21), 1.21 (3H, s, CH3ꢀ19); APCIꢀMS
(+) m/z 667 [M+Na]+.
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Preparation of 15α,27ꢀDiacetoxyphysachenolide D (33). Acetylation of 15αꢀ
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amorphous powder;
[
α]
25 +51 (c 0.04, MeOH); H NMR (400 MHz, CDCl3) δ 6.74 (1H, ddd, J =
D
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10.0, 5.2, 2.8 Hz, Hꢀ3), 5.84 (1H, dd, J = 10.0, 2.0 Hz, Hꢀ2), 5.52 (1H, d, J = 5.6 Hz, Hꢀ6), 5.18 (1H,
dd, J = 8.8, 8.4 Hz, Hꢀ15β), 4.92 (1H, m, Hꢀ22), 4.89 (1H, d, J = 12.0 Hz, Hꢀ27), 4.85 (1H, d, J =
12.0 Hz, Hꢀ27), 4.73 (1H, d, J = 12.0 Hz, Hꢀ18), 4.23 (1H, d, J = 11.6 Hz, Hꢀ18), 3.24 (1H, brd, J =
21.2 Hz,, Hꢀ4β), 2.80 (1H, dd, J = 21.2, 4.8 Hz,, Hꢀ4α), 2.15 (3H, s, 18ꢀOAc), 2.08 (3H, s, 15ꢀOAc),
2.06 (6H, s, 27ꢀOAc and CH3ꢀ28), 1.39 (3H, s, CH3ꢀ21), 1.21 (3H, s, CH3ꢀ19); APCIꢀMS (+) m/z 667
[M+Na]+.
TRAILꢀSensitization Assay. The renal cancer cell lines ACHN, Cakiꢀ1, SN12C,
TK10 and UOꢀ31 were obtained from the NCI Frederick. Cells were plated at 5000 cells per well
in 96 well flatꢀbottomed microtiter plates. Plates were incubated overnight at 37 oC. Cells were
treated with compound for 2 h, 50 ng/mL of TRAIL was then added to appropriate wells and the
plates were incubated for a further 18 h at 37 oC. Viable cell number was determined by the
addition of CellTiter 96® AQueous One Solution Cell Proliferation Assay solution (MTS) and
plates were incubated for 2 h and then absorbance at 490 nM was measured. Growth inhibition
was calculated as in the following GI =[(MediaꢀTreatment)/Media)]*100. The assay was
performed in RPMI with 5% FCS, 2.0 mM Lꢀglutamine, 1 x nonessential amino acids, 1.0 mM
sodium pyruvate, 100 U/mL penicillin, 100
2ꢀmercaptoethanol.
ꢀ
g/mL streptomycin, 10 mM HEPES and 5 x 10ꢀ5 M
Immunoblotting. ACHN cells were grown in RPMI (RPMI, 5% FBS, Penꢀstrep,
NEAA, HEPES, Glutamax, Sodium Pyruvate, 2ME), 1.5 x 106 cells/well were plated in Costar
6ꢀwell plates and then incubated overnight at 37 oC . On day 2 compounds were added, followed
by overnight incubation. On day 3 either cells were treated with media or TRAIL (200 ng/mL)
29
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