Strongylophorine-26, an inhibitor of cancer cell invasion: SAR revealed by synthesis of analogues

Article abstract of DOI:10.1021/np060481l

The absolute configuration of strongylophorine-26 (1) was determined to be 4S, 5R, 8R, 9S, 10S, 13S, 14S by single-crystal X-ray diffraction analysis of the derivative 7 prepared from the co-occurring metabolite strongylophorine-8 (4) and chemical interconversion to the bislactone 8. Synthetic analogues (+)- and (-)-3 have been prepared in order to explore the structure-activity relationship for the anti-invasion pharmacophore of stronglylophorine-26. These studies revealed the unanticipated importance of the A ring lactone moiety for the anti-invasion activity of 1.

Full text of DOI:10.1021/np060481l

736
J. Nat. Prod. 2007, 70, 736-740
Strongylophorine-26, an Inhibitor of Cancer Cell Invasion: SAR Revealed by Synthesis of
Analogues
,§
Kaoru Warabi,† Brian O. Patrick,‡ Pamela Austin, Calvin D. Roskelley, Michel Roberge,§ and Raymond J. Andersen*,†
Departments of Chemistry, Earth & Ocean Sciences, Cellular & Physiological Sciences, and Biochemistry & Molecular Biology, UniVersity of
British Columbia, VancouVer, B.C. V6T 1Z1, Canada
ReceiVed September 30, 2006
The absolute configuration of strongylophorine-26 (1) was determined to be 4S, 5R, 8R, 9S, 10S, 13S, 14S by single-
crystal X-ray diffraction analysis of the derivative 7 prepared from the co-occurring metabolite strongylophorine-8 (4)
and chemical interconversion to the bislactone 8. Synthetic analogues (+)- and (-)-3 have been prepared in order to
explore the structure-activity relationship for the anti-invasion pharmacophore of stronglylophorine-26. These studies
revealed the unanticipated importance of the A ring lactone moiety for the anti-invasion activity of 1.
The ability of cells to invade adjacent tissues is a biological
phenomenon that is crucial for angiogenesis and metastasis. Under
the control of angiogenic factors produced by tumor cells, vascular
endothelial cells invade solid tumor masses to establish new blood
vessels that supply nutrients and oxygen to tumor cells. These newly
formed blood vessels can also provide a conduit for metastatic
spread, a process that requires the tumor cells themselves to become
invasive. Thus, inhibitors of cell invasion may be useful in cancer
therapy by preventing both neoangiogenesis and metastasis.1 In the
course of our ongoing search for anti-invasive natural products from
marine organisms, strongylophorine-26 (1) was found in the
lipophilic extract of the marine sponge Petrosia (Strongylophora)
corticata as an inhibitor of cell invasion by MDA-MB-231 breast
carcinoma cells.2 Investigation of the biological mechanism revealed
that 1 induces nonpolarized lamellipodial extensions, resulting in
inhibition of cell motility, and that the anti-invasive activity of 1
depends on the small GTPase Rho.3 Other inhibitors of cell
migration such as fumagillin, TNP-470, thronbospondin-1, endosta-
tin, EMAP II, and dihydromotuporamine C4,5 all cause an induction
of actin stress fibers. By contrast, 1 decreases actin stress fibers
and increases focal adhesions while causing a dense meshwork of
actin filaments to form at the cell periphery, indicating a distinct
mechanism of action.
Our interest in identifying the structural features responsible for
this unique cellular response and the molecular target of the
compound required the availability of larger amounts of 1 than were
available from the natural sponge source, so we turned to synthesis
to provide the material. At the outset, our hypothesis was that the
quinone substructure was an essential part of the anti-invasive
pharmacophore, that the diterpenoid skeleton and the tertiary alcohol
at C-13 were also likely important, but that the A ring lactone was
probably not critical. On the basis of this hypothesis, the strongy-
lophorine-26 analogue 3 was chosen as the synthetic target. This
analogue had the advantage of being much easier to synthesize than
1 because installation of the A ring lactone was viewed as a
significant challenge. As part of our synthetic efforts, we planned
to investigate the relative bioactivities of the two enantiomers of 3
to determine if the configuration of the terpenoid fragment was
important. In order to know which enantiomer of 3 corresponded
to the natural product, it was necessary to first determine the
absolute configuration of strongylophorine-26 (1). Herein, we
describe the determination of the absolute configuration of 1, the
synthesis of its analogues (+)- and (-)-3, and some unanticipated
SAR for its anti-invasive pharmacophore.
Results and Discussion
The absolute configuration of 1 was determined via chemical
degradation and interconversion experiments using strongylopho-
rine-8 (4), which was isolated along with 1 from P. corticata.2
Treatment of 4 with tert-butyldimethylsilyl chloride followed by
benzyl bromide yielded 7 as needle-like crystals (Scheme 1). Single-
crystal X-ray diffraction analysis of 7 revealed that its relative
configuration was 4S*, 5R*, 8R*, 9S*, 10S*, 13S*, and 14S*
(Supporting Information). The absolute configuration of 7 was
determined on the basis of the refined Flack parameter value, 0.12-
(9).6 Ideally this value should be 0 for the correct configuration or
1 for its enantiomer. Values between 0 and 1 may indicate racemic
twinning; however, in this case modeling the structure as a racemic
twin did not improve the refinement (i.e., R1 values) at all. The
slightly elevated Flack value is likely due to weak anomalous
dispersion factors for the heaviest atom, Si. Therefore, the absolute
configuration of 4 is 4S, 5R, 8R, 9S, 10S, 13S, and 14S. Since
strongylophorine-8 (4) and strongylophorine-26 (1) come from the
same extract and they have identical diterpenoid fragments, it was
assumed that they had the same absolute configuration. In order to
confirm this assumption, strongylophorine-8 (4) was oxidatively
degraded by treatment with RuCl3 and NaIO4, yielding the optically
* Corresponding author. Tel: 604 822 4511. Fax: 604 822 6091.
E-mail: randersn@interchange.ubc.ca.
† Chemistry and EOS.
23
active bislactone 8 ([R]D -46 (c 0.13 CH2Cl2)). When strongy-
lophorine-26 (1) was chemically degraded using the same RuCl3
and NaIO4 treatment, the reaction product was identical by 1H NMR
comparison. The small amount of bislactone 8 obtained from 1
‡ Chemistry.
Cellular & Physiological Sciences.
§ Biochemistry & Molecular Biology.
10.1021/np060481l CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/04/2007

Inhibitor of Cancer Cell InVasion
Journal of Natural Products, 2007, Vol. 70, No. 5 737
Scheme 1a
Analysis of COSY, HMQC, and HMBC correlations for 3 indicated
the existence of a tautomeric equilibrium between the open form 3
and the two cyclic forms 3a/b (Scheme 4).9 The open tautomer,
the major cyclic form, and the minor cyclic form were present in
the ratio 6.1:2.2:1, respectively. Since the same equilibrium was
earlier observed for strongylophorine-26 (1), it appears that the
absence of the A ring γ-lactone of 1 does not affect the reactivity
of the quinone moiety.
In the CD spectrum, (+)-3 showed a positive Cotton effect
around 280 nm, which can be attributed to the configuration of the
chiral centers around the quinone moiety (Supporting Information).
Since strongylophorine-26 (1) also exhibited a positive Cotton effect
around 280 nm in the CD spectrum (Supporting Information), (+)-3
must have the same absolute configuration as 1, while (-)-3 must
be the antipode.
a
Reagents and conditions: (a) TBDMSCl, i-Pr2NEt, pyridine, THF, rt, 59%;
(b) BnBr, NaH, THF, 0 °C to rt, 81%; (c) RuCl3, NaIO4, CCl4, CH3CN, H2O,
In the initial study of the structure and bioactivity of strongy-
lophorine (1), it was found that strongylophorine-8 (4) and the
corresponding quinone 2 were active in the cell-based anti-invasion
assay, but with IC50’s roughly an order of magnitude greater than
1, while ilimaquinone (5) was completely inactive.2 It was assumed
that the similar activities of 2 and 4 resulted from the hydroquinone
4 being oxidized to the quinone 2 during the assay. On the basis of
relative potencies of 1 and 2, it was apparent that the methoxy
substituent on the quinone in 1 enhanced the anti-invasive activity.
The lack of activity in ilimaquinone (5) seemed to suggest that the
structure of the terpenoid fragment in 1 and 2 was also important.
When the anti-invasive activity of synthetic (+)- and (-)-3 was
examined in the cell-based assay using MDA-MB-231 cells, it was
found that neither enantiomer showed anti-invasion activity at
concentrations below their IC50’s for cytotoxicity (≈0.3 µg/mL for
both enantiomers). This result suggests that removal of the A ring
lactone from 1 has diminished its selectivity for binding to the anti-
invasion molecular target, and as a consequence, the synthetic
analogues 3 display only cytotoxicity to cancer cells. Therefore, in
strongylophorine-26 (1) the A ring lactone and the methoxy-
substituted quinone moieties are both indispensable components
of its anti-invasion pharmacophore, in contrast to our initial
hypothesis.
rt.
Scheme 2a
a
Reagents and conditions: (a) Br2, MeOH, rt; (b) H2O2, NaOH, H2O, rt; (c)
BOMCl, DMAP, i-Pr2NEt, CH2Cl2, rt, 27% for 2 steps.
precluded an accurate determination of the magnitude of its specific
rotation; however, it was possible to ascertain that it had the same
sign of optical rotation as the bislactone 8 obtained from strongy-
lophorine-8 (4). Therefore, strongylophorine-26 (1) has been
assigned the absolute configuration 4S, 5R, 8R, 9S, 10S, 13S, and
14S.
A biomimetic approach was adopted for the synthesis of the
strongylophorine analogue 3 missing the A ring lactone. The
tricyclic diterpenoid fragment of 3 was constructed from gera-
nylgeranylacetate (12) via polyene cyclization, while the quinone
moiety was synthesized from vanillin (9) (Schemes 2 and 3).
Bromination of vanillin (9) was carried out as described in the
literature,7 yielding 5-bromovanillin (10). Baeyer-Villiger oxidation
of 10 under alkaline conditions generated the bromohydroquinone,
whose phenolic groups were subsequently protected with BOM,
affording the bromobenzene 11.
The racemic diol 13 was synthesized following a literature
precedent8 involving cyclization of geranylgeranylacetate (12) with
mercury ditrifluoroacetate and N,N-dimethylaniline, followed by
treatment with KBr, and reduction in sequence (Scheme 3). The
primary hydroxyl group of 13 was protected as a TBDMS ether,
while the tertiary hydroxyl group was protected with BOM.
Deprotection of the primary hydroxyl group of 15 with tetrabuty-
lammonium fluoride followed by PCC oxidation yielded the
aldehyde 17, the precursor for the subsequent coupling reaction.
Treatment of 11 with tert-butyllithium generated the correspond-
ing phenyllithium reagent, which was coupled with the aldehyde
17 to furnish a mixture of the alcohols 18a and 18b (C-15 epimers)
in the ratio 1:1. After acylation of the secondary hydroxyl groups
of 18a and 18b with trifluoroacetic anhydride, the resulting ester
mixture was hydrogenated to afford the hydroquinone, which
spontaneously oxidized to the quinone 3 in air. In order to obtain
the individual enantiomers of 3, chiral HPLC separation of one of
the epimers of 18 was performed on Chiralpak IA (DAICEL),
resulting in two distinct peaks corresponding to (+)- and (-)-18a.
Acylation of (+)- and (-)-18a followed by hydrogenation in a
similar manner to racemic 18 afforded (+)- and (-)-3, respectively.
The 1H NMR spectrum of quinone 3 showed two sets of minor
signals in the regions of δ 2.35-2.75, 3.70-3.72, and 5.23-5.94,
which mirrored the 1H NMR data of strongylophorine-26 (1).
Experimental Section
General Experimental Procedures. Synthetic reagents were ob-
tained from commercial sources and were used without further
purification. THF was distilled from sodium/benzophenone under Ar
gas before use. CH2Cl2 was distilled from CaH2 under N2 gas. Chemical
reactions were monitored on Si gel 60 F-254 precoated aluminum plates
(Merk). Normal-phase chromatography was carried out on 120-230
mesh Si gel. Measurement of melting points was performed on a Fisher-
Johns melting point apparatus. Optical rotations were measured with a
JASCO P-1010 polarimeter. UV absorptions were recorded on a Waters
2487 dual λ absorbance detector. CD spectra ware obtained using a
JASCO J-700 spectropolarimeter. Proton and carbon NMR spectra were
obtained using Bruker AM-400, AMX-500, or AV-600 spectrometers.
ESIMS and EIMS spectra were obtained using an ESI micromass LCT
spectrometer and a Kratos MS-50 mass spectrometer, respectively.
Compound 6. Strongylophorine-8 (4) (22.9 mg, 53.8 µmol) was
dissolved in a mixed solvent system of THF (0.5 mL), N,N-diisopro-
pylethylamine (1.5 mL), and pyridine (2.5 mL). To this solution,
t-BuMe2SiCl (157.8 mg, 1.047 mmol) was added. After the reaction
solution was stirred at room temperature for 12 h, additional t-BuMe2-
SiCl (70.0 mg, 0.464 mmol) was added and the reaction solution was
stirred for an additional 7.5 h. The reaction was quenched with saturated
aqueous NaHCO3, and the resulting solution was extracted with EtOAc.
The organic layer was washed with brine and dried over MgSO4, passed
through a cotton filter to remove MgSO4, and concentrated in vacuo.
The residue was purified on a Si gel column, eluted in a stepwise
gradient manner with EtOAc/hexanes (0:100 to 40:60) to yield 6 (17.1
mg, 59% yield) as a colorless solid: 1H NMR (DMSO-d6) δ 9.16 (s,
17-OH), 6.69 (d, J ) 2.8 Hz, H21), 6.53 (d, J ) 8.6 Hz, H18), 6.44
(dd, J ) 2.8, 8.6 Hz, H19), 5.36 (s, 13-OH), 4.57 (d, J ) 12, H24),
3.97 (d, J ) 12 Hz, H24), 2.60 (d, J ) 15 Hz, H15), 2.48 (m, H15),

738 Journal of Natural Products, 2007, Vol. 70, No. 5
Warabi et al.
Scheme 3a
a
Reagents and conditions: (a) Hg(OTf)2, N,N-dimethylaniline, MeNO2, then KBr.; (b) NaOH, NaBH4, EtOH/CH2Cl2/H2O (5:5:2); (c) TBDMSCl, DMAP, Et3N,
CH2Cl2, rt, 62%; (d) BOMCl, DMAP, i-Pr2NEt, CH2Cl2, rt, 95%; (e) TBAF, THF, 60-70 °C, 96%; (f) PCC, AcONa, CH2Cl2; (g) 11, t-BuLi, THF, -78 °C, 65%
(18a:18b ) 1:1); (h) H2, Pd(OH)2, EtOH/AcOEt (1:4), rt; (i) [O], 50% for 2 steps from 18a.
remove MgSO4, the filtrate was concentrated in vacuo. The residue
was subjected to Si gel column chromatography, eluted in a stepwise
gradient manner with EtOAc/hexanes (0:100 to 40:60) to afford 7 (16.2
mg, 81% yield) as colorless needles: mp 195-196 °C; HRESIMS [M
+ Na]+ m/z 655.3781 (C39H56O5NaSi, calcd 655.3795).
Scheme 4. Possible Tautomeric Equibilium of 3
Compound 8 from Strongylophorine-8 (4). To strongylophorine-8
(4) (17.5 mg, 41.4 mmol) in a mixed solvent system of CCl4 (0.5 mL),
CH3CN (0.5 mL), and H2O (0.5 mL) were added catalytic amounts of
NaIO4 (167.1 mg, 0.781 mmol) and RuCl3. The reaction solution was
stirred at 40 °C for 25 h and then diluted with H2O (10 mL). The
resulting solution was extracted with Et2O, and the organic layer was
concentrated in vacuo. The residue was fractionated with a Sepak
(Waters Sepak 12 cm3 Si gel 2 g) eluted using a stepwise gradient of
EtOAc/hexanes (40:60 to 100:0). The fraction containing 8 was
subjected to normal-phase HPLC (Alltech Econosil Si 5 µm) eluted
with EtOAc/hexanes (44:56) to afford 8 (0.2 mg, 1% yield) as a
23
1
colorless solid: [R]D -46 (c 0.13, CH2Cl2); H NMR (CD2Cl2) δ
4.74 (dd, J ) 2.0, 12 Hz, H-19), 4.03 (d, J ) 12 Hz, H-19), 2.43 (dd,
J ) 15, 16 Hz, H-15), 2.23 (dd, J ) 7.0, 16 Hz, H-15), 2.12 (m, H-1),
2.10 (m, H-12), 2.03 (m, H-11), 1.97 (dd, J ) 7.0, 15, H-14), 1.82 (m,
H-3), 1.78 (m, H-6), 1.72 (m, H-2), 1.68 (m, H-12), 1.65 (m, H-2),
1.57 (m, H-7), 1.52 (m, H-3), 1.36 (m, H-6), 1.33 (m, H-5), 1.33 (s,
H-17), 1.24 (m, H-9), 1.24 (m, H-11), 1.18 (m, H-7), 1.16 (m, H-1),
1.16 (s, H-20), 1.05 (s, H-18); HREIMS m/z 346.21465 (C21H30O4, calcd
246.21441).
Compound 8 from Strongylophorine-26 (1). Chemical degradation
of strongylophorine-26 (1) (3.5 mg, 7.7 µmol) in a similar manner to
strongylophorine-8 described above yielded 8 (0.1 mg, 4% yield).
2.08 (d, J ) 12, H1), 1.81 (d, J ) 13 Hz, H12), 1.73 (d, J ) 14 Hz,
H7), 1.69 (d, J ) 13 Hz, H11), 1.63 (m, H2), 1.61 (m, H3), 1.49 (m,
H6), 1.47 (m, H14), 1.43 (m, H12), 1.43 (m, H2), 1.41 (m, H3), 1.19
(m, H5), 1.16 (m, H6), 1.13 (s, H22), 1.03 (m, H1), 1.01 (m, H9), 1.00
(s, H25), 0.99 (s, H23), 0.92 (s, H4′, H5′, H6′), 0.62 (dd, J ) 10, 10
Hz, H7), 0.12 (s, H2′), 0.12 (s, H1′); HRESIMS [M + Na]+ m/z
565.3329 (C32H50O5NaSi, calcd 565.3325).
Compound 7. To compound 6 (17.1 mg, 31.5 µmol) in THF (2.0
mL) on an ice bath were added NaH (60% dispersion in mineral oil,
101.4 mg) and benzyl bromide (0.5 mL, 4.2 mmol). The reaction
solution was stirred at room temperature for 16 h. The reaction was
quenched with saturated aqueous NaHCO3 on an ice bath, and then
the solution was extracted with EtOAc. The organic layer was washed
with saturated aqueous NH4Cl and brine, successively, and then dried
over MgSO4. After the solution was passed through a cotton filter to
Compound 11. To the compound 10 (1.5244 g, 0.6980 mmol) in
H2O (25 mL) were added KOH (587 mg, 10.5 mmol) in H2O (3.8 mL)
and aqueous H2O2 (50% (w/w), 1.7 mL). The reaction solution was
stirred at 90 °C for 5.3 h. After being cooled to room temperature, the
reaction solution was acidified with aqueous HCl and then extracted
with Et2O. The organic layer was concentrated under reduced pressure
and then dried in vacuo. To the residue were added 4-(dimethyamino)-
pyridine (5.1 mg, 0.041 mmol), CH2Cl2 (16.0 mL), and N,N-diisopro-
pylethylamine (10.0 mL). After sonication for a few minutes, benzyl
chloromethyl ether (techn. ca. 60%, 5.4 mL) was added to the solution.
The reaction solution was stirred at room temperature for 4.5 h. After
quenching with saturated aqueous NaHCO3, the solution was extracted
with Et2O. The organic layer was washed with brine and then
concentrated under reduced pressure. The residue was subjected to Si

Inhibitor of Cancer Cell InVasion
Journal of Natural Products, 2007, Vol. 70, No. 5 739
gel chromatography, eluted in a stepwise gradient manner with EtOAc/
hexanes (0:100 to 13:87) to furnish 11 (805 mg, 27% yield for 2 steps)
as a colorless oil: 1H NMR (acetone-d6) δ 7.33-7.27 (10H, H4′/H5′/
H6′/H7′/H8′/H4′′/H5′′/H6′′/H7′′/H8′′), 6.92 (d, J ) 2.7 Hz, 1H, H6),
6.77 (d, J ) 2.7 Hz, 1H, H4), 5.33 (s, 2H, H1′′), 5.21 (s, 2H, H1′),
4.94 (s, 2H, H2′), 4.74 (s, 2H, H2′′), 3.83 (s, 3H, H7); HREIMS m/z
column and eluted with Et2O. The filtrate was concentrated under
reduced pressure. The residue (102 mg) was used for the next reaction
without purification.
To the aromatic compound 11 in THF (3.0 mL) at -78 °C was
added dropwise 1.7 M t-BuLi/pentane (0.3 mL). The solution was stirred
at -78 °C for 30 min. To the resulting solution was added dropwise
the residue containing 17 (102 mg) in THF (2.0 mL), and the solution
was stirred at -78 °C for 3 h. The reaction was quenched with saturated
aqueous NH4Cl, and the solution was stirred at room temperature
overnight. After addition of brine to the quenched reaction solution,
the resulting solution was extracted with Et2O, and the organic layer
was concentrated under reduced pressure. The residue was purified by
Si gel column chromatography eluted in a stepwise manner with EtOAc/
hexanes (0:100 to 20:80) to furnish 18a (19 mg, 32% yield in 2 steps)
and 18b (20 mg, 33% yield for 2 steps). Compound 18a: colorless
oil; 1H NMR (C6D6) δ 7.37 (d, J ) 2.7 Hz, 1H, H21), 7.36 (d, J ) 7.1
Hz, 2H, H4′, H8′), 7.29 (d, J ) 7.2 Hz, 2H, H4′′, H8′′), 7.24 (d, J )
7.1 Hz, 2H, H4′′′, H8′′′), 7.18-7.04 (9H, H5′, H6′, H7′, H5′′, H6′′,
H7′′), 6.59 (d, J ) 2.7, 1H, H19), 5.96 (dd, J ) 3.5, 5.7 Hz, 1H, H15),
5.28 (d, J ) 5.6 Hz, 1H, H1′′), 5.22 (d, J ) 5.6 Hz, 1H, H1′′), 5.11 (d,
J ) 7.0 Hz, 1H, H1′′′), 5.09 (d, J ) 7.0 Hz, 1H, H1′′′), 4.84 (d, J )
12 Hz, 1H, H2′), 4.76 (d, J ) 12 Hz, 1H, H2′), 4.72 (d, J ) 7.6 Hz,
1H, H1′), 4.60 (s, 2H, H2′′), 4.58 (s, 2H, H2′′′), 4.28 (d, J ) 7.6 Hz,
1H, H1′), 3.21 (s, 3H, H27), 3.06 (d, J ) 5.7 Hz, 1H, 15-OH), 2.74 (d,
J ) 12 Hz, 1H, H7), 2.39 (d, J ) 3.5 Hz, 1H, H14), 2.01 (d, J ) 13
Hz, 1H, H12), 1.73 (ddd, J ) 2.8, 13, 13 Hz, 1H, H12), 1.62 (s, 3H,
H22), 1.52 (m, 1H, H1), 1.49 (m, 1H, H11), 1.41 (m, 1H, H6), 1.41 (s,
3H, H23), 1.33 (m, 1H, H6), 1.30 (m, 1H, H3), 1.29 (m, 1H, H7), 1.21
(m, 1H, H11), 1.07 (ddd, J ) 3.1, 13, 13 Hz, 1H, H3), 0.86 (m, 1H,
H9), 0.80 (s, 3H, H24), 0.77 (s, 3H, H25), 0.71 (s, 3H, H26), 0.67 (dd,
J ) 2.5, 11 Hz, 1H, H5), 0.63 (ddd, J ) 2.9, 13, 13 Hz, 1H, H1);
HRESIMS [M + Na]+ m/z 829.4656 (C51H66O8Na, calcd 829.4655).
Compound 18b: colorless oil; 1H NMR (C6D6) δ 7.59 (d, J ) 2.7 Hz,
1H, H21), 7.47 (d, J ) 7.1 Hz, 2H, H4′′, H8′′), 7.38 (d, J ) 7.1 Hz,
2H, H4′, H8′), 7.23-7.03 (9H, H5′, H6′, H7′, H5′′, H6′′, H7′′), 7.19
(2H, H4′′′, H8′′′), 6.64 (d, J ) 2.7 Hz, 1H, H19), 5.80 (dd, J ) 6.3, 10
Hz, 1H, H15), 5.59 (d, J ) 10 Hz, 1H, H1′′), 5.46 (d, J ) 10 Hz, 1H,
H1′′), 5.16 (d, J ) 12 Hz, 1H, H2′′), 5.14 (d, J ) 7.1 Hz, 1H, H1′′′),
5.10 (d, J ) 7.1 Hz, 1H, H1′′′), 5.08 (d, J ) 12 Hz, 1H, H2′′), 4.82 (d,
J ) 7.6 Hz, 1H, H1′), 4.73 (d, J ) 10 Hz, 1H, 15-OH), 4.73 (d, J )
7.6 Hz, 1H, H1′), 4.67 (d, J ) 12 Hz, 1H, H2′), 4.60 (d, J ) 12 Hz,
1H, H2′), 4.55 (s, 2H, H2′′′), 2.45 (d, J ) 6.3 Hz, 1H, H14), 2.05 (m,
1H, H7), 1.94 (ddd, J ) 2.7, 3.3, 12 Hz, 1H, H12), 1.72 (s, 3H, H22),
1.65 (ddd, J ) 2.7, 12, 13 Hz, 1H, H12), 1.51 (m, 1H, H11), 1.45 (dd,
J ) 13, 13 Hz, 1H, H1), 1.44 (m, 1H, H2), 1.30 (m, 1H, H11), 1.24
(m, 1H, H3), 1.20 (m, 1H, H6), 1.14 (s, 3H, H23), 1.13 (m, 1H, H6),
1.09 (m, 1H, H2), 0.99 (ddd, J ) 4.1, 14, 14 Hz, H3), 0.73 (m, 1H,
H7), 0.72 (m, 1H, H8), 0.71 (s, 3H, H26), 0.70 (s, 3H, H24), 0.56 (m,
1H, H1), 0.56 (s, 3H, H25), 0.45 (m, 1H, H5); HRESIMS [M + Na]+
m/z 829.4659 (C51H66O8Na, calcd 829.4655).
460.07127 (C23H23O581Br, calcd 460.07084), m/z 458.07291 (C23H23O579
-
Br, calcd 458.07289).
Compound 14. To compound 13 (48.0 mg, 0.156 mmol) in CH2-
Cl2 (4.0 mL) were added 4-(dimethylamino)pyridine (8.3 mg, 0.068
mmol), triethylamine (4.0 mL), and t-BuMe2SiCl (473 mg, 3.14 mmol).
The reaction solution was stirred at room temperature for 2 h, and then
additional t-BuMe2SiCl (146 mg, 0.967 mmol) was added to the
solution. After 1 h, the reaction was quenched with saturated aqueous
NaHCO3 and brine. The resulting mixture was extracted with Et2O,
and the organic layer was concentrated under reduced pressure. The
residue was subjected to Si gel column chromatography eluted in a
stepwise gradient manner with Et2O/hexanes (0:100 to 10:90) to furnish
14 (40.5 mg, 62% yield) as a colorless solid: 1H NMR (CDCl3) δ
4.00 (dd, J ) 10, 10 Hz, 1H, H15), 3.89 (dd, J ) 3.9, 10 Hz, 1H,
H15), 1.85 (ddd, J ) 3.4, 3.4, 13 Hz, 1H, H12), 1.77 (ddd, J ) 3.5,
3.5, 12 Hz, 1H, H1), 1.70 (m, 1H, H7), 1.61 (m, 1H, H14), 1.60 (m,
1H, H6), 1.60 (m, 1H, H11), 1.54 (m, 1H, H2), 1.52 (m, 1H, H12),
1.41 (m, 1H, H6), 1.37 (m, 1H, H3), 1.35 (m, 1H, H2), 1.30 (s, 3H,
H16), 1.22 (m, 1H, H11), 1.17 (m, 1H, H1), 1.15 (m, 1H, H3), 0.95
(m, 1H, H9), 0.91 (s, 9H, H4′/H5′/H6′), 0.86 (s, 3H, H20), 0.84 (m,
1H, H5), 0.83 (m, 1H, H7), 0.82 (s, 3H, H17), 0.81 (s, 3H, H18), 0.81
(s, 3H, H19), 0.11 (s, 3H, H1′), 0.10 (s, 3H, H2′); HRESIMS [M +
Na]+ m/z 445.3471 (C26H50O2NaSi, calcd 445.3478).
Compound 15. To compound 14 (41 mg, 0.096 mmol) in CH2Cl2
(10.0 mL) were added 4-(dimethylamino)pyridine (1.9 mg, 0.016
mmol), N,N-diisopropylethylamine (2.0 mL), and benzyl chloromethyl
ether (techn. ca. 60%, 0.8 mL). The solution was stirred at 50-70 °C
for 21 h. After cooling to room temperature, the reaction was quenched
with saturated aqueous NaCO3 and brine. The resulting solution was
extracted with Et2O, and then the organic layer was concentrated under
reduced pressure. The residue was purified by Si gel column chroma-
tography eluted in a stepwise gradient manner with Et2O/hexanes (0:
100 to 3:97) to afford 15 (49 mg, 95% yield) as a colorless solid: 1H
NMR (CDCl3) δ 7.32 (m, 5H, H4′′/H5′′/H7′′/H8′′), 4.93 (d, J ) 7.7
Hz, 1H, H1′′), 4.78 (d, J ) 7.6 Hz, 1H, H1′′), 4.66 (d, J ) 12 Hz, 1H,
H2′′), 4.55 (d, J ) 12 Hz, 1H, H2′′), 3.88 (dd, J ) 1.7, 11 Hz, 1H,
H15), 3.71 (dd, J ) 5.7, 11, 1H, H15), 1.90 (m, 2H, H2), 1.85 (m, 1H,
H12), 1.83 (m, 1H, H7), 1.66 (m, 1H, H12), 1.65 (m, 1H, H1), 1.50
(m, 2H, H6), 1.44 (d, J ) 4.9 Hz, 1H, H14), 1.37 (m, 2H, H11), 1.35
(m, 1H, H3), 1.17 (s, 3H, H16), 1.13 (m, 1H, H7), 1.11 (m, 1H, H3),
0.93 (s, 3H, H17), 0.87 (m, 1H, H9), 0.87 (s, 9H, H4′/H5′/H6′), 0.84
(s, 3H, H20), 0.83 (m, 1H, H5), 0.79 (m, 1H, H1), 0.79 (s, 3H, H18),
0.79 (s, 3H, H19), 0.03 (s, 3H, H1′), 0.02 (s, 3H, H2′); HREIMS m/z
542.41501 (C34H58O3Si, calcd 542.41552).
Compound 16. To compound 15 (46 mg, 0.084 mmol) in THF (4.0
mL) was added 1 M tetrabutylammonium fluoride/THF (1.0 mL). The
solution was stirred at 60-70 °C for 21 h. After cooling to room
temperature, the solution was diluted with H2O, and then the THF was
removed under reduced pressure. To the resulting aqueous solution,
brine was added, and then the solution was extracted with Et2O. The
organic layer was concentrated under reduced pressure. The residue
was purified by Si gel column chromatography eluted in a stepwise
gradient manner with EtOAc/hexanes (0:100 to 20:80) to afford 16
(35 mg, 96% yield) as a colorless solid: 1H NMR (CDCl3) δ 7.35-
7.30 (m, 5H, H4′/H5′/H6′/H7′/H8′), 4.86 (s, 2H, H2′), 4.60 (s, 2H, H1′),
3.87 (dd, J ) 8.5, 11 Hz, 1H, H15), 3.73 (dd, J ) 2.6, 11 Hz, 1H,
H15), 2.02 (ddd, J ) 3.1, 3.1, 12 Hz, 1H, H12), 1.88 (ddd, J ) 3.0,
3.0, 13 Hz, 1H, H7), 1.65 (m, 1H, H12), 1.63 (m, 1H, H2), 1.63 (m,
1H, H11), 1.61 (m, 1H, H1), 1.58 (m, 1H, H14), 1.55 (m, 1H, H6),
1.37 (s, 3H, H16), 1.35 (m, 1H, H2), 1.32 (m, 1H, H3), 1.22 (m, 1H,
H7), 1.19 (m, 1H, H11), 1.11 (m, 1H, H3), 0.90 (d, J ) 12 Hz, 1H,
H9), 0.84 (s, 3H, H20), 0.83 (s, 3H, H5), 0.80 (m, 1H, H1), 0.79 (s,
3H, H17), 0.78 (s, 3H, H19), 0.77 (s, 3H, H18); HREIMS m/z
428.32859 (C28H44O3, calcd 428.32905).
Compound 3. To the alcohol 18a (4 mg, 5 µmol) in pyridine (1.0
mL) on ice was added trifluoroacetic anhydride (100 µL, 719 µmol).
After the reaction solution was stirred on ice for 50 min, the reaction
was quenched with saturated aqueous NaHCO3. To the quenched
solution was added brine, and then the resulting solution was extracted
with Et2O/toluene (1:1). The organic layer was concentrated under
reduced pressure, and the resulting residue was used for the next step
without further purification. The residue was dissolved in EtOAc/EtOH
(1:4) (2.0 mL). To this solution was added Pd(OH)2/C (21 mg).
Hydrogenation was undertaken using a hydrogen gas pressure of 450
psi. The reaction solution was stirred at room temperature for 16 h.
The reaction solution was filtered though a Celite 545 column to remove
the Pd(OH)2/C powder. The column was washed with EtOAc (10 mL),
which was combined with the filtrate. The combined organic solution
was concentrated under reduced pressure. The residue was purified by
Si gel column chromatography eluted in a stepwise gradient manner
with EtOAc/hexanes (0:100 to 40:60) to afford 3 (0.6 mg, 50% yield
over 2 steps) as a yellow oil: UV (MeOH) λmax (log ꢀ) 244 nm (3.86),
1
278 nm (3.81); H NMR (acetone-d6) δ 6.55 (ddd, J ) 1.4, 1.4, 2.5
Hz, 1H, H21), 5.87 (d, J ) 2.5 Hz, 1H, H19), 3.81 (s, 3H, H27), 3.19
(s, 1H, 13-OH), 2.57 (dd, J ) 1.4, 5.0 Hz, 1H, H15), 2.56 (dd, J )
1.4, 6.6 Hz, 1H, H15), 1.80 (ddd, J ) 1.3, 1.3, 6.3 Hz, 1H, H12), 1.69
(m, 1H, H1), 1.67 (m, 1H, H2), 1.67 (m, 1H, H7), 1.67 (m, 1H, H14),
1.59 (m, 1H, H6), 1.50 (m, 1H, H2), 1.47 (m, 1H, H12), 1.38 (m, 1H,
Compound 18a,b. To compound 16 (32 mg, 0.074 mmol) in CH2-
Cl2 (5.0 mL) on an ice bath were added CH3COONa (52 mg, 0.63
mmol) and PCC (130 mg, 0.60 mmol). The solution was stirred on the
ice bath for 4.5 h. The reaction solution was filtered through a Florisil

740 Journal of Natural Products, 2007, Vol. 70, No. 5
Warabi et al.
H6), 1.35 (m, 1H, H3), 1.30 (m, 2H, H11), 1.18 (s, 3H, H22), 1.16 (m,
1H, H3), 1.08 (m, 1H, H7), 0.95 (m, 1H, H9), 0.92 (s, 3H, H23), 0.85
(m, 1H, H5), 0.85 (s, 3H, H24), 0.83 (m, 1H, H1), 0.83 (s, 3H, H26),
0.82 (3H, H25); HREIMS m/z 428.29235 (C27H40O4, calcd 428.29266).
Chiral Separation of the Compound 18a. A 14 mg portion of the
fraction containing 18a was subjected to normal-phase chiral HPLC
(DAICEL Chiralpak IA) eluted with EtOAc/hexanes/triethylamine (13:
87:0.03), furnishing (-)-18a (3.7 mg) and (+)-18a (3.4 mg) at retention
times of 29.4 and 37.0 min, respectively. (-)-18a: [R]D24 -10 (c 1.0,
References and Notes
(1) Eccles, S. A. Int. DeV. Biol. 2004, 48, 583-598.
(2) Warabi, K.; McHardy, L. M.; Matainaho, L.; Van Soest, R.;
Roskelley, C. D.; Roberge, M.; Andersen, R. J. J. Nat. Prod. 2004,
67, 1387-1389.
(3) McHardy, L. M.; Warabi, K.; Andersen, R. J.; Roskelley, C. D.;
Roberge, M. Mol. Cancer Ther. 2005, 4, 772-8.
(4) McHardy, L. M.; Sinotte, R.; Troussard, A.; Sheldon, C.; Church,
J.; Williams, D. E.; Andersen, R. J.; Dedhar, S.; Roberge, M.;
Roskelley, C. D. Cancer Res. 2004, 64, 1468-1474.
(5) Keezer, S. M.; Ivie, S. E.; Krutzsch, H. C.; Tandle, A.; Libutti, S.
K.; Roberts, D. Cancer Res. 1993, 63, 6405-6412.
(6) Bernardinelli, G.; Flack, H. D. Acta Crystallogr. 1985, A41, 500-
511.
24
CH2Cl2). (+)-18a: [R]D +10 (c 1.0, CH2Cl2).
Compound (+)-3. Treatment of (-)-18a (2.2 mg, 2.7 µmol) in a
similar manner to racemic 18a afforded (+)-3 (0.4 mg, 35% yield):
23
[R]D +16 (c 0.27, EtOAc).
Compound (-)-3. Treatment of (+)-18a (2.0 mg, 2.5 µmol) in a
similar manner to racemic 18a afforded (-)-3 (0.8 mg, 70% yield):
(7) Furstner, A.; Stelzer, F.; Rumbo, A.; Krause, H. Chem.-Eur. J. 2002,
8, 1856-1871.
23
[R]D -16 (c 0.33, EtOAc).
(8) Nishizawa, M.; Takenaka, H.; Hayashi, Y. J. Org. Chem. 1986, 51,
806-813.
Acknowledgment. Financial support was provided by grants from
the National Cancer Institute of Canada (R.J.A, M.R., C.D.R.), the
Michael Smith Foundation for Health Research (C.D.R, M.R., R.J.A),
and NSERC (R.J.A.).
1
(9) Partial H and 13C chemical shift values were assigned as follows:
C-15 [δ 2.35, dd, J ) 2.5, 13 Hz and 2.72, ddd, J ) 1.8, 13, 14 Hz
(major); 2.35, d, J ) 15 Hz and 2.75, m (minor)], C-19 [δ 5.25, d,
J ) 1.6 Hz; 99.7 (major); 5.23, d, J ) 1.9 Hz; 98.6 (minor)], C-21
[δ 5.76, dd, J ) 1.8, 1.6 Hz; 122.5 (major); 5.94, brd; 125.6 (minor)],
C-27 [δ 3.70, s (major); 3.72, s (minor)].
Supporting Information Available: 13C NMR data for synthetic
compounds; experimental details and data for single-crystal X-ray
diffraction analysis of 7; CD spectra for 1 and 3. This material is
available free of charge via the Internet at http://pubs.acs.org.
NP060481L