Design, synthesis, and biological evaluation of estrone-derived hedgehog signaling inhibitors

Article abstract of DOI:10.1016/j.tet.2011.10.028

The design, synthesis, and biological evaluation of new analogs of the naturally occurring compound cyclopamine, a hedgehog signaling inhibitor, are described. Structure-activity relationship studies lead to an evolving model for the pharmacophore of this medically promising compound class of anti-cancer chemotherapeutic agents.

Full text of DOI:10.1016/j.tet.2011.10.028

Tetrahedron 67 (2011) 10261e10266
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Design, synthesis, and biological evaluation of estrone-derived hedgehog
signaling inhibitors
,
a
b
b
b,
Jeffrey D. Winkler a , Andre K. Isaacs , Chaomei Xiang , Valerie Baubet , Nadia Dahmane
*
*
ꢀ
ꢀ
a Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA
b The Wistar Institute, Philadelphia, PA 19104, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 August 2011
Received in revised form 9 October 2011
Accepted 10 October 2011
Available online 19 October 2011
The design, synthesis, and biological evaluation of new analogs of the naturally occurring compound
cyclopamine, a hedgehog signaling inhibitor, are described. Structureeactivity relationship studies lead
to an evolving model for the pharmacophore of this medically promising compound class of anti-cancer
chemotherapeutic agents.
Ó 2011 Elsevier Ltd. All rights reserved.
Dedicated with both great affection and
admiration to Professor Gilbert Stork, a re-
markable scientist and mentor who con-
tinues to inspire us all
Keywords:
Cyclopamine
Steroidal alkaloid
Cancer chemotherapy
Hedgehog signaling
Structureeactivity relationships
1. Introduction
SHH signaling was first linked to cancer with the identification
of mutations in the PTCH1 gene in Gorlin syndrome patients.8 It was
More than 40 years ago, Binns and co-workers established that
the alkaloid cyclopamine 1 (Fig. 1a) was responsible for the birth
defects observed in calves from livestock that were fed diets rich in
the corn lily, Veratrum californicum (Fig. 1b).1,2 The observed phe-
notype included anophthalmia, cyclopia, and severe craniofacial
effects (Fig. 1c).
It was later established that cyclopamine 1 acts by inhibiting the
Sonic Hedgehog (SHH) cellular signaling pathway, which is critical
for tissue growth and differentiation, thus playing a pivotal role in
embryogenesis.3,4 Activation of the SHH-signal transduction path-
way is initiated by the binding of the SHH ligand to the cellular
membrane receptor Patched (PTCH1), which relieves the PTCH1-
mediated inhibition of the transmembrane protein Smoothened
(SMO) (Fig. 1d).5,6 Activated SMO transduces the signal to the nu-
cleus to regulate gene expression via Gli transcription factors.
Beachy and co-workers have established that 1 disrupts this
pathway by inhibition of SMO.7
subsequently shown that this pathway is also active in the majority
of sporadic basal cell carcinomas.9 In addition, activation of the SHH
pathway has been linked to brain tumors, including medulloblas-
tomas and gliomas,12 melanoma,13 lung adenocarcinoma,14 as well
as prostate,15 small cell lung,16 and pancreatic cancer.17 Treatment
of cancer cells with cyclopamine 1 induces a decrease in pro-
liferation, an increase of apoptosis and/or a decrease of metasta-
sis.10,18 The teratogenicity associated with cyclopamine has not
hampered interest in this natural product as an important lead
structure in the development of cancer chemotherapeutic agents
that act via inhibition of SHH signaling.19
In spite of the attractive pharmacological profile against
a number of cancer xenografts, in vivo evaluation of cyclopamine
has been hampered by its poor aqueous solubility (ca. 5 mg/ml) and
acid lability. Under acidic conditions, cyclopamine 1 readily con-
verts to veratramine 2, via cleavage of the spirotetrahydrofuran
ring, followed by aromatization of the D ring.20 Unlike cyclopamine,
2 does not act as an SHH antagonist, and causes hemolysis by tar-
geting other receptors21 (Fig. 2).
Two strategies have been reported to address the issues of water
solubility and acid lability of 1: (1) the covalent modification of 1 to
* Corresponding authors. E-mail address: winkler@sas.upenn.edu (J.D. Winkler).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.10.028

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J.D. Winkler et al. / Tetrahedron 67 (2011) 10261e10266
25
Fig. 3. Structures of hedgehog signaling inhibitors IPI-926 (Infinity Pharmaceuticals) 3
and GDC-0449 (Curis/Genentech) 4.
2. Results and discussion
We have opted to explore a third approach to the identification
of novel SHH signaling inhibitors, which is not dependent on the
availability of 1, and yet generates new lead compounds that closely
resemble 1 in both structure and function. The difference in tera-
togenicity between cyclopamine 1 and the close structural analog
tomatidine 5 (Fig. 4; non-teratogenic) has been attributed to the
difference in the orientation of the nitrogen atom relative to the
steroid plane in 1 and 5. The C-nor-D-homo framework of 1 can
thus be viewed as a scaffold that orients the E/F heterobicyclic
moiety orthogonal to the steroidal ring system, with the F-ring
nitrogen atom on the
tetrahydrofuran ring of 5 lies in the steroid plane and the nitrogen
atom is on the -face of the steroid plane.
a
-face of the steroid plane.26 In contrast, the
b
Fig. 1. (a) Structure of cyclopamine 1; (b) V. californicum;10 (c) cyclopia;11 and (d)
a simplified schematic of SHH signaling showing the effect of 1 on SMO.
Fig. 2. Conversion of cyclopamine 1 to the D-ring aromatic compound veratramine 2
under acidic conditions.
produce structurally related and metabolically stable lead struc-
tures, i.e., IPI-926 3,22 in which the D ring of 1 is expanded to
a seven-membered ring, as pioneered by Tremblay and co-workers
at Infinity Pharmaceuticals; and (2) the screening of libraries of
diverse chemical structures in the hope of discovering drug-like
structures that will interfere with SHH signaling.23 The most
noteworthy success to date using this approach is GDC-0449 4
(Fig. 3), a compound that is currently in Phase II clinical trials.24 The
first approach, however, relies on the availability of the natural
product 1, which is expensive, and recent results indicate an ac-
quired resistance to inhibition of SHH signaling in an MB patient
treated with the GDC-0449, providing the impetus for the de-
velopment of new SHH signaling antagonists.25
Consequently, there is an urgent need to identify readily avail-
able potent inhibitors of SHH as lead structures for the de-
velopment of new cancer chemotherapies. We report herein the
design and synthesis of cyclopamine-like structures derived from
readily available steroidal precursors that function as potent
cyclopamine mimetics. Outlined herein are the results of our
structureeactivity relationship studies on this novel compound
class.
Fig. 4. Structures and energy-minimized structures of cyclopamine 1, tomatidine 5,
estrone-derived analog 6, and 7, the C-17 epimer of 6.27
We reasoned that the C-nor-D-homo steroidal ring system of 1
functions as a scaffold for the orientation of the heterobicyclic
framework of the EF rings relative to the C-3b oxygen functionality
in 1. Replacement of the C-nor-D-homo steroidal system with the

J.D. Winkler et al. / Tetrahedron 67 (2011) 10261e10266
10263
androstane ring system and further stereochemical simplification
2.2. Synthesis and biological evaluation of truncated analogs
of cyclopamine 1
via aromatization of rings A and F leads to the novel estrone-
derived analog 6.27
The energy-minimized structures in Fig. 4 suggest an important
role for the C-17 stereochemistry common to both 1 and 3, which,
unlike 5 (and 7, the C-17 epi analog of 6), share the orientation of
These results necessitate a revision of the binding model that we
originally advanced (Fig. 4).27,28 The potent activity of 8 suggests
that the C-3 hydroxyl common to both 6 and 7 is not required for
biological activity, and brings into question the importance of the
intact steroidal framework. To examine the effect of truncating the
steroid, we have examined the deletion of portions of the tetracy-
clic steroidal ring system common to 6, 7, and 8.
the C-17 oxygen substituent on the
contrast, the C-17 oxygen atom of 7, the C-17 epimer of 6, is ori-
ented on the -face of the steroid plane, which leads to the orien-
tation of the F-ring nitrogen atom of 7 on the -face of the steroid
b-face of the steroid plane. In
a
b
plane, the same orientation that is found in tomatidine 5, a natu-
rally occurring compound, which displays no activity as a hedgehog
signaling inhibitor.
Toward that end, we have prepared 13 (Scheme 1), an analog
lacking the AB ring system present in 6, 7, and 8. Using the same
annelation strategy that was employed for the syntheses of the
previously described analogs, addition of the conjugate base of 10
to the known thioketal 930 led to the formation of carbinol 11,
which on BuchwaldeHartwig cyclization generated 12, containing
the dihydrofuropyridine that constitutes the EF ring system of
cyclopamine 1. Dithioketal deprotection of 12 gave the desired
truncated analog 13 in good yield.
2.1. Synthesis and biological evaluation of estrone-derived
analogs 6, 7, and 8 of cyclopamine 1
To test the hypothesis that the three-point recognition of the C-3
oxygen, C-17 oxygen, and C-21 nitrogen heteroatoms as oriented in
1 is required for recognition at SMO, we have synthesized both 627
and 7.28 As previously described, both 6 and 7 are potent inhibitors
of SHH signaling as evaluated by inhibition of ligand-induced SHH
signaling activity in a luciferase-based assay and by inhibition of
SHH-induced proliferation of mouse granule neuron precursors,
with activities comparable or superior to that of cyclopamine 1 at
concentrations as low as 5 mM.
These results, the comparable potency of 6 and the C-17 epimer
7, challenged our hypothesis that the three-point recognition of the
C-3 oxygen and each of the other E and F-ring heteroatom func-
tionalities in 1 (and 6) is required for recognition at SMO, the cel-
lular target of cyclopamine, since structures with either orientation
at C-17, i.e., both 6 and 7, are potent inhibitors of SHH signaling. The
relative orientations of the tetrahydrofuran oxygen and pyridine
nitrogen relative to the steroid plane do not appear to be important
features for recognition of these cyclopamine analogs at SMO,
suggesting that the C-3 oxygen functionality may not be required
for recognition at SMO.
To establish the role, if any, of the C-3 oxygen functionality that is
present in 6 and 7 on the biological activity of these estrone-derived
analogs of cyclopamine 1, we prepared the C-3 deoxy compound 8
(Fig. 5) as previously described.28 Biological evaluation of 8 using the
same GLI-luciferase assay29 described for 6 and 7 revealed that 8 is
a potent inhibitor of SHH signaling. In this assay, the C-3 deoxy an-
alog 8 led to a strong inhibition of SHH signaling activity (80% in-
Scheme 1. Syntheses of des-AB analog 13.
Biological evaluation of 13 using the same SHH-Light2 cells
luciferase-based assay29 that was used to evaluate the biological
activity of 6, 7, and 8 reveals that the tetracyclic analog 20, lacking
the steroidal A and B rings contained in all of the previously de-
scribed analogs, has no effect on SHH signaling, and suggests that
the AB ring system is important for SHH signaling inhibitory ac-
tivity. This finding prompted us to examine the preparation of an-
alogs that would more closely resemble the structures of the
previously prepared estrone-based systems, i.e., containing the
aromatic A ring that is present in 6, 7, and 8. The synthesis of des-B
(lacking the steroidal B ring common to 6, 7, and 8) analogs 15 and
16 is outlined in Scheme 2.
hibition at 5 mM; compared to 70% inhibition in the same assay with
6). We have also reported that the C-3 deoxy analog 8 is ca. 2ꢀ more
potent than cyclopamine 1 at reducing DAOY medulloblastoma cell
viability, an important measure of SHH inhibitory activity, and
a significant illustration of the potential of these structures for the
development of brain cancer chemotherapeutics.28
Fig. 5. Structures of estrone-derived cyclopamine analog 6, the C-17 epi analog 7, and
the C-3 deoxy analog 8.
Scheme 2. Syntheses of des-B analogs 15 and 16.

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J.D. Winkler et al. / Tetrahedron 67 (2011) 10261e10266
Reaction of the dienol triflate 14, derived from 13, via Suzuki
0.6
0.5
0.4
0.3
0.2
0.1
0
coupling of 14 with both phenylboronic acid and p-hydroxy-phe-
nylboronic acid, respectively, provided 15 and 16, both of which
lack the B-ring present in cyclopamine. Based on the observation
that the C-3 deoxy analog 8 (Fig. 5) was more potent than the C-3
hydroxy compound 6 (and 7),28 we were surprised to find that 16
(R¼OH) is more potent than 15 (lacking the C-3 hydroxy group) in
the previously described luciferase-based assay for hedgehog sig-
naling activity, as shown in Fig. 6.
12
10
8
Control
1
16
Fig. 8. Analog 16 reduces DAOY medulloblastoma cell viability. DAOY human medul-
loblastoma cells were treated with either carrier DMSO (control), cyclopamine
(10 M) or 16 (10 M) for 3 days. The histogram measures cell viability assessed by the
1
6
m
m
MTT assay (absorbance at 570 nm) (asterisk indicates p<0.05). Similar results were
obtained with U87GBM cells (not shown).
4
Evaluation of the SAR of this lead compound by examination of
the C-17 epi compound 7 and the C-3 deoxy analog 8 established
2
that the two-point binding model (using the C-3b hydroxyl and the
heterobicyclic EF ring system of 1 (and 6)) at the cellular receptor
SMO is not sufficient to explain the surprising level of potency
observed for 7 and 8.28
0
Control
SHH
SHH + 1
SHH + 15
SHH + 16
This important finding led us to examine the synthesis and bi-
ological evaluation of truncated structures, such as 13, lacking both
the A and B rings common to the previously described structures.
We have found such a structural modification too extreme to retain
SHH inhibitory activity, but we report that the addition of the ar-
omatic A ring to 13 leads to potent SHH signaling inhibitors.
The introduction of the aromatic A ring, that is present in 6, 7,
and 18, leads to novel des-B structures 15 and 16, i.e., lacking the
steroidal B ring. Biological evaluation of 15 and 16 reveals that, in
contrast to 6 (C-3 hydroxy) and 8 (C-3 deoxy), in which removal of
the C-3 hydroxyl leads to more potent inhibitory activity, the C-3
hydroxylated analog 16 is decidedly more potent than the C-3 de-
oxy compound 15. The basis for this difference is currently under
investigation in our laboratories. Biological evaluation of 16 es-
tablishes that it is more potent than cyclopamine 1 in the inhibition
of SHH-induced proliferation of GNPs (Fig. 7) and ca. equipotent
with 1 in the DAOY medulloblastoma cell viability assay (Fig. 8).
These findings suggest that partial structures of estrone-based
analogs of 1 are sufficient to generate potent SHH signaling in-
hibitors. Further work on the development of more potent com-
pounds is currently underway in our laboratory and our results will
be reported in due course.
Fig. 6. Luciferase-based assay for SHH activity: treatment of SHH-Light2 cells with
recombinant SHH (200 ng) resulted in the strong induction of reporter activity, which
was blocked by co-treatment with either cyclopamine 1 or with 15 or 16 at 5
P<0.001 [SHH vs SHHþ1; SHH vs SHHþ15/16].
mM, both
Further investigation with 16 revealed that it is a potent SHH
signaling inhibitory compound, as demonstrated in the GNP pro-
liferation assay as shown in Fig. 7, where it is ca. 3ꢀ more potent
than cyclopamine 1. We have also established that 16 is ca. equi-
potent with cyclopamine 1 in the DAOY medulloblastoma cell vi-
ability assay, as illustrated in Fig. 8.
7
6
5
4
3
2
1
0
4. Experimental section
4.1. General methods
Control
SHH
SHH + 1
SHH + 16
Solvents used for extraction and purification were HPLC grade
from Fisher. Unless otherwise indicated, all reactions were run un-
der an inert atmosphere of Argon. Anhydrous tetrahydrofuran, ethyl
ether, and toluene were obtained via passage through an activated
alumina column.31 Commercial reagents were used as received.
Deuterated solvents were obtained from Cambridge Isotope labs.
Fig. 7. Estrone-derived analog 16 inhibits SHH-induced proliferation of granule neuron
precursors (GNPs).
3. Conclusions
We have demonstrated that it is possible to replace the C-nor-D-
homo ring system of cyclopamine 1 with an estrone-derived ste-
roidal ring system and to prepare a cyclopamine analog 6 that is
a potent SHH signaling inhibitor as measured in both the luciferase
and GNP (granule neuron precursor) assays.27
Merck pre-coated silica gel plates (250 mm, 60 F254) were used for
analytical TLC. Spots were visualized using 254 nm ultraviolet light,
with either anisaldehyde or potassium permanganate stains as vi-
sualizing agents. Chromatographic purifications were performed on
Sorbent Technologies silica gel (particle size 32e63 m
). 1H and 13C

J.D. Winkler et al. / Tetrahedron 67 (2011) 10261e10266
10265
NMR spectra were recorded at 500 MHz and 125 MHz, respectively,
in CDCl3 on a Bruker AM-500 or DRX-500 spectrometer. Chemical
hexanes) yielded 13 as a white solid (23 mg, 85%): mp 133e135 ꢁC.
½
a 1D9:6
ꢃ
ꢂ56.4 (c 1.0, CHCl3). 1H NMR (CDCl3):
¼8.04 (s, 1H), 7.02 (m,
d
shifts are reported relative to internal chloroform (
d
d
7.26 for 1H,
2H), 5.88 (s, 1H), 3.41 (d, J¼17.0 Hz, 1H), 2.98 (d, J¼17.0 Hz, 1H), 2.82
(m, 1H), 2.53 (m, 3H), 2.37 (dd, J¼5.0, 13.0 Hz, 1H), 2.05 (m, 2H), 1.63
77.0 for 13C). Infrared spectra were recorded on a NaCl plate using
a PerkineElmer 1600 series Fourier transform spectrometer. High
resolution mass spectra were obtained at the University of Penn-
sylvania Mass Spectrometry Service Center on an Autospec high
resolution double-focusing electrospray ionization/chemical ioni-
zation spectrometer with either DEC 11/73 or OPUS software data
system. Melting points were obtained on a Thomas Hoover capillary
melting point apparatus and are uncorrected.
(m, 1H), 1.40 (s, 3H). 13C NMR (CDCl3):
141.9, 124.3, 122.6, 115.3, 95.7, 47.9, 39.1, 34.7, 33.0, 28.9, 26.3, 18.7.
FTIR (thin film) 3428, 2930, 1666, 1430, 1258, 1004 cmꢂ1. HRMS (ES)
calcd for C18H17NO2: 255.1259 (Mþ), found 256.1332 (MHþ).
d
¼198.1, 172.1, 153.2, 150.0,
4.1.3. (10R,7a0S)-7a0-Methyl-20,60,70,7a0-tetrahydro-3H-spiro[furo
[3,2-b]pyridine-2,10-inden]-50-yl trifluoromethanesulfonate (14). A
solution of 13 (64 mg, 0.25 mmol) in dry CH2Cl2 (1.2 mL) was cooled
4.1.1. (10R,7a0S)-10-((3-Bromopyridin-2-yl)methyl)-7a0-methyl-
10,20,30,60,70,7a0-hexahydrospiro[[1,3]dithiolane-2,50-inden]-10-ol
(11). To a solution of diisopropylamine (2.6 mL, 18.6 mmol) in dry
Et2O (6 mL) stirred at 0 ꢁC under argon was added dropwise a so-
lution of 2.5 M n-BuLi in hexanes (7.4 mL, 18.6 mmol). The solution
was stirred at 0 ꢁC for 30 min. The flask was cooled to ꢂ20 ꢁC and
a solution of 2-methyl-3-bromopyridine (3.19 g, 18.6 mmol) in Et2O
(9 mL) was added dropwise. The resulting red mixture was stirred
at ꢂ20 ꢁC, under argon, for 2 h. A solution of thiolane 9 (1.78 g,
7.42 mmol) in THF (11 mL) was added dropwise and kept stirring at
ꢂ20 ꢁC for 1 h. The reaction flask was allowed to warm up to room
temperature and was quenched slowly with H2O (30 mL). The
mixture was extracted with EtOAc (3ꢀ75 mL), washed with satu-
rated NH4Cl (30 mL), saturated NaHCO3 (30 mL), brine (30 mL), and
dried with Na2SO4. The solvent was removed under reduced pres-
sure and the crude product was purified by silica gel chromatog-
raphy (10% ethyl acetate in hexanes) to yield 11 as an off-white solid
to ꢂ20 ꢁC and triethylamine (42
mL, 0.30 mmol) was added drop-
wise to the stirring solution. After a period of 5 min, tri-
fluoromethanesulfonic anhydride (50 mL, 0.30 mmol) was added
dropwise and the solution was allowed to warm up to 0 ꢁC over
a 1 h period. The reaction mixture was diluted with CH2Cl2 (10 mL)
and quenched with brine (5 mL). The layers were separated and the
aqueous layer was extracted with CH2Cl2 (10 mL). The combined
organic layers were dried with Na2SO4 and the solvent was re-
moved under reduced pressure. Purification by silica gel chroma-
tography (33% ethyl acetate in hexanes) gave 14 as an orange oil
(65 mg, 67%). 1H NMR (CDCl3):
d
¼8.03 (m, 1H), 7.02 (m, 2H), 6.23
(J¼3.0 Hz, 1H), 5.64 (s, 1H), 3.51 (d, J¼16.5 Hz, 1H), 3.08 (d,
J¼17.0 Hz, 1H), 2.96 (d, J¼17.0 Hz, 1H), 2.64 (dd, J¼3.0, 17.0 Hz, 2H),
2.43 (dd, J¼5.5, 18.0 Hz, 1H), 1.89 (dt, J¼5.5, 12.0 Hz, 1H), 1.54 (dd,
J¼5.5, 12.0 Hz, 1H), 1.19 (s, 3H). 13C NMR (CDCl3):
¼153.1, 150.7,
d
150.6, 141.9, 141.7, 123.7, 122.5, 115.4, 115.1, 97.1, 47.1, 44.6, 39.2, 27.6,
26.1, 17.6. FTIR (thin film) 2935, 1423, 1212 cmꢂ1. HRMS (ES) calcd
for C17H16F3NO4S: 387.0752 (Mþ), found 388.0821(MHþ).
(2.66 g, 87%, mp 71e73 ꢁC). ½a D21:2
ꢃ
ꢂ91.9 (c 1.0, CHCl3). 1H NMR
(CDCl3):
d
¼8.41 (dd, J¼1.5, 4.5 Hz, 1H), 7.88 (dd, J¼1.5, 8.0 Hz, 1H),
7.06 (dd, J¼4.5, 8.0 Hz, 1H), 6.31 (s, 1H), 5.58 (s, 1H), 3.43e3.35 (m,
3H), 3.25e3.19 (m, 1H), 3.11 (d, J¼15.5 Hz, 1H), 2.98 (d, J¼15.5 Hz,
1H), 2.50e2.44 (m, 1H), 2.34 (m, 1H), 2.26e2.17 (m, 2H), 1.89e1.82
(m, 1H), 1.77 (m, 1H), 1.61e1.58 (m, 1H), 1.48 (m, 1H), 1.18 (s, 3H). 13C
4.1.4. (10R,7a0S)-7a0-Methyl-50-phenyl-20,60,70,7a0-tetrahydro-3H-
spiro[furo[3,2-b]pyridine-2,10-indene] (15). To a solution of triflate
14 (16 mg, 0.04 mmol) in a 600
added phenylboronic acid (5 mg, 0.04 mmol), Pd(PPh3)4 (2.4 mg,
0.002 mmol) followed by a 0.5 N solution of Na2CO3 (80 L,
mL of a 1:1 THF/PhCH3 mixture were
NMR (CDCl3):
d
¼159.3, 147.4, 146.6, 140.8, 124.4, 122.7, 122.6, 82.7,
m
66.0, 47.1, 40.4, 40.2, 39.7, 38.9, 33.5, 29.6, 26.0, 19.8. FTIR (thin film)
0.04 mmol). The mixture was heated to reflux for 3 h. The reaction
flask was allowed to cool to room temperature and then diluted
with H2O (5 mL). The mixture was partitioned with CH2Cl2
(3ꢀ10 mL) and the combined organic layers were washed with
brine (10 mL). The organic layer was dried with Na2SO4 and the
solvent was removed under reduced pressure. Purification by silica
gel chromatography (33% ethyl acetate in hexanes) yielded 15 as
3362, 2922, 1428, 1066, 1033 cmꢂ1
. HRMS (ES) calcd for
C18H22BrNOS2: 411.0326 (Mþ), found 412.0388 (MHþ).
4.1.2. (10R,7a0S)-7a0-Methyl-20,30,70,7a0-tetrahydro-3H-spiro[furo
[3,2-b]pyridine-2,10-inden]-50(60H)-one (13). A resealable Schlenk
tube was charged with alcohol 11 (90 mg, 0.2 mmol), Pd(OAc)2
(10 mg, 0.04 mmol), BINAP (27 mg, 0.04 mmol) and Cs2CO3 (107 mg,
0.3 mmol). Dry toluene (3 mL) was added and the tube was capped
underargon and the resulting mixturewas allowed tostirat 80 ꢁC for
3 h. The mixture was allowed to cool to room temperature, filtered
through Celite, concentrated under reduced pressure and purified
by silica gel chromatography (15% ethyl acetate in hexanes) to yield
a yellow oil (12 mg, 92%). ½a D23:6
ꢃ
ꢂ111.2 (c 0.36, CHCl3). 1H NMR
(CDCl3):
d
¼8.03 (t, J¼2.8 Hz, 1H), 7.48 (d, J¼8.5 Hz, 2H), 7.34 (t,
J¼7.8 Hz, 2H), 7.26 (m, 1H), 7.02 (m, 2H), 6.63 (s, 1H), 5.52 (s, 1H),
3.61 (d, J¼17.0 Hz, 1H), 3.11 (d, J¼16.5 Hz, 1H), 2.98 (d, J¼17.0 Hz,
1H), 2.66 (m, 2H), 2.61 (dd, J¼3.0, 16.5 Hz, 1H), 1.88 (m, 1H), 1.63 (m,
1H),1.22 (s, 3H). 13C NMR (CDCl3):
d¼153.4, 151.4, 146.0, 141.3,140.8,
12 as a white solid (43 mg, 60%): mp 134e136 ꢁC. ½a D19:2
ꢃ
ꢂ29.7 (c 1.0,
138.5, 128.4, 127.5, 125.2, 122.3, 120.0, 119.4, 115.2, 98.2, 47.0, 44.1,
39.2, 28.4, 25.6, 17.9. FTIR (thin film) 2928, 1429, 993 cmꢂ1. HRMS
(ES) calcd for C22H21NO: 315.1623 (Mþ), found 316.1719 (MHþ).
CHCl3). 1H NMR (CDCl3):
d
¼8.02 (dd, J¼2.0, 4.0 Hz,1H), 6.98 (m, 2H),
5.63 (s,1H), 3.42e3.33 (m, 4H), 3.22e3.16 (m,1H), 2.90 (d, J¼17.0 Hz,
1H), 2.60e2.54 (m, 1H), 2.45e2.39 (m, 1H), 2.31e2.15 (m, 3H),
1.95e1.91 (m,1H),1.75 (td, J¼3.5,13.5 Hz,1H),1.33 (dt, J¼3.5,13.5 Hz,
4.1.5. 4-((10R,7a0S)-7a0-Methyl-20,60,70,7a0-tetrahydro-3H-spiro[furo
[3,2-b]pyridine-2,10-inden]-50-yl)phenol (16). To a solution of triflate
1H),1.22 (s, 3H).13C NMR (CDCl3):
122.2, 114.9, 96.1, 65.3, 46.4, 40.5, 40.0, 39.7, 38.3, 34.7, 29.2, 24.9,
19.8. FTIR (thin film) 2923, 1429, 1001 cmꢂ1. HRMS (ES) calcd for
C18H21NOS2: 331.1064 (Mþ), found 332.1129 (MHþ).
d
¼153.5,150.8,144.0,141.4,125.8,
14 (19 mg, 0.05 mmol) in a 740
added phenylboronic acid (7 mg, 0.05 mmol), Pd(PPh3)4 (3 mg,
0.003 mmol) followed by a 0.5 N solution of Na2CO3 (100 L,
mL of a 1:1 THF/PhCH3 mixture were
m
To a solution of thioketal 12 (35 mg, 0.11 mmol) in MeOH (1.4 mL),
H2O (0.2 mL) and CH2Cl2 (0.7 mL) was added THF (0.1 mL) followed
by bis(trifluoroacetoxy)iodobenzene (68 mg, 0.16 mmol) at room
temperature. After 10 min, the solution was poured into a saturated
NaHCO3 solution (5 mL) and extracted with CH2Cl2 (3ꢀ10 mL). The
combined organic layers werewashed with brine (10 mL), dried over
anhydrous Na2SO4, filtered, and concentrated under reduced pres-
sure. Purification by silica gel chromatography (50% ethyl acetate in
0.05 mmol). The mixture was heated to reflux for 3 h. The reaction
flask was allowed to cool to room temperature and then diluted
with H2O (5 mL). The mixture was partitioned with CH2Cl2
(3ꢀ10 mL) and the combined organic layers were washed with
brine (10 mL), dried with Na2SO4 and the solvent was removed
under reduced pressure. Purification by silica gel chromatography
(33% ethyl acetate in hexanes) yielded 16 as a yellow oil (9 mg, 56%).
½
a 2D3:9
ꢃ
ꢂ35.2 (c 0.64, CHCl3). 1H NMR (CDCl3):
¼8.04 (t, J¼3.0 Hz,
d

10266
J.D. Winkler et al. / Tetrahedron 67 (2011) 10261e10266
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NMR (CDCl3):
d
¼156.5, 154.1, 151.2, 145.7, 140.4, 137.6, 132.3, 126.3,
122.7, 118.3, 117.8, 115.7, 115.6, 98.6, 47.5, 44.0, 39.2, 28.2, 25.3, 17.9.
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000 cells/well and cultured in DMEM/F12 (Gibco, 11330), B27
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Acknowledgements
We gratefully acknowledge the generous financial support of
the NIH (CA-134983) to J.D.W. and N.D.
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Supplementary data
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