Chemical and biological studies of nakiterpiosin and nakiterpiosinone

Article abstract of DOI:10.1021/ja908626k

Nakiterpiosin and nakiterpiosinone are two related C-nor-D-homosteroids isolated from the sponge Terpios hoshinota that show promise as anticancer agents. We have previously described the asymmetric synthesis and revision of the relative configuration of

Full text of DOI:10.1021/ja908626k

Published on Web 12/10/2009
Chemical and Biological Studies of Nakiterpiosin and
Nakiterpiosinone
Shuanhu Gao,† Qiaoling Wang,† Lily Jun-Shen Huang,‡ Lawrence Lum,‡ and
Chuo Chen*,†
Departments of Biochemistry and Cell Biology, The UniVersity of Texas Southwestern Medical
Center at Dallas, 5323 Harry Hines BouleVard, Dallas, Texas 75390-9038
Received October 15, 2009; E-mail: Chuo.Chen@UTSouthwestern.edu
Abstract: Nakiterpiosin and nakiterpiosinone are two related C-nor-D-homosteroids isolated from the sponge
Terpios hoshinota that show promise as anticancer agents. We have previously described the asymmetric
synthesis and revision of the relative configuration of nakiterpiosin. We now provide detailed information
on the stereochemical analysis that supports our structure revision and the synthesis of the originally
proposed and revised nakiterpiosin. In addition, we herein describe a refined approach for the synthesis of
nakiterpiosin, the first synthesis of nakiterpiosinone, and preliminary mechanistic studies of nakiterpiosin’s
action in mammalian cells. Cells treated with nakiterpiosin exhibit compromised formation of the primary
cilium, an organelle that functions as an assembly point for components of the Hedgehog signal transduction
pathway. We provide evidence that the biological effects exhibited by nakiterpiosin are mechanistically
distinct from those of well-established antimitotic agents such as taxol. Nakiterpiosin may be useful as an
anticancer agent in those tumors resistant to existing antimitotic agents and those dependent on Hedgehog
pathway responses for growth.
Introduction
Terpios is a genus of thin, encrusting sponges that harbor
large amounts of symbiotic bacteria.1 These sponges are known
to aggressively compete with corals for space by epizoism.
During 1981-1985, large patches (up to 1000 m in length) of
the cyanobacteriosponge Terpios hoshinota were observed in
Okinawa. They killed large populations of corals in that region
and were known as “black disease”. Uemura and co-workers2
hypothesized that T. hoshinota could secrete toxic compounds
and kill the covered corals. In a search for these toxins, they
isolated 0.4 mg of nakiterpiosin and 0.1 mg of nakiterpiosinone
from 30 kg of the sponges. Both compounds inhibited the
growth of P388 mouse leukemia cells with a mean inhibitory
concentration (IC50) of 10 ng/mL. The structures of these
compounds were originally assigned as 3 and 4 on the basis of
NMR experiments (Figure 1). They bear a unique molecular
skeleton and several unusual functional groups. To date, they
are the only C-nor-D-homosteroids isolated from a marine
source. We recently proposed a revision of their relative
configurations to those shown in 1 and 2 by analyzing
spectroscopic data for their fragments. We further synthesized
Figure 1. Structures of C-nor-D-homosteroids 1-6.
1 and 3 and confirmed that the structure of nakiterpiosin should
be 1.3 We present herein the details of our synthetic studies
and spectroscopic analysis of nakiterpiosin (1) and 6,20,25-epi-
nakiterpiosin (3). In addition, we report in this article an
improved synthesis of 1, the first synthesis of nakiterpiosinone
(2), and the results of our preliminary biological studies of 1.
C-nor-D-homosteroids are skeletally rearranged steroids in
which the C ring is contracted and the D ring expanded by one
carbon. The first known, and arguably best known, members
† Department of Biochemistry.
‡ Department of Cell Biology.
(1) (a) Plucer-Rosario, G. Coral Reefs 1987, 5, 197–200. (b) Ru¨tzler, K.;
Smith, K. P. Sci. Mar. 1993, 57, 381–393. (c) Ru¨tzler, K.; Smith,
K. P. Sci. Mar. 1993, 57, 395–403. (d) Liao, M.-H.; Tang, S.-L.; Hsu,
C.-M.; Wen, K.-C.; Wu, H.; Chen, W.-M.; Wang, J.-T.; Meng, P.-J.;
Twan, W.-H.; Lu, C.-K.; Dai, C.-F.; Soong, K.; Chen, C.-A. Zool.
Stud. 2007, 46, 520. (e) Lin, W.-j. M.S. Thesis, National Sun Yat-sen
University, Kaohsiung, Taiwan, R.O.C., 2009.
(2) (a) Teruya, T.; Nakagawa, S.; Koyama, T.; Suenaga, K.; Kita, M.;
Uemura, D. Tetrahedron Lett. 2003, 44, 5171–5173. (b) Teruya, T.;
Nakagawa, S.; Koyama, T.; Arimoto, H.; Kita, M.; Uemura, D.
Tetrahedron 2004, 60, 6989–6993.
(3) Gao, S.; Wang, Q.; Chen, C. J. Am. Chem. Soc. 2009, 131, 1410–
1412.
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Gao et al.
Table 1. Probing the C-6 Configurations of Nakiterpiosin and Nakiterpiosinone
of this family of natural products are cyclopamine (5) and
veratramine (6). The discovery of these veratrum alkaloids is a
rather interesting story.4 Ranchers in the Rocky Mountain region
had long been puzzled by a mysterious birth defect in their
sheep. They found that 1-20% of the lambs were born as
“chattos” (or “monkey faces”) when the mother sheep grazed
in the national forests in central Idaho during the summer. The
Poisonous Plant Research Laboratory of the U.S. Department
of Agriculture was contacted in 1954 to investigate this
“malformed lamb disease”. After 11 years of work, they found
that ewes that grazed on corn lily (Veratrum californicum) on
the 14th day of gestation would give birth to cyclopic lambs,
while the ewes were left unaffected. They further found that 5
was responsible for the one-eye face malformation and that 6
led to leg deformity.4a However, it was not until 30 years later
that the molecular target of 5 was identified to be Smoothened
(Smo).5 Suppression of Hedgehog (Hh) signaling by inhibition
of Smo with small molecules has since been pursued as a new
strategy for cancer treatment.6 The semisynthesis of 5 and 6
was first accomplished by Masamune and Johnson in 19677a-e
and recently by Giannis.7g A formal synthesis was also reported
by Kutney in 1975.7f
(Table 1).12 On the other hand, we found that the corresponding
C-6 epimers (8 and 10) and the natural products exhibited similar
H-6 splitting patterns. We therefore concluded that the C-6
stereocenters in nakiterpiosin and nakiterpiosinone have the R
configuration.
In regard to the stereochemistry of the side chain of
nakiterpiosin and nakiterpiosinone, we first focused on the C-20/
C-22/C-23 relative configuration. We synthesized all four
possible diastereomers (11-14)11 and compared their NMR
spectra with those of the natural products. We found that the
(4) (a) Binns, W.; James, L. F.; Keeler, R. F.; Balls, L. D. Cancer Res.
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James, L. F. J. Nat. Toxins 1999, 8, 63–80.
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Natl. Acad. Sci. U.S.A. 2009, 106, 2623–2628. (e) Rohatgi, R.;
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U.S.A. 2009, 106, 3196–3201.
(6) (a) Scalesa, S. J.; de Sauvage, F. J. Trends Pharmacol. Sci. 2009, 30,
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2008, 455, 406–410. (g) Tremblay, M. R.; et al. J. Med. Chem. 2008,
51, 6646–6649.
Results and Discussion
Revision of the Relative Stereochemistry. The unique mo-
lecular structures and strong P388 growth inhibition activity of
nakiterpiosin 1 and nakiterpiosinone 2 prompted us to initiate
a research program to explore their laboratory synthesis8 and
biological functions. At the onset of this project, we noticed
that the C-20 and C-25 configurations of the originally proposed
structures for nakiterpiosin and nakiterpiosinone (i.e., 3 and 4,
respectively)2 were opposite to those of cyclopamine 5 and
veratramine 6 (Figure 1) and other “normal” steroids. In
addition, the C-6 and C-20 configurations of 3 and 4 could not
be easily rationalized on the basis of biogenesis analysis.10 We
further considered that the reported nuclear Overhauser effect
data for the natural products2 provided little support for the
proposed stereochemistry. Most importantly, the 1H NMR
spectra of our synthetic intermediates leading to 3 displayed
significantly different H-6 and H-21 splitting patterns than the
natural products. We therefore decided to first study the relative
stereochemistry of nakiterpiosin and nakiterpiosinone using
model systems.
(7) (a) Masamune, T.; Takasugi, M.; Murai, A.; Kobayashi, K. J. Am.
Chem. Soc. 1967, 89, 4521–4523. (b) Masamune, T.; Takasugi, M.;
Murai, A. Tetrahedron 1971, 27, 3369–3386. (c) Johnson, W. S.;
deJongh, H. A. P.; Coverdale, C. E.; Scott, J. W.; Burckhardt, U. J. Am.
Chem. Soc. 1967, 89, 4523–4524. (d) Johnson, W. S.; Cox, J. M.;
Graham, D. W.; Whitlock, H. W., Jr. J. Am. Chem. Soc. 1967, 89,
4524–4526. (e) Masamune, T.; Mori, Y.; Takasugi, M.; Murai, A.;
Ohuchi, S.; Sato, N.; Katsui, N. Bull. Chem. Soc. Jpn. 1965, 38, 1374–
1378. (f) Kutney, J. P.; Cable, J.; Gladstone, W. A. F.; Hanssen, H. W.;
Nair, G. V.; Torupka, E. J.; Warnock, W. D. C. Can. J. Chem. 1975,
53, 1796–1817. (g) Giannis, A.; Heretsch, P.; Sarli, V.; Sto¨ssel, A.
Angew. Chem., Int. Ed. 2009, 48, 7911–7914.
(8) For synthetic studies of nakiterpiosin, see: (a) Ito, T.; Ito, M.; Arimoto,
H.; Takamura, H.; Uemura, D. Tetrahedron 2007, 48, 5465–5469. (b)
Takamura, H.; Yamagami, Y.; Ito, T.; Ito, M.; Arimoto, H.; Kadota,
I.; Uemura, D. Heterocycles 2009, 77, 351–364.
(9) For a review of structure revisions of natural products through synthetic
studies, see: Nicolaou, K. C.; Snyder, S. A. Angew. Chem., Int. Ed.
2005, 44, 1012–1044.
(10) For reviews of halogenated natural product biosynthesis, see: (a)
Vaillancourt, F. H.; Yeh, E.; Vosburg, D. A.; Garneau-Tsodikova, S.;
Walsh, C. T. Chem. ReV. 2006, 106, 3364–3378. (b) Neumann, C. S.;
Fujimori, D. G.; Walsh, C. T. Chem. Biol. 2008, 15, 99–109. (c) Butler,
A.; Walker, J. V. Chem. ReV. 1993, 93, 1937–1944.
(11) For details, see the Supporting Information.
In order to probe the C-6 stereochemistry of the natural
products, we synthesized a series of compounds (7-10)11 that
bear the A,B,C ring systems of nakiterpiosin and nakiterpiosi-
none. We found that the JH6-H7 values of 7 and 9, which bear
the same C-6 configuration as nakiterpiosin and nakiterpiosi-
none, were very different from those of the natural products
(12) The chemical shifts of H-6 in 7-10 and the residual H2O are too
close in CD3OD for the accurate measurement of the coupling
constants. We therefore recorded their 1H NMR spectra in CDCl3.
We reasoned that the rigid polycyclic ring systems of 7-10 would
adopt conformations similar to those of the natural products even in
different solvents. Therefore, the coupling constants should not be
significantly influenced by the NMR solvent.
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Studies of Nakiterpiosin and Nakiterpiosinone
A R T I C L E S
Figure 2. Probing the C-20/C-22/C-23 configurations of nakiterpiosin and nakiterpiosinone.
Table 2. Studies of the C-25 Configurations of Nakiterpiosin and Nakiterpiosinone
3JH-H coupling constants and 1H and 13C chemical shifts of 11
(syn-syn), 12 (syn-anti) and 13 (anti-syn) were considerably
different from those of the natural products (Figure 2). In
particular, the JH20-H21 values for 11 (syn-syn) and 12
(syn-anti) indicated a gauche instead of an anti H-20/H-21
conformation. Only 14 (anti-anti) exhibited 1H and 13C NMR
spectra similar to those of the natural products.13 We therefore
revised the C-20 stereochemistry of nakiterpiosin and nakiter-
piosinone to be S.
significantly different. We therefore determined that the C-25
stereocenters in nakiterpiosin and nakiterpiosinone have the S
configuration.
The revised structures of nakiterpiosin 1 and nakiterpiosinone
2 have the same configurations at C-20 and C-25 as 5 and 6.
The C-6 and C-20 configurations are also consistent with the
stereochemical outcomes of enzymatic halogenation of the
steroid (Figure 4).9 The C-21 chlorine atoms of nakiterpiosin
are likely introduced by nonheme iron halogenase through
radical chlorination. It was expected that this reaction would
lead to retention of the C-20 configuration. On the other hand,
the C-6 bromine atom is likely introduced by vanadium-
dependent bromoperoxidase through bromoetherification. It is
therefore expected that the C-5,6 bromohydrin would exist in
an anti stereochemical relationship.
After revising the C-20 stereochemistry, we turned our
attention to the C-25 configuration of nakiterpiosin and nak-
iterpiosinone and synthesized the two model substrates 15 and
16.11 We found that the coupling constants and 1H and 13C NMR
chemical shifts of (25S)-16 (anti-anti-trans) but not (25R)-15
(anti-anti-cis) matched well with those of the natural products
(Table 2 and Figure 3). In particular, the JH23-H24a, JH23-H24b
,
On the basis of these results, we proposed the revision of
the relative stereochemistry of nakiterpiosin and nakiterpiosinone
to that shown for 1 and 2, respectively. Indeed, we found that
the 1H and 13C NMR spectra of our synthetic sample of 1 agree
with those of the natural product. In contrast, those of synthetic
and JH23-H25 values for 15 and the natural products were
(13) The 13C NMR spectrum of nakiterpiosin was misreferenced by ca.
-2 ppm in the original reports. We adjusted the reported data by +2.0
ppm in this article.
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Gao et al.
Figure 3. Probing the C-25 configurations of nakiterpiosin and nakiterpiosinone.
aromaticity and would therefore have an activation energy
significantly higher than that of the divinyl systems.22 However,
we believed that a suitable set of reaction conditions could be
found to realize this highly efficient plan. We also expected
that 17 could be used as a versatile intermediate for synthesizing
not only 1 but also 2.
It should be noted that the carbonylative cross-coupling of
18 and 19 is also considerably challenging because of their
sensitive functionalities and congested reaction sites. Compared
with the recent advancement in transition-metal-catalyzed
cross-coupling,24,25 catalytic carbonylative cross-coupling is
relatively underdeveloped.15 Suppression of the direct coupling
reactions when more nucleophilic coupling components are used
remains challenging. In general, the Stille and Suzuki carbo-
nylative coupling reactions are more successful because of the
lower rate of transmetalation. We later found that the nearly
Figure 4. Biogenetic analysis of nakiterpiosin.
(16) For the development of carbonylative Stille coupling, see: (a) Tanaka,
M. Tetrahedron Lett. 1979, 20, 2601–2602. (b) Beletskaya, I. P. J.
Organomet. Chem. 1983, 250, 551–564. (c) Baillargeon, V. P.; Stille,
J. K. J. Am. Chem. Soc. 1983, 105, 7175–7176. (d) Sheffy, F. K.;
Godschalx, J. P.; Stille, J. K. J. Am. Chem. Soc. 1984, 106, 4833–
4840. (e) Goure, W. F.; Wright, M. E.; Davis, P. D.; Labadie, S. S.;
Stille, J. K. J. Am. Chem. Soc. 1984, 106, 6417–6422. (f) Crisp, G. T.;
Scott, W. J.; Stille, J. K. J. Am. Chem. Soc. 1984, 106, 7500–7506.
(g) Merrifield, J. H.; Godschalx, J. P.; Stille, J. K. Organometallics
1984, 3, 1108–1112. (h) Echavarren, A. M.; Stille, J. K. J. Am. Chem.
Soc. 1988, 110, 1557–1565.
Figure 5. Retrosynthetic analysis for nakiterpiosin.
3 and the natural product are significantly different. We thus
revised the relative stereochemistry of nakiterpiosin to be that
indicated in 1, which has the same configuration at C-20 and
C-25 as cyclopamine 5 and veratramine 6.
(17) For further examples of carbonylative Stille coupling, see: (a) Smith,
A. B., III; Cho, Y. S.; Ishiyama, H. Org. Lett. 2001, 3, 3971–3974.
(b) Ceccarelli, S.; Piarulli, U.; Gennari, C. J. Org. Chem. 2000, 65,
6254–6256. (c) Kang, S.-K.; Ryu, H.-C.; Lee, S.-W. J. Chem. Soc.,
Perkin Trans. 1 1999, 2661–2663. (d) Xiang, A. X.; Watson, D. A.;
Ling, T.; Theodorakis, E. A. J. Org. Chem. 1998, 63, 6774–6775. (e)
Angle, S. R.; Fevig, J. M.; Knight, S. D.; Marquis, R. W.; Overman,
L. E. J. Am. Chem. Soc. 1993, 3966–3976.
Synthetic Plans. After determining the correct stereochemistry
of nakiterpiosin and nakiterpiosinone, we sought to develop a
general strategy to target these two steroid derivatives. Our first-
generation approach3 is outlined in Figure 5. We opted to
construct the central cyclopentanone ring at a late stage for
convergence reasons. We envisioned that a carbonylative cross-
coupling reaction14-20 together with a Nazarov cyclization
reaction21-23 could be used to effectively assemble this cyclo-
pentanone ring. We recognized that the Nazarov cyclization of
vinyl aryl ketone 17 would involve a disruption of the
(18) For examples of carbonylative Negishi coupling, see: (a) Wang, Q.;
Chen, C. Tetrahedron Lett. 2008, 49, 2916–2921. (b) Jackson,
R. F. W.; Turner, D.; Block, M. H. J. Chem. Soc., Perkin Trans. 1
1997, 865–870. (c) Yasui, K.; Fugami, K.; Tanaka, S.; Tamaru, Y. J.
Org. Chem. 1995, 60, 1365–1380. (d) Tamaru, Y.; Ochiai, H.; Yoshida,
Z.-i. Tetrahedron Lett. 1984, 25, 3861–3864. (e) Tamaru, Y.; Ochiai,
H.; Yamada, Y.; Yoshida, Z.-i. Tetrahedron Lett. 1983, 24, 3869–
3872.
(19) For examples of carbonylative Suzuki coupling, see: (a) O’Keefe,
B. M.; Simmons, N.; Martin, S. F. Org. Lett. 2008, 10, 5301–5304.
(b) Dai, M.; Liang, B.; Wang, C.; You, Z.; Xiang, J.; Dong, G.; Chen,
J.; Yang, Z. AdV. Synth. Catal. 2004, 346, 1669–1673. (c) Ishiyama,
T.; Kizaki, H.; Hayashi, T.; Suzuki, A.; Miyaura, N. J. Org. Chem.
1998, 63, 4726–4731. (d) Ishikura, M.; Terashima, M. J. Org. Chem.
1994, 59, 2634–2637. (e) Suzuki, A. Pure Appl. Chem. 1994, 66, 213–
222. (f) Kondo, T.; Tsuji, Y.; Watanabe, Y. J. Organomet. Chem.
1988, 345, 397–403. (g) Wakita, Y.; Yasunaga, T.; Akita, M.; Kojima,
M. J. Organomet. Chem. 1986, 301, C17–C20.
(14) For a review of palladium-catalyzed cross-coupling reactions in natural
product synthesis, see: Nicolaou, K. C.; Bulger, P. G.; Sarlah, D.
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neutral conditions of the Stille carbonylative reaction are crucial
to the successful implementation of this strategy for the coupling
of highly acid- and base-sensitive 18 and 19.
kaiyama aldol reaction.28,29 It should be noted that intramo-
lecular Diels-Alder reactions of furan derivatives are normally
exo-selective and much less kinetically and thermodynamically
favored because of the aromaticity of furan and the ring strain
of the product.26b The exo selectivity should lead to the desired
relative stereochemistry between the oxo bridge and angular
methyl group. We planned to use the C-6 stereogenic center to
control the diastereoselectivity of this reaction. The use of a
substituent group on the tether R to the diene to control the
diastereoselectivity was first studied by Roush, Taber, and
Boeckman.27 Since the C-6 bromine atom resides at the axial
position, we anticipated that introduction of the bromine atom
into the cycloaddition product with an inversion of configuration
would set up the correct C-6 configuration.
In terms of the vinylogous Mukaiyama aldol reaction, we
planned to use the C-20 stereocenter to control the installation
of the C-22 and C-23 stereocenters. We were able to use this
versatile approach to obtain a diverse array of C-20/C-22/C-23
diastereomers (11-16: syn-syn, syn-anti, anti-syn, anti-anti)
for the previously described model studies.11 The absolute
stereochemistries of 18 and 19 were established by the Noyori
reduction30 and Sharpless epoxidation31 reactions.
First-Generation Synthesis of Nakiterpiosin. We previously
reported our first synthetic approach to 1 (Scheme 1).3 This
sequence commenced with the Friedel-Crafts acylation of furan
(20) with succinic anhydride (21)32 and the formation of
Weinreb amide 23 from acid 22. Subsequently, a Noyori
reduction30 was used to set the C-6 stereochemistry. While a
significant amount of dehydrated lactone byproduct was formed
under the conventional NEt3/HCOOH azeotropic conditions,30a,b
we found that the in-water protocol developed by Xiao30c-e
provided significant enhancement of the reaction rate and
allowed low catalyst loading. Alcohol 24 was obtained in 91%
ee without the formation of the undesired lactone. The dieneno-
For the synthesis of the coupling fragments, we envisioned
that the electrophilic coupling component 17 could be synthe-
sized by an intramolecular Diels-Alder reaction26,27 and the
nucleophilic coupling component 18 by a vinylogous Mu-
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S.; Hiyama, T. Tetrahedron 1992, 48, 2113–2126. (g) Satoh, T.; Itaya,
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M. Angew. Chem., Int. Ed. 2008, 47, 4887–4891.
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2005, 2193–2206. (d) Habermas, K. L.; Denmark, S. E.; Jones, T. K.
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Marcus, A. P.; Lee, A. S.; Davis, R. L.; Tantillo, D. J.; Sarpong, R.
Angew. Chem., Int. Ed. 2008, 47, 6379–6383. (b) He, W.; Herrick,
I. R.; Atesin, T. A.; Caruana, P. A.; Kellenberger, C. A.; Frontier,
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Seiple, I. B.; Trauner, D. J. Am. Chem. Soc. 2006, 128, 11022–11023.
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Crandall, J. K.; Haseltine, R. P. J. Am. Chem. Soc. 1968, 90, 6251–
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(c) Smith, A. B., III; Agosta, W. C. J. Am. Chem. Soc. 1973, 95, 1961–
1968. (d) Leitich, J.; Heise, I.; Werner, S.; Kru¨ger, C.; Schaffner, K.
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I.; Rust, J.; Schaffner, K. Eur. J. Org. Chem. 2001, 2719–2726.
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(b) Frisch, A. C.; Beller, M. Angew. Chem., Int. Ed. 2005, 44, 674–
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2000, 100, 3187–3204.
(28) For reviews, see: (a) Brodmann, T.; Lorenz, M.; Scha¨ckel, R.; Simsek,
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Mini-ReV. Org. Chem. 2008, 5, 1–18. (c) Denmark, S. E.; Heemstra,
J.; John, R.; Beutner, G. L. Angew. Chem., Int. Ed. 2005, 44, 4682–
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Casiraghi, G. Chem. Soc. ReV. 2000, 29, 109–118.
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see: (a) Chen, X.; Engle, K. M.; Wang, D.-H.; Yu, J.-Q. Angew. Chem.,
Int. Ed. 2009, 48, 5094–5115. (b) Li, C.-J. Acc. Chem. Res. 2009, 42,
335–344. (c) Lewis, J. C.; Bergman, R. G.; Ellman, J. A. Acc. Chem.
Res. 2008, 41, 1013–1025. (d) Park, Y. J.; Park, J.-W.; Jun, C.-H.
Acc. Chem. Res. 2008, 41, 222–234. (e) Alberico, D.; Scott, M. E.;
Lautens, M. Chem. ReV. 2007, 107, 174–238. (f) Seregin, I. V.;
Gevorgyan, V. Chem. Soc. ReV. 2007, 36, 1173–1193. (g) Yu, J.-Q.;
Giri, R.; Chen, X. Org. Biomol. Chem. 2006, 4, 4041–4047. (h) Dick,
A. R.; Sanford, M. S. Tetrahedron 2006, 62, 2439–2463. (i) Daugulis,
O.; Zaitsev, V. G.; Shabashov, D.; Pham, Q.-N.; Lazareva, A. Synlett
2006, 3382–3388. (j) Goj, L. A.; Gunnoe, T. B. Curr. Org. Chem.
2005, 9, 671–685. (k) Ritleng, V.; Sirlin, C.; Pfeffer, M. Chem. ReV.
2002, 102, 1731–1769. (l) Dyker, G. Angew. Chem., Int. Ed. 1999,
38, 1698–1712.
(29) For examples of the vinylogous Mukaiyama aldol reaction of 2-si-
loxyfuran, see: (a) Ollevier, T.; Bouchard, J.-E.; Desyroy, V. J. Org.
Chem. 2008, 73, 331–334. (b) Evans, D. A.; Dunn, T. B.; Kværnø,
L.; Beauchemin, A.; Raymer, B.; Olhava, E. J.; Mulder, J. A.; Juhl,
M.; Kagechika, K.; Favor, D. A. Angew. Chem., Int. Ed. 2007, 46,
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4698–4703. (c) Lo´pez, C. S.; Alvarez, R.; Vaz, B.; Faza, O. N.; de
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Lera, A. R. J. Org. Chem. 2005, 70, 3654–3659. (d) Kong, K.; Romo,
D. Org. Lett. 2006, 8, 2909–2912. (e) Evans, D. A.; Kozlowski, M. C.;
Murry, J. A.; Burgey, C. S.; Campos, K. R.; Connell, B. T.; Staples,
R. J. J. Am. Chem. Soc. 1999, 121, 669–685. (f) Evans, D. A.; Burgey,
C. S.; Kozlowski, M. C.; Tregay, S. W. J. Am. Chem. Soc. 1999, 121,
686–699. (g) Casiraghi, G.; Colombo, L.; Rassu, G.; Spanu, P. J. Org.
Chem. 1991, 56, 6523–6527. (h) Rassu, G.; Spanu, P.; Casiraghi, G.;
Pinna, L. Tetrahedron 1991, 47, 8025–8030. (i) Jefford, C. W.; Jaggi,
D.; Boukouvalas, J. Tetrahedron Lett. 1987, 28, 4037–4040.
(30) (a) Hashiguchi, S.; Fujii, A.; Takehara, J.; Ikariya, T.; Noyori, R. J. Am.
Chem. Soc. 1995, 117, 7562–7563. (b) Fujii, A.; Hashiguchi, S.;
Uematsu, N.; Ikariya, T.; Noyori, R. J. Am. Chem. Soc. 1996, 118,
2521–2522. (c) Wu, X.; Li, X.; Hems, W.; King, F.; Xiao, J. Org.
Biomol. Chem. 2004, 2, 1818–1821. (d) Wu, X.; Li, X.; King, F.;
Xiao, J. Angew. Chem., Int. Ed. 2005, 44, 3407–3411. (e) Ohkuma,
T.; Utsumi, N.; Tsutsumi, K.; Murata, K.; Sandoval, C.; Noyori, R.
J. Am. Chem. Soc. 2006, 128, 8724–8725.
(26) For reviews, see: (a) Takao, K.-i.; Munakata, R.; Tadano, K.-i. Chem.
ReV. 2005, 105, 4779–4807. (b) Keay, B. A.; Hunt, I. R. AdV.
Cycloaddit. 1999, 6, 173–210. (c) Roush, W. R. AdV. Cycloaddit. 1990,
2, 91–146. (d) Craig, D. Chem. Soc. ReV. 1987, 16, 187–238.
(27) For the development of tether-controlled diastereoselective intramo-
lecular Diels-Alder reactions, see: (a) Roush, W. R. J. Org. Chem.
1979, 44, 4008–4010. (b) Roush, W. R.; Hall, S. E. J. Am. Chem.
Soc. 1981, 103, 5200–5211. (c) Roush, W. R.; Kageyama, M.; Riva,
R.; Brown, B. B.; Warmus, J. S.; Moriarty, K. J. J. Org. Chem. 1991,
56, 1192–1210. (d) Taber, D. F.; Gunn, B. P. J. Am. Chem. Soc. 1979,
101, 3992–3993. (e) Taber, D. F.; Saleh, S. A. J. Am. Chem. Soc.
1980, 102, 5085–5088. (f) Boeckman, R. K., Jr.; Napier, J. J.; Thomas,
E. W.; Sato, R. I. J. Org. Chem. 1983, 48, 4152–4154. (g) Boeckman,
R. K., Jr.; Barta, T. E. J. Org. Chem. 1985, 50, 3421–3423.
(31) (a) Katsuki, T.; Sharpless, K. B. J. Am. Chem. Soc. 1980, 102, 5974–
5976. (b) Gao, Y.; Klunder, J. M.; Hanson, R. M.; Masamune, H.;
Ko, S. Y.; Sharpless, K. B. J. Am. Chem. Soc. 1987, 109, 5765–5780.
(c) Katsuki, T.; Martin, V. S. Org. React. 1996, 48, 1–300.
(32) Chen, L.; Yin, B.-l.; Xu, H.-h.; Chiu, M.-h.; Wu, Y.-l. Chin. J. Chem.
2004, 22, 92–99.
9
J. AM. CHEM. SOC. VOL. 132, NO. 1, 2010 375

A R T I C L E S
Gao et al.
Scheme 1. Synthesis of the Electrophilic Coupling Component 30
type rearrangement using Yamamoto’s conditions.33 We pre-
viously reported that aldehyde 38 was obtained with significant
erosion of enantiomeric purity (71% ee).3 We have since
determined that the loss of ee occurred during workup of the
reaction and purification of 38 by column chromatography.
Quenching the reaction with 1 N HCl aqueous solution instead
of sodium fluoride afforded 38 with 90% ee. Aldehyde 38 should
be used directly without purification. We also found that while
low conversions were observed when catalytic amounts of
Yamatomo’s aluminum salt were used for the reaction of 37,
only 5 mol % Cr(TPP)(OTf)33e was needed to drive this reaction
to completion. However, a signification amount of ketone was
also obtained (aldehyde/ketone ) 3:1).
The previously reported problem of the subsequent viny-
lougous Mukaiyama aldol reaction was also solved. The
bismuth(III) triflate-catalyzed reaction between purified 38 (71%
ee) and 3-methyl-2-(triisopropylsiloxy)furan yielded 39 with
only 56% conversion, 11:1 dr, and 60% ee.3 We recently found
that 2 equiv of tin(II) triflate promoted this reaction with 83%
conversion. Alcohol 39 was obtained from crude 38 as the only
diastereomer in 80% isolated yield and 90% ee. For large-scale
reactions, we found that boron trifluoride diethyl etherate was
a more economical Lewis acid. Full conversion could be
obtained and no loss of ee was observed. It provided 39 in
comparable yields despite having a diastereoselectivity of only
4:1. Results from the survey of fourteen different Lewis acids
can be found in the Supporting Information.
Table 3. Influence of the Lewis Acid on the Intramolecular
Diels-Alder Reaction of 25
entry
Lewis acid
equiv
25/26
note
1
2
3
4
5
Me2AlCl
Me2AlCl
Me2AlCl
MeAlCl2
MeAlCl2
2.5
1.0
0.1
1.0
0.1
0:100
30:70
100:0
15:85
100:0
with decomposition
phile was then introduced into 24 by addition of isopropenyl
Grignard reagent, giving enone 25 and setting the stage for the
Diels-Alder cyclization.
It has been shown that the conversion in the intramolecular
furan Diels-Alder reaction is highly dependent on the relative
basicity of the reactant and product and the stoichiometry of
the Lewis acids.26b We found that for the reaction of 25, 2.5
equiv of Me2AlCl provided the best yield of 26 (Table 3),
indicating that the reaction was driven by the complexation of
Me2AlCl to 26. The reaction proceeded with good stereochem-
ical control and gave only the desired exo product with an
equatorial hydroxyl group. When the C-6 hydroxyl group of
25 was protected as a triethylsilyl (TES) ether, the reaction
proceeded with the opposite enantiomeric preference and
provided the exo product with an axial TES ether group.11
Since the cycloaddition product 26 was not stable and
underwent a retro-Diels-Alder reaction upon gentle heating or
exposure to other Lewis acids, we dihydroxylated the olefin
before the introduction of the bromine atom to prevent the retro-
Diels-Alder reaction. The steric congestion around the C-6
position necessitated the use of an electron-deficient arylsul-
fonate group (27) for 6-OH activation. Thus, treatment of 28
with lithium bromide in warm acetone allowed the introduction
of the bromine atom with inversion of configuration and
concomitant acetonide protection to give 29. The use of an
electron-deficient sulfonate was crucial to the success of this
SN2 reaction. 4-Toluenesulfonate (Ts) was unreactive and
4-nitrophenylsulfonate (Ns) less reactive under these reaction
conditions. The absolute and relative configurations of 29 were
confirmed by X-ray analysis.11 Finally, enol triflate installation
completed the synthesis of the electrophilic coupling component
30.
We previously used the Crabtree catalyst29b,34 to direct the
hydrogenation of 39 and set the desired C-25 configuration of
40 with moderate diastereoselectivity (6:1 dr). We later found
that 40 could be obtained as the only diastereomer by conjugate
reduction with L-Selectride. Overall, these new protocols are
much more cost-effective and provide significantly improved
yields and stereoselectivities of the desired products.
To invert the C-22 configuration, we oxidized 40 with
Dess-Martin periodinane and reduced the resulting ketone with
sodium borohydride. We then protected the C-22 alcohol as a
TBS ether, selectively removed the C-21 TBS protecting group,
and oxidized the primary alcohol. Introduction of the C-21 gem-
dichloride was achieved by reacting aldehyde 45 with Cl2/
The synthesis of the nucleophilic coupling component 47
(Scheme 2) started with reduction of acid 31 to aldehyde 33
followed by Horner-Wadsworth-Emmons reaction and reduc-
tion of the resulting ester to afford 35. Asymmetric installation
of the C-20 stereocenter was achieved by Sharpless epoxidation
with 92% ee.31 The resulting alcohol was protected as a tert-
butyldimethylsilyl (TBS) ether and then subjected to the pinacol-
(33) (a) Maruoka, K.; Ooi, T.; Yamamoto, H. J. Am. Chem. Soc. 1989,
111, 6431–6432. (b) Maruoka, K.; Ooi, T.; Nagahara, S.; Yamamoto,
H. Tetrahedron 1991, 47, 6983–6998. (c) Maruoka, K.; Ooi, T.;
Yamamoto, H. Tetrahedron 1992, 48, 3303–3312. (d) Maruoka, K.;
Murase, N.; Bureau, R.; Ooi, T.; Yamamoto, H. Tetrahedron 1994,
50, 3663–3672. (e) Suda, K.; Kikkawa, T.; Nakajima, S.-i.; Takanami,
T. J. Am. Chem. Soc. 2004, 126, 9554–9555.
(34) (a) Crabtree, R. Acc. Chem. Res. 1979, 12, 331–337. (b) Crabtree,
R. H.; Davis, M. W. Organometallics 1983, 2, 681–682.
9
376 J. AM. CHEM. SOC. VOL. 132, NO. 1, 2010

Studies of Nakiterpiosin and Nakiterpiosinone
A R T I C L E S
Scheme 2. Synthesis of the Nucleophilic Coupling Component 47
Scheme 3. Model Studies of the Carbonylative Coupling/Nazarov
Cyclization Strategy
use of catalytic amounts of Pd(PPh3)4 gave only small amounts
of the desired product. No reaction occurred when Pd(OAc)2
was used in combination with AsPh3, P(furyl)3, PCy3, and
P(t-Bu)3 under otherwise identical conditions. The use of
Pd(OAc)2/P(OMe)3 led mainly to decomposition. It is also worth
noting that 49 was highly sensitive to both acids and bases.
Addition of lithium chloride, prolonged heating, and employ-
ment of base-promoted carbonylative Suzuki coupling protocols
led to the elimination of the bromine atom.
The sensitive nature and aromaticity of 49 made the imple-
mentation of the Nazarov cyclization strategy challenging.
Normally, Lewis acid-promoted Nazarov cyclizations of aryl
vinyl ketones require harsh reaction conditions or activated
substrates.22 Indeed, exposure of 49 to various Lewis acids
resulted only in substrate decomposition. However, we found
that irradiation of a solution of 49 in acetonitrile at 350 nm
readily delivered the desired annulation product 50 in its enol
form, which tautomerized to the ketone form as a 1:1 mixture
of C-9 diastereomers upon addition of ammonium chloride.
Treating this mixture with diisopropylamine in warm methanol
gave the desired diastereomer 50, the structure of which was
confirmed by X-ray analysis.11 The reaction can also be carried
out in methanol to directly give the C-9 diastereomeric mixture
of 50 with slightly lower yield. The photo-Nazarov cyclization
reaction of aryl vinyl ketones was first reported by Smith and
Agosta.23c Subsequent mechanistic studies by Leitich and
Schaffner23d,e revealed the reaction mechanism to be a thermal
electrocyclization induced by photolytic enone isomerization.
The mildness of the reaction conditions and the selective
activation of the enone functional group were key to the success
of this reaction.
P(OPh)3.35 The hydrazone method developed by Myers36 was
not compatible with the other functional groups of 45. The
relative and absolute stereochemistries of 46 were determined
by single-crystal X-ray analysis after TBS deprotection.11 The
synthesis of the nucleophilic coupling component 47 was
concluded by a palladium-catalyzed stannylation of 46. Despite
the high catalyst loading and moderate yield, this protocol
continues to be the best method for the introduction of the
trimethylstannane to 47.
After the routes to both cross-coupling fragments had been
established, our next goal was to realize the carbonylative
coupling and Nazarov cyclization strategy to assemble the C
ring of 1 and 2. We used several model systems carrying a
simplified side chain on the E ring to study these two key
reactions (Scheme 3). We found that the carbonylative coupling
of 30 and 48 could be achieved with a modified Stille’s protocol
to afford 49.16 After an extensive survey of the reaction
conditions, we concluded that Pd(PPh3)4 is the best promoter,
that copper(I) chloride additive37 is crucial, and that dimethyl
sulfoxide (DMSO) is the ideal solvent for this reaction. The
To complete the synthesis of 1, we carried out the carbony-
lative coupling of 30 and 47 under the optimized conditions to
give 51, which was effectively transformed to 53 upon pho-
tolysis and C-9 epimerization (Scheme 4). The previous
(35) (a) Spaggiari, A.; Vaccari, D.; Davoli, P.; Torre, G.; Prati, F. J. Org.
Chem. 2007, 72, 2216–2219. (b) Hoffmann, R. W.; Bovicelli, P.
Synthesis 1990, 657–659.
(37) (a) Han, X.; Stoltz, B. M.; Corey, E. J. J. Am. Chem. Soc. 1999, 121,
7600–7605. (b) Farina, V.; Kapadia, S.; Krishnan, B.; Wang, C.;
Liebeskind, L. S. J. Org. Chem. 1994, 59, 5905–5911. (c) Liebeskind,
L. S.; Fengl, R. W. J. Org. Chem. 1990, 55, 5359–5364. (d) Marino,
J. P.; Long, J. K. J. Am. Chem. Soc. 1988, 110, 7916–7917.
(36) (a) Furrow, M. E.; Myers, A. G. J. Am. Chem. Soc. 2004, 126, 5436–
5445. For an example of the use of the original Barton-Pross-
Sternhell protocol, see: (b) Kropp, P. J.; Pienta, N. J. J. Org. Chem.
1983, 48, 2084–2090.
9
J. AM. CHEM. SOC. VOL. 132, NO. 1, 2010 377

A R T I C L E S
Gao et al.
Scheme 4. Completion of the Synthesis of Nakiterpiosin (1)
Scheme 5. An Improved Synthesis of Nakiterpiosin (1)
stereochemical issues involving 51 were solved (99.5% ee and
dr 10:1) because 47 could be prepared with high enantiomeric
purity.
With the central C ring in place, the remaining task was to
assemble the A ring of 1. We first removed the acetonide
protecting group of 53 and cleaved the diol of 54 to afford
bishemiacetal 55. Selective reduction of the less hindered
hemiacetal of 55 followed by TBS deprotection38 concluded
the synthesis of 1. A single crystal suitable for X-ray analysis11
was recently obtained by evaporation of the solvent from a 30%
ethyl acetate/hexanes solution of 1, allowing unambiguous
determination of its relative and absolute configurations.
To confirm our proposal for the structure revision of 1, we
compared the 1H and 13C NMR spectra of our synthetic 1 with
those of the natural product.2 We were pleased to find that the
chemical shift differences for all peaks were within 0.03 and
1
0.2 ppm for the H and 13C signals, respectively, and that the
1
splitting patterns for all of the H signals were also consistent
with those of the natural product. While the optical rotation of
the natural product was not reported, the Mosher ester analysis
of our synthetic 1 was consistent with the data reported for the
natural product. The ∆δSR of H-4 was found to be +0.03 ppm
in the 4-Mosher ester of 1, compared to +0.04 ppm for that of
H-4 in the 4,22-di-Mosher ester of the natural product. We also
determined that nakiterpiosin 1 is levorotatory in methanol with
[R]2D0 ) -33° (c 0.067, MeOH).
Second-Generation Synthesis of Nakiterpiosin. While the
sequence shown in Scheme 4 successfully delivered 1, it
required an additional six steps after the convergent fragment
coupling reaction. We anticipated that all of the functional
groups of 1 could tolerate the carbonylative coupling and photo-
Nazarov cyclization reactions. We therefore decided to perform
the C-ring construction on a fully elaborated system to improve
the overall efficiency of the synthesis of 1 (Scheme 5).
In ketone 29, we introduced the A-ring functionality using
the chemistry previously described, generating 10. We then
protected the hemiacetal and installed the triflate to give 57.
We also removed the TBS protecting group of 47 prior to the
carbonylative coupling. Pleasingly, the fragment coupling and
photo-Nazarov cyclization proceeded in good yields. Subsequent
TES deprotection provided 1 with significantly improved
efficiency. However, it was unfortunate that photolysis of des-
TES-58 resulted in significant amounts of decomposition.
Synthesis of 6,20,25-epi-Nakiterpiosin. To unambiguously
determine the relative stereochemistry of nakiterpiosin, we also
synthesized the originally proposed structure (6,20,25-epi-
nakiterpiosin, 3) and compared its NMR spectra with those of
the natural product. Several approaches were explored in an
attempt to introduce the C-6 bromine atom. We first sought to
use the C-6 bromine atom as the stereochemistry-controlling
element for the intramolecular Diels-Alder reaction and set its
equatorial configuration. However, nucleophilic substitution of
the C-6 hydroxyl group of 24 under various conditions failed
to deliver the desired product. The use of a chelating leaving
group39 did not result in retention of configuration, and an SN2
instead of SNi reaction occurred. Equally fruitless was the effort
to replace the hydroxyl group of 26 or its derivatives with a
bromine atom through radical chemistry. Eventually, we found
that the desired C-6 configuration could be set through isomer-
ization of vinyl bromide 62 (Scheme 6).
(39) (a) Lepore, S. D.; Mondal, D. Tetrahedron 2007, 63, 5103–5122. (b)
Braddock, D. C.; Pouwer, R. H.; Burton, J. W.; Broadwith, P. J. Org.
Chem. 2009, 74, 6042–6049.
(38) For a convenient workup method, see: Kaburagi, Y.; Kishi, Y. Org.
Lett. 2007, 9, 723–726.
9
378 J. AM. CHEM. SOC. VOL. 132, NO. 1, 2010

Studies of Nakiterpiosin and Nakiterpiosinone
A R T I C L E S
Scheme 6. Synthesis of 64 (6-epi-30)
Scheme 7. Synthesis of 6,20,25-epi-Nakiterpiosin (3)
Starting from 26, the olefin was first dihydroxylated and the
resulting diol protected as an acetonide. The C-6 hydroxyl group
of 59 was then protected and the ketone group reduced to afford
60. Subsequent TBS protection, TES deprotection, and alcohol
oxidation provided 61. The vinyl bromide was then introduced
by the Barton-Myers method to give 62.36,40 Removal of the
TBS protecting group and alcohol oxidation under Dess-Martin
conditions induced olefin isomerization to yield enone 63.
Finally, conjugate reduction yielded a crystalline ketone, al-
lowing unambiguous assignment of the C-6 configuration.11
Conversion of the ketone to the enol triflate furnished electro-
philic coupling component 64 (6-epi-30).
To set the syn-anti-cis configuration of the side chain, an
anti-selective vinylogous aldol reaction was need. Despite the
fact that efforts to reverse the syn selectivity failed, we found
that a useful amount of the desired diastereomer could be
obtained with any of the following Lewis acids: boron trifluoride
diethyl etherate, zirconium(IV) chloride, ytterbium(III) triflate,
or scandium(III) triflate.11 Reaction of ent-38 and 3-methyl-2-
(triisopropylsiloxy)furan (Scheme 7) gave syn-anti-65 as the
minor diastereomer (syn-syn/syn-anti ) 4:1). The C-25
configuration was then set by catalytic hydrogenation using
Adams’ catalyst without debromination. Protection of the
alcohol as a TBS ether gave the desired syn-anti-cis-66.
With the protocols previously described for the synthesis of 47,
the gem-dichloromethyl and stannyl groups were then introduced
to provide 68. The relative stereochemistry of 68 was confirmed
by X-ray analysis of a racemic sample after removal of the TBS
protecting group.11 Its carbonylative coupling with 64 and the
subsequent photolysis reaction proceeded uneventfully to yield 70.
Interestingly, cleavage of the diol gave 71 as a bisaldehyde instead
of a bisacetonide. Nevertheless, reduction of 71 with Et3SiH in
the presence of BF3 ·OEt2 induced the acetal formation. After TBS
deprotection, 6,20,25-epi-nakiterpiosin 3 was obtained as an
equilibrium mixture of the C-4 aldehyde and hemiacetal forms.
Through variable-temperature NMR experiments, we determined
the ∆H and ∆S values for this equilibrium to be -2.5 kcal/mol
and -6.0 cal mol-1 K-1, respectively.11 By comparing the 1H and
13C NMR spectra of 1, 3, and the natural product,2 we confirmed
that the structure of nakiterpiosin should be 1 instead of 3 (Table
4 and Figures 6 and 7).
Synthesis of Nakiterpiosinone. After completion of the
synthesis of 1, we turned our attention to the related steroid
nakiterpiosinone 2. Initially, we planned to selectively oxidize
the C-4 hydroxyl group of diol 28 or 54 to accomplish this goal.
However, we encountered considerable difficulties in utilizing
(40) Barton, D. H. R.; O’Brien, R. E.; Sternhell, S. J. Chem. Soc. 1962,
470–476.
9
J. AM. CHEM. SOC. VOL. 132, NO. 1, 2010 379

A R T I C L E S
Table 4. Selected H NMR Chemical Shifts and Coupling Constants of Nakiterpiosin
Gao et al.
1
H no.
natural samplea
synthetic 1b
synthetic 3c
H no.
natural samplea
synthetic 1b
synthetic 3c
6
4.70 (dd)
4.71 (dd)
4.74 (dd)
21
6.32 (d)
6.32 (d)
6.66 (d)
J ) 2.7, 1.4 Hz
7a 2.28 (m)
J ) 3.4, 2.5 Hz
2.29 (ddd)
J ) 12.3, 5.1 Hz
2.22 (ddd)
J ) 10.3 Hz
4.39 (dd)
J ) 10.0 Hz
4.41 (dd)
J ) 3.6 Hz
4.52 (dd)
22
J ) 13.7, 12.7, 3.4 Hz J ) 12.3, 12.0, 12.0 Hz
2.75 (ddd) 2.94 (ddd)
J ) 8.0, 3.8 Hz
24a 1.71 (ddd)
J ) 7.9, 3.2 Hz
1.72 (ddd)
J ) 10.2, 2.3 Hz
2.00 (m)
7b 2.74 (ddd)
J ) 13.4, 2.7, 1.4 Hz J ) 13.7, 2.5, 2.3 Hz J ) 12.0, 5.1, 2.7 Hz
3.58 (m) 3.55 (ddd) 3.03 (ddd)
J ) 12.7, 9.4, 2.3 Hz J ) 12.0, 9.1, 2.7 Hz
J ) 12.8, 8.4, 8.2 Hz J ) 13.2, 8.2, 8.1 Hz
8
24b 2.28 (m)
2.29 (ddd)
2.00 (m)
J ) 13.2, 4.8, 3.9 Hz
a Recorded at 800 MHz, as reported by Uemura et al. (ref 2). b Recorded at 600 MHz. c Recorded at 500 MHz.
1
Figure 6. Differences in the chemical shifts in the H NMR spectra of 1, 3, and the natural product.
Figure 7. Differences in the chemical shifts in the 13C NMR spectra of 1, 3, and the natural product.
this strategy to synthesize 2. First, despite the fact that we could
selectively protect the C-3 hydroxyl group and oxidize the C-4
hydroxyl group of 28, we were not able to perform C-3
epimerization without considerable decomposition. Next, our
attempts to introduce a C-3 hydroxyl group to the more hindered
ꢀ-face of 26 by Pre´vost-Woodward dihydroxylation41 were
complicated by the Wagner-Meerwein rearrangement of the
[2.2.1] bicyclic system (Scheme 8).42,43 Equally unsuccessful
was the oxidation of diol 28 to the diketone because of diol
cleavage or decomposition.44 We therefore sought to install the
C-3 stereocenter by an alternative approach.
Scheme 8. Rearrangement of the [2.2.1] Bicyclic System
Since the ꢀ-face of the A ring of 2 is significantly less
hindered than its R-face, we decided to set the desired C-3
stereochemistry by reduction of the corresponding ketone
(Scheme 9). Starting from 27, the enol triflate was first installed,
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Studies of Nakiterpiosin and Nakiterpiosinone
A R T I C L E S
Scheme 9. Synthesis of Nakiterpiosinone (2)
and the olefin of 74 was then hydroborylated with 9-bora[3.3.1]bi-
cyclononane (9-BBN) to afford alcohol 75 as the only regioi-
somer after oxidative cleavage of the borane. Subsequently, the
alcohol was oxidized to the ketone, and the bromine atom was
introduced. The nucleophilic bromination reaction of 76 was
much more difficult than that of 28. Nevertheless, 77 could be
obtained in good yield by reacting 76 with lithium bromide in
2-heptanone at 120 °C.
We next found R-oxidation of ketone 77 to be particularly
challenging. Oxidation by the Rubottom method, Davis reagent,
or selenium(IV) oxide was unsuccessful.45 However, reaction
of 77 with PhI(OAc)2 under basic conditions gave 78 in good
yield.46 While oxidation of the C-4 ketone regioisomer of 77
may deliver the desired protected hydroxyketone directly, we
found that hydroborylation of 74 with the complementary
regioselectivity did not proceed with good efficiency. Depro-
tection of the dimethyl acetal of 78 and subsequent reduction
of the resulting ketone 79 gave cis diol 80 with the desired C-3
configuration. The structure of 80 was confirmed by X-ray
structural analysis of its acetonide derivative. Selective protec-
tion of the C-3 hydroxyl group of 80 afforded the electrophilic
coupling component 81. Carbonylative coupling of 81 with 47
and the subsequent photo-Nazarov cyclization proceeded smoothly
to give 83. Interestingly, ketone 83 with the desired 9S
configuration was obtained directly as the only diastereomer.
Finally, oxidation of the hydroxyl group and removal of the
TES and TBS protecting groups concluded the synthesis of 2.
1
The H NMR spectrum of 2 agreed with that of the natural
product,2b,11 confirming the structure of nakiterpiosinone to
be 2.
Biological Studies of Nakiterpiosin. Uemura and co-workers2
initially reported that nakiterpiosin and nakiterpiosinone inhibit
the growth of P388 mouse leukemia cells with an IC50 of 10
ng/mL. However, the limited amount of material that could be
isolated from the sponge hampered further efforts to investigate
the anticancer potential of these compounds. Having resolved
the resupply issue through total synthesis, we are now pursuing
further mechanistic studies focused on the anti-cell-proliferation
properties associated with 1.
The steroid skeleton of 1 is related to that of cyclopamine 5,
a potent Hh pathway antagonist. 5 inhibits Hh signaling by
influencing the movement of Smo,5a-c a seven-pass transmem-
brane protein, in and out of the primary cilium.5d,e The primary
cilium is a microtubule-based assembly-point organelle for Hh
pathway components.47 Indeed, 1 suppressed Hh signaling at
submicromolar concentrations in NIH3T3 mouse fibroblasts
(Figure 8A). Surprisingly, as part of our efforts to evaluate the
effects of 1 on the movement of Smo with respect to the primary
Figure 8. Effects of nakiterpiosin on Hh signaling and primary cilia. (A) Hh pathway response of Light2 cells (NIH3T3 cells with an Hh-specific firefly
luciferase reporter) in the presence of nakiterpiosin. (B) NIH3T3 cells treated with nakiterpiosin, which show a compromised ability to form primary cilia
as detected by antiacetylated tubulin antibody.
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A R T I C L E S
Gao et al.
Figure 9. DNA profiles of nakiterpiosin-treated HeLa cells.
cilium, we observed that cell populations treated with nakiter-
poisin show a loss of the primary cilium (Figure 8B).
Because the formation and retraction of the primary cilium
is cell-cycle- and microtubule-dependent,48 we suspected 1 to
be an antimitotic agent. Indeed, flow cytometry experiments
suggested that 1 induces cell-cycle arrest in the G2/M phase
(Figure 9). We first determined the cell-growth IC50 of 1 in HeLa
cells to be 375 nM. Subsequently, we incubated the HeLa cells
with 1 at this concentration for 16 h and found the population
of cells in the G2/M phase to be significantly increased relative
to the DMSO control. Since the most common mechanism for
G2/M arrest is the targeting of the microtubule assembly and it
has been reported that JK184, a microtubule depolymerizing
agent,49 effectively regulates Hh signaling, we next examined
the effect of 1 on microtubule dynamics. However, we found
that in contrast to taxol and nocodazole, tubulin polymerization
was not influenced in the presence of 5 µM 1 in an in vitro
assay (Figure 10). Taken together, these results suggest that 1
may be useful in treatment of tumors resistant to existing
antimitotic chemotherapeutic agents. Lastly, because of the
frequency of mutations in Smo that render it resistant to Hh
pathway antagonists currently under clinical development,5c
considerable effort is being devoted to the identification of small
molecules that act upstream or downstream of Smo.50 The ability
Figure 10. In vitro tubulin polymerization assays.
of 1 to block Hh pathway responses suggests that it may be
particularly useful against Hh-dependent tumors.
Summary
Through extensive spectroscopic analysis and convergent total
synthesis, we have established the stereochemistry of nakiter-
piosin (1) and nakiterpiosinone (2). Our synthetic route provides
1 in 21 steps with 5% overall yield along the longest linear
sequence. Our preliminary biological studies show that 1 is
antimitotic and has functions distinct from those of other well-
established antimitotic agents such as taxol. Nakiterpiosin may
be an alternative chemotherapeutic agent against tumors that
are resistant to antitubulin agents and dependent on Hh signaling.
(41) (a) Emmanuvel, L.; Shaikh, T. M. A.; Sudalai, A. Org. Lett. 2005, 7,
5071–5074. (b) Woodward, R. B.; Brutcher, F. V., Jr. J. Am. Chem.
Soc. 1958, 80, 209–211.
(42) (a) Winstein, S.; Trifan, D. S. J. Am. Chem. Soc. 1949, 71, 2953. (b)
Winstein, S.; Trifan, D. S. J. Am. Chem. Soc. 1952, 74, 1147–1154.
(c) Winstein, S.; Trifan, D. S. J. Am. Chem. Soc. 1952, 74, 1154–
1160. (d) Winstein, S.; Clippinger, E.; Howe, R.; Vogelfanger, E.
J. Am. Chem. Soc. 1965, 87, 376–377. (e) Keay, B. A.; Rogers, C.;
Bontront, J.-L. J. J. Chem. Soc., Chem. Commun. 1989, 1782–1784.
(43) (a) Grob, C. A. Acc. Chem. Res. 1983, 16, 426–431. (b) Brown, H. C.
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Acknowledgment. Financial support was provided by the NIH
(NIGMS R01-GM079554 to C.C., NHLBI R01-HL089966 to L.J.-
S.H., and NIGMS R01-GM076398 to L.L.), the Welch Foundation
(I-1596 to C.C.), and UT Southwestern. L.L. is a Virginia
(44) (a) Burns, N. Z.; Baran, P. S. Angew. Chem., Int. Ed. 2008, 47, 205–
208. (b) Moorthy, J. N.; Singhal, N.; Senapati, K. Org. Biomol. Chem.
2007, 5, 767–771.
(48) (a) Quarmby, L. M.; Parker, J. D. K. J. Cell Biol. 2005, 169, 707–
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(45) (a) Rubottom, G. M.; Vazquez, M. A.; Pelegrina, D. R. Tetrahedron
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H. W. J. Org. Chem. 1975, 40, 3427–3429. (c) Davis, F. A.; Chen,
B. C. Chem. ReV. 1992, 92, 919–934. (d) Ruano, J. L. G.; Alema´n, J.;
Fajardo, C.; Parra, A. Org. Lett. 2005, 7, 5493–5496. (e) White, J. D.;
Wardrop, D. J.; Sundermann, K. F. Org. Synth. 2002, 79, 125–129.
(46) Moriarty, R. M.; Prakash, O. Acc. Chem. Res. 1986, 19, 244–250.
(47) Eggenschwiler, J. T.; Anderson, K. V. Annu. ReV. Cell DeV. Biol.
2007, 23, 345–373.
(49) Cupido, T.; Rack, P. G.; Firestone, A. J.; Hyman, J. M.; Han, K.;
Sinha, S.; Ocasio, C. A.; Chen, J. K. Angew. Chem., Int. Ed. 2009,
48, 2321–2324.
(50) Hyman, J. M.; Firestone, A. J.; Heine, V. M.; Zhao, Y.; Ocasio, C. A.;
Han, K.; Sun, M.; Rack, P. G.; Sinha, S.; Wu, J. J.; Solow-Cordero,
D. E.; Jiang, J.; Rowitch, D. H.; Chen, J. K. Proc. Natl. Acad. Sci.
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A R T I C L E S
Murchison Linthicum Scholar in Medical Research, and C.C. is a
Southwestern Medical Foundation Scholar in Biomedical Research.
We thank Prof. John MacMillan and Dr. Ana Paula Espindola for
assistance with the NMR experiments, Dr. Vincent Lynch (UT
Austin) and Dr. Radha Akella for X-ray analysis, and Dr. Michael
G. Roth for helpful discussions. We also thank Prof. Daisuke
Supporting Information Available: Complete refs 6e and 6g,
1H and 13C NMR spectra of 2, calculation of the thermodynamic
properties of 3, details of the optimization of reaction param-
eters, experimental procedures, crystallographic data (CIF), and
characterization data. This material is available free of charge
via the Internet at http://pubs.acs.org.
1
Uemura (Keio University) for providing copies of the H and 13C
NMR spectra of the natural products.
JA908626K
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