Total synthesis and biological evaluation of tyroscherin

Article abstract of DOI:10.1021/ol101801u

Figure Presented. The efficient synthesis and biological evaluation of both the reported and revised structures of tyroscherin have been achieved. Central to our synthesis is a cross metathesis reaction that generated the trans-olefin regioselectively. Th

Full text of DOI:10.1021/ol101801u

ORGANIC
LETTERS
2010
Vol. 12, No. 19
4308-4311
Total Synthesis and Biological
Evaluation of Tyroscherin
Hyun Seop Tae,† John Hines,† Ashley R. Schneekloth,‡ and Craig M. Crews*,†,‡,§
Department of Molecular, Cellular, and DeVelopmental Biology, Department of
Chemistry, and Department of Pharmacology, Yale UniVersity,
New HaVen, Connecticut 06511
craig.crews@yale.edu
Received August 2, 2010
ABSTRACT
The efficient synthesis and biological evaluation of both the reported and revised structures of tyroscherin have been achieved. Central to our
synthesis is a cross metathesis reaction that generated the trans-olefin regioselectively. This synthetic strategy enabled the facile manipulation
of tyroscherin stereochemistry, facilitating the generation of all 16 tyroscherin diastereomers and a photoactivatable tyroscherin-based affinity
probe for future mode of action studies.
Natural products continue to provide promising leads for drug
candidates as well as a powerful arsenal of small molecule
probes to dissect complex biological processes.1 The potent
cytotoxic natural product tyroscherin (1, Figure 1) was
isolated from the culture of a fungus identified as Pseudall-
escheria sp. by the research groups of Hayakawa and
Watanabe in 2004.2 Tyroscherin was originally reported to
inhibit insulin-like growth factor-1 (IGF-1)-induced and fetal
bovine serum (FBS)-induced growth of MCF-7 human breast
cancer cells with IC50 values of 30 nM and 6.2 µM,
respectively.2,3
† Department of Molecular, Cellular, and Developmental Biology.
‡ Department of Chemistry.
The reported highly selective inhibition of IGF1-induced
growth of MCF-7 cancer cells and unique structural features
of tyroscherin led us to embark on its total synthesis
independently of, and in parallel to, the more recently
reported studies by the Watanabe group and the Maier
group.4,5 Our motivation was to investigate the structure-
activity relationship (SAR) of tyroscherin, followed by efforts
to understand its mode of action and IGF-specificity.
§ Department of Pharmacology.
(1) For recent reviews on the natural products, see: (a) Clardy, J.; Walsh,
C. Nature 2004, 432, 829–837. (b) Paterson, I.; Anderson, E. A. Science
2005, 310, 451–453. (c) Ganesan, A. Curr. Opin. Chem. Biol. 2008, 12,
306–317. (d) Li, J. W.-H; Vederas, J. C. Science 2009, 325, 161–165. (e)
Harvey, A. L. Drug DiscoVery Today 2008, 13, 894–901. (f) Koehn, F. E.;
Carter, G. T. Nat. ReV. Drug DiscoVery 2005, 4, 206–220. (g) Wilson, R. M.;
Danishefsky, S. J. J. Org. Chem. 2006, 71, 8329–8351. (h) Driggers, E. M.;
Hale, S. P.; Lee, J.; Terrett, N. K. Nat. ReV. Drug DiscoVery 2008, 7, 608–
624. (i) Tietze, L. F.; Bell, H. P.; Chandrasekhar, S. Angew. Chem. Int. Ed.
2003, 42, 3996–4028. (j) Schreiber, S. L. Nat. Chem. Biol. 2005, 1, 64–66.
(k) Morton, D.; Leach, S.; Cordier, C.; Warriner, S.; Nelson, A. Angew.
Chem. Int. Ed. 2009, 48, 104–109. (l) Nicolaou, K. C.; Chen, J. S.; Edmonds,
D. J.; Estrade, A. A. Angew. Chem. Int. Ed. 2009, 48, 660–719.
(2) Hayakawa, Y.; Yamashita, T.; Mori, T.; Nagai, K.; Shin-Ya, K.;
Watanabe, H. J. Antibiot. 2004, 57, 634–638
.
(3) Yamanouchi Pharmaceutical Co., Ltd., Jpn. JP 2005-272357.
10.1021/ol101801u  2010 American Chemical Society
Published on Web 09/10/2010

Herein, we report a concise total synthesis of the originally
proposed tyroscherin structure, whose spectral data did not
match that of the reported natural product. Subsequent
syntheses of the other 15 stereoisomers of tyroscherin
allowed for the correct structural assignment of tyroscherin,
consistent with a recent reassignment by Watanabe and co-
workers.4 A full biological evaluation of the correct ty-
roscherin was performed, revealing discrepancies with the
previously observed selectivity for IGF-induced growth. In
addition, we report the synthesis of a photoactivatable
tyroscherin-based affinity reagent that will allow for the
identification of tyroscherin binding protein(s). Similar
affinity chromatographic approaches have proven successful
in mode of action studies of other biologically active natural
products.6
The originally reported structure of tyroscherin (1) was
retrosynthetically disconnected at the C6-C7 bond, yielding
two olefinic precursors, 5 and 6, which we envisioned could
be coupled via a cross metathesis (Figure 1).7 The target
would be synthesized through a cross metathesis of two key
fragments 5 and 6, which were chosen to provide convenient
access to the minimum stereoarray needed to support future
structure-activity relationship (SAR) and mode of action
studies.
Scheme 1. Synthesis of Fragment 5
magnesium bromide to the resulting Weinreb amide gave
the ketone 7 in excellent yield. Reduction of the ketone in 7
with L-selectride resulted in a single diastereomer of the
desired secondary alcohol 8. Protection of the hydroxyl group
as an acetate afforded the desired fragment 5. The config-
uration of the newly installed hydroxyl group at C3 was
determined by NOE experiments on the oxazolidinone
obtained after reaction of 8 with KHMDS, and the absolute
configuration was confirmed using the modified Mosher’s
method.8
Fragment 6 was prepared with readily available (1S, 2S)-
(+)-pseudoephedrine propionamide according to the proce-
dure of Smith and co-workers.9 Initial attempts to obtain the
trans-olefin 10 at C6-C7 by cross metathesis between
alcohol 9 and olefin 6 resulted in an unsatisfactory (less than
25%) yield (Scheme 2). At the same time, we undertook
Scheme 2. Synthesis of the Proposed Structure of Tyroscherin 1
Figure 1. Retrosynthetic analysis of the reported tyroscherin 1.
The fragment 5 was obtained via a five-step sequence
starting with Boc-D-Tyr(t-Bu)-OH (Scheme 1). N-Methyla-
tion, EDCI-mediated coupling of the N-methylated acid with
N,O-dimethylhydroxylamine, and the addition of 3-butenyl-
cross metathesis reactions of 5 with 6 to construct the
E-olefin framework of 1. By screening various reaction
conditions, it was found that the use of 1.0 equiv of olefin 5
and 2.0 equiv of olefin 6 in the presence of 5 mol % of the
second generation Grubbs catalyst 11 afforded the best results
(Supporting Information).10
(4) (a) Katsuta, R.; Shibata, C.; Ishigami, K.; Watanabe, H.; Kitahara,
T. Tetrahedron Lett. 2008, 49, 7042–7045. (b) Ishigami, K.; Katsuta, R.;
Shibata, C.; Hayakawa, Y.; Watanabe, H.; Kitahara, T. Tetrahedron 2009,
65, 3629–3638.
(5) Ugele, M.; Maier, M. E. Tetrahedron 2010, 66, 2653–2641.
(6) (a) Chen, L.; Yang, S.; Zhang, J. J.; Huang, X.-Y. Nature 2010,
464, 1062–1066. (b) Ulanovskaya, O. A.; Janjic, J.; Suzuki, M.; Sabharwal,
S. S.; Schumacker, P. T.; Kron, S. J.; Kozmin, S. A. Nat. Chem. Biol. 2008,
4, 418–421. (c) Kotake, Y.; Sagane, K.; Owa, T.; Mimori-Kiyosue, Y.;
Shimizu, H.; Uesugi, M.; Ishihama, Y.; Iwata, M.; Mizui, Y. Nat. Chem.
Biol. 2007, 3, 570–575. (d) Wulff, J. E.; Siegrist, R.; Myers, A. G. J. Am.
Chem. Soc. 2007, 129, 14444–14451. (e) Sato, S.-i.; Murata, A.; Shirakawa,
T.; Uesug, M. Chem. Biol. 2010, 17, 616–623.
Having established suitable conditions for the cross
metathesis, we proceeded next toward the completion of the
(8) See Supporting Information.
(9) (a) Smith, A. B., III; Basu, K.; Bosanac, T. J. Am. Chem. Soc. 2007,
129, 14872–14874. (b) Smith, A. B., III; Basu, K.; Bosanac, T. J. Am. Chem.
Soc. 2009, 131, 2348–2358. (c) For more details on the synthesis results of
6, see Supporting Information.
(10) This protecting group on the C3 alcohol had an important influence
on the selectivity and yield of the cross metathesis reaction. The best E:Z
ratio was obtained with the C3 acetate compared to other esters or the free
hydroxyl.
(7) (a) Chatterjee, A. K.; Choi, T. L.; Sanders, D. P.; Grubbs, R. H.
J. Am. Chem. Soc. 2003, 125, 11360–11370. (b) Connon, S. J.; Blechert,
S. Angew. Chem. Int. Ed. 2003, 42, 1900–1923. (c) Harrison, B. A.;
Gierasch, T. M.; Neilan, C.; Pasternak, G. W.; Verdine, G. L. J. Am. Chem.
Soc. 2002, 124, 13352–13353.
Org. Lett., Vol. 12, No. 19, 2010
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synthesis of 1. Cross metathesis of fragment 5 with fragment
6 gave exclusively the E-isomer of the adduct 4 in 89% yield
(Scheme 2). Treatment of TBDPS ether 4 with TBAF
afforded the alcohol, and mesylation of this primary alcohol
followed by treatment with MeMgBr (2.5 equiv) and CuI
(0.4 equiv) resulted in the addition of the terminal methyl
group and the simultaneous cleavage of the acetyl group.
Finally, global deprotection using TFA afforded the originally
proposed structure of tyroscherin 1. Although similar, the
1H and 13C NMR spectra of synthetic tyroscherin 1 did not
match the reported data for the natural product, with notable
differences for H2 and H3 (tyroscherin numbering). Further,
the optical rotation obtained for 1, [R]20D ) +21.5 (c 0.346,
MeOH), was very similar in magnitude but opposite in sign
to the value for the reported structure of the natural product,
[R]20 ) -21 (c 0.365, MeOH).11
D
At this point, we hypothesized that the originally reported
structure of tyroscherin 1 was incorrect; indeed, this has been
subsequently corroborated in the literature.4 We suspected
that the error lay in the assignment of the stereochemistry
and therefore turned our focus to the syntheses of diastere-
omers of 1. To assign the correct structure of tyroscherin
and to establish extensive SAR studies, we synthesized all
possible diastereomers of 1 using the same synthetic strategy
we employed to generate tyroscherin 1.12 This was achieved
with eight stereochemically different fragments. Tyroscherin
2 was synthesized by the same methods as tyroscherin 1
(Scheme 3). Tyroscherin 2 matched in all spectral and
physical data to the published natural product.2,4
Figure 2. Activity of Tyroscherin 2 on MCF-7 cells. Results
obtained using [3H]-thymidine incorporation assay (upper panel)
and using colorimetric MTS conversion assay (lower panel) are
shown. Similar IC50 values were obtained with IGF and fetal bovine
serum (2.23 µM vs 7.13 µM, respectively) (upper panel). Ty-
roscherin 2 displayed equivalent potency to inhibit cell growth
driven by IGF, basic FGF, EGF, or fetal bovine serum (lower panel).
Scheme 3. Synthesis of the Revised Structure of Tyroscherin 2
measure MCF-7 cell passage through S phase to quantify cell
proliferation, we obtained similar results using a tetrazolium
salt based assay (MTS assay) (Figure 2, lower panel). In
addition, to eliminate the possibility of differences between the
different MCF-7 cell lines, we tested tyroscherin 2 in our MTS
assay using the same MCF-7 cells that were used previously
(kindly provided by Dr. Shin-Ichiro Takahashi) under identical
mitogen and culture conditions as reported.4
Nevertheless, we failed to observe any selective growth
inhibition when cells were incubated with IGF vs FBS.
Finally, given the heterogeneous and relatively undefined
nature of serum as a mitogen, we chose to test tyroscherin
2 further against other purified growth factors, namely, bFGF
and EGF. However, tyroscherin 2 exhibited the same potency
to inhibit bFGF- and EGF-driven proliferation (2.77 µM and
3.02 µM, respectively) as it did against IGF-driven prolifera-
tion. Having identified a correct structure of tyroscherin 2,
the next aim was to design a chemical probe for identification
of its binding protein(s). In general, three structural compo-
nents are important in the successful design of affinity probes:
the potential ligand structure, an appropriate linker, and a
tag suitable for detection. On the basis of our preliminary
SAR analysis (data not shown) and optimization of linker
length, we chose to synthesize a biotinylated tyroscherin 3
using the same synthetic strategy we employed to generate
reported tyroscherin 1 and revised tyroscherin 2.
Interestingly, while we confirmed the recently revised
structure of tyroscherin 2, we were unable to corroborate its
reported IGF-selective inhibitory activity. As shown in Figure
2 (upper panel), we found that tyroscherin 2 inhibits IGF-
and FBS-induced cell proliferation with nearly equivalent
IC50 values (2.23 µM vs 7.13 µM) as opposed to the reported
1800-fold selectivity for IGF-induced growth over FBS-
stimulated proliferation.3
While our results were obtained using the more rigorous and
sensitive method of [3H]-thymidine incorporation to specifically
(11) For 1 as free amine, [R] ) +21.5 (c 0.346, MeOH), whereas for 1
as HCl salt, [R]20D ) +7.3 (c 0.325, MeOH) and for 1 as TFA salt, [R]20
D
) +7.8 (c 0.330, MeOH). The large discrepancy in optical rotation values
is perhaps due to the equivalent of amine salt in C2.
(12) The syntheses of 8 key fragments and 16 stereoisomers and the
biological results will be reported elsewhere.
4310
Org. Lett., Vol. 12, No. 19, 2010

Scheme 4
.
Synthesis of Acid 18
Scheme 6. Synthesis of Biotinylated Tyroscherin 3
In summary, we have accomplished a concise total
synthesis of the proposed and revised structure of tyroscherin
in 12 steps from commercially available materials, and we
have synthesized a revised structure-based molecular probe
for finding tyroscherin-binding proteins, using an efficient
cross metathesis strategy to construct the trans-olefin. This
convergent synthesis should be easily applicable to the
construction of various analogues for extensive SAR studies.
The main advantages of our synthesis are its brevity and its
efficiency, as well as its flexibility: a unified strategy was
used to access all desired diastereomers. Through our total
synthesis of all tyroscherin diastereomers, we have provided
additional evidence that the originally reported structure is
incorrect, which has been recently acknowledged by the
original team that reported the natural product structure. Our
results confirm that tyroscherin 2 (2S,3R,8R,10R) matches
the reported natural product spectral data. However, our
findings contradict the reported IGF-specificity of growth
inhibition previously reported for tyroscherin. Studies to
identify tyroscherin’s in ViVo biological targets and elucidate
its mode of action are underway.
The synthesis of acid 18 proceeded smoothly (Scheme 4).
Sequential protection of secondary alcohol, simultaneous
removal of Boc and t-Bu groups, and selective protection of
the amine gave phenol 17. Alkylation of 17 with methyl
4-bromobutyrate and hydrolysis of the ester provided
acid 18.
Scheme 5. Synthesis of Benzophenone-biotin 21
The benzophenone-biotin 21 was obtained via cycload-
dition of benzophenone-alkyne 19 (prepared by alkylation
of 4,4′-dihydroxy benzophenone with propargyl bromide
followed by Mitsunobu reaction of monoalkylated phenol
with Boc-protected aminoethoxy ethanol) and biotinylated
azide 20 (prepared by coupling of 11-azido-3,6,9-trioxaun-
decan-1-amine and biotin-ONp) (Scheme 5).13 The conver-
gent step of the synthesis of biotinylated tyroscherin 3 is
depicted in Scheme 6. The removal of Boc group in 21 using
TFA provided the amine as a TFA salt, which was coupled
to acid 18, using HATU and DIEA in DMF. Finally,
subsequent TBS deprotection of the silyl ether, followed by
the removal of Boc group by TFA in CH2Cl2, afforded
biotinylated tyroscherin 3.13
Acknowledgment. This work was supported by a Korea
Research Foundation Grant (KRF-2005-214-C00218) (H.S.T.),
the National Science Foundation (A.R.S.), and the National
Institutes of Health (GM062120) (C.M.C.). The authors
gratefully acknowledge Shin-Ichiro Takashashi (University
of Tokyo) for providing MCF-7 cells and David Spiegel
(Yale University) and members of the Crews lab for helpful
discussions throughout the preparation of this manuscript.
Supporting Information Available: Representative ex-
perimental procedures, spectral data, and analytical data for
new compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
(13) For more details on the syntheses of 19, 20, 21, and 3, see
Supporting Information.
OL101801U
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