Discovery and total synthesis of a new estrogen receptor heterodimerizing actinopolymorphol A from actinopolymorpha rutilus

Article abstract of DOI:10.1021/ol1013526

(Equation Presented). Estrogen receptor ERα and ERβ heterodimerization has been implicated in cancer chemoprevention. The discovery, structural elucidation, and total synthesis of a new natural product, actinopolymorphol A (1), from Actinopolymorpha rutil

Full text of DOI:10.1021/ol1013526

ORGANIC
LETTERS
2010
Vol. 12, No. 15
3525-3527
Discovery and Total Synthesis of a New
Estrogen Receptor Heterodimerizing
Actinopolymorphol A from
Actinopolymorpha rutilus
Sheng-Xiong Huang,† Emily Powell,‡ Scott R. Rajski,† Li-Xing Zhao,⊥
Cheng-Lin Jiang,⊥ Yanwen Duan,# Wei Xu,‡ and Ben Shen*,†,§,|
DiVision of Pharmaceutical Sciences, McArdle Laboratory for Cancer Research,
UniVersity of Wisconsin National CooperatiVe Drug DiscoVery Group, and,
Department of Chemistry, UniVersity of Wisconsin-Madison, Madison, Wisconsin
53705, Yunnan Institute of Microbiology, Yunnan UniVersity, Kunming, Yunnan
650091, China, and Hunan Engineering Research Center of Combinatorial
Biosynthesis and Natural Product Drug DiscoVery, Changsha, Hunan 410329, China
bshen@pharmacy.wisc.edu
Received June 11, 2010
ABSTRACT
Estrogen receptor ERr and ERꢀ heterodimerization has been implicated in cancer chemoprevention. The discovery, structural elucidation,
and total synthesis of a new natural product, actinopolymorphol A (1), from Actinopolymorpha rutilus (YIM45725) that preferentially induces
ERr/ꢀ heterodimerization is reported. Total synthesis of 1 has allowed us to determine its absolute stereochemistry and that of a previously
known deacetylated congener, and 1 represents the first member of a new class of natural products not previously recognized to modulate
ER function.
The estrogen receptors (ERs) are ligand-inducible transcrip-
tion factors implicated in a wide array of biological processes.
Binding of 17ꢀ-estradiol (E2) and other estrogenic ligands
triggers receptor dimerization and subsequent control of gene
transcription. ERs exist in two forms, ERR and ERꢀ, which
have opposing roles in regulating estrogen action: ERR
promotes, whereas ERꢀ inhibits E2-dependent cell growth.1
Notably, the coexpression of ERꢀ with ERR leads to reduced
proliferation of breast cancer cells and ∼60% of all breast
tumors express both receptors.2 Coexpression of ERꢀ has
† Division
of
Pharmaceutical
Sciences,
University
of
Wisconsin-Madison.
‡ McArdle Laboratory for Cancer Research, University of
Wisconsin-Madison.
⊥ Yunnan University.
# Hunan Engineering Research Center of Combinatorial Biosynthesis and
Natural Product Drug Discovery.
(1) Helguero, L. A.; Faulds, M. H.; Gustafsson, J. A.; Haldosen, L. A.
Oncogene 2005, 24, 6605–6616.
§ University of Wisconsin National Cooperative Drug Discovery Group,
University of Wisconsin-Madison.
(2) Pettersson, K.; Delaunay, F.; Gustafsson, J. A. Oncogene 2000, 19,
4970–4978.
| Department of Chemistry, University of Wisconsin-Madison.
10.1021/ol1013526  2010 American Chemical Society
Published on Web 07/01/2010

been correlated with a more favorable prognosis relative to
tumors expressing ERR alone, and many data now suggest
that ERR/ꢀ heterodimers function to regulate distinct E2-
responsive genes.3 However, facile homodimerization has
obscured a clear understanding of heterodimer function.
We recently reported a bioluminescent resonance energy
transfer (BRET) assay to monitor the ERR/ꢀ heterodimer
and its respective homodimer formation in live cells, setting
the stage to screen for homo- and heterodimer selective ER
modulators.4 We now report the discovery and total synthesis
of a new natural product, actinopolymorphol A (1), that
preferentially induces ERR/ꢀ heterodimerization relative to
either homodimer. Actinopolymorphol A represents the first
member of a new class of natural products not previously
recognized to modulate ER function.
During the course of our efforts to identify novel natural
products from microorganisms from diverse and unique
ecological niches, we first subjected the crude extract from
a recently identified Actinopolymorpha rutilus (YIM45725)5
originating from a soil sample from the Yunnan province of
China to the BRET assay. Initial assays clearly indicated
the presence of potential ERR/ꢀ heterodimerization inducers
in the crude extract. Subsequent large-scale fermentation (5
L) and BRET assay-guided fractionation afforded an active
fraction, from which 1, actinopolymorphol B (2), and
actinopolymorphol C (3) were purified. The 1H and 13C NMR
spectra of purified 1, 2, and 3 were fully assigned on the
basis of extensive 1D and 2D NMR (gCOSY, gHSQC, and
gHMBC) and APCI-MS and high-resolution ESI-MS analy-
ses (Figure 1B and also see the Supporting Information).
Dereplication revealed 1, 2, and 3 to be new natural products
(Figure 1A) although a relationship to previously reported
natural products (Figure 1C)6-10 is clearly evident for 1 and
2.
Figure 1. Actinopolymorphol A (1), B (2), and C (3) from ERR/ꢀ
heterodimerizing fraction of A. rutilus crude extract: (A) structures
of 1, 2, and 3; (B) COSY and HMBC correlations observed for 1,
2, and 3; and (C) the sattabacin, sattazolin, and kurasoin classes of
natural products bearing molecular scaffolds similar to 1 or 2.6-10
heterodimerization may have bearing on the scope of
previously reported bioactivities for this class of natural
products (Figure 1C).6,7,9,10
Compound 1 was isolated as a colorless oil, for which
HR-ESIMS established the molecular formula as C15H20O4
(287.12567 [M + Na]+, calcd for C15H20O4Na, 287.12538)
possessing six degrees of unsaturation. Analysis of the NMR
spectra revealed chemical shifts indicative of a ketone (δC
207.8), acetoxyl (δC 170.9, s; δC 19.3, q), one 1,4-disubsti-
tuted benzene ring [(δH 6.68, 2H; δC 115.1, d) and (δH 7.02,
2H; δC 130.3, d)], a secondary alcohol methine (δH 5.08, δC
79.6, d), one methine (δH 2.03, δC 43.7, d), two methylenes
(δC 47.8, t; δC 35.7, t), and two secondary methyl groups
[(δH 0.82, 3H; δC 21.7, q) and (δH 0.84, 3H; δC 21.7, q)]
(Table S1, Supporting Information). On the basis of the
gHMBC correlations from two methylene protons to corre-
sponding carbons and the gCOSY correlations, the con-
nectivity of every carbon was readily established (Figure 1B).
Third, we established the structure of 1 by total synthesis
using the optically pure starting material (S)-2-hydroxy-3-
(4-hydroxyphenyl)propionic acid (98% purity) (4, Figure
2A). Conversion of 4 to methyl ester 5 was effected by using
methanolic HCl followed by treatment with TBSCl and
imidazole to afford diprotected methyl ester 6. The methyl
ester underwent smooth conversion to the Weinreb amide 7
by treatment with N,O-dimethylhydroxylamine hydrochloride
in the presence of (CH3)2CHMgCl. Grignard reaction of 7
with (CH3)2CHCH2MgCl proceeded extraordinarily slowly
over the course of 24 h but ultimately afforded the dipro-
tected isobutyl ketone 8 in 67% yield from the Weinreb
amide. Subsequent cleavage of both silyl ethers with tet-
Second, we determined, using the BRET assay and pure
1, 2, and 3, that 1 was solely responsible for ERR/ꢀ
heterodimerization induction (EC50 ) 19 µM) and 2 and 3
were devoid of any ER dimerization inducing activity.11
Consequently, further studies focused exclusively on 1.
Significantly, none of the previously reported natural products
to which 1 bears a structural resemblance have been noted
as ER modulators. Thus, the finding that 1 induces ERR/ꢀ
(3) Chang, E. C.; Frasor, J.; Komm, B.; Katzellenbogen, B. S. Endo-
crinology 2006, 147, 4831–4842.
(4) Powell, E.; Xu, W. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 19012–
19017.
(5) Wang, Y.-X.; Zhang, Y. Q.; Xu, L. H.; Li, W. J. Int. J. Syst. EVol.
Microbiol. 2008, 58, 2443–2446.
(6) Sattabacins and sattazolins: Lampis, G.; Deidda, D.; Maullu, C.;
Madeddu, M. A.; Pompei, R.; Monache, R. D.; Satta, G. J. Antibiot. 1995,
48, 967–972
(7) Soraphinol: Li, X.; Yu, T. K.; Kwak, J. H.; Son, B. Y.; Seo, Y.;
Zee, O. P.; Ahn, J. W. J. Microbiol. Biotechnol. 2008, 18, 520–522
(8) Kurasoins: Andrus, M. B.; Hicken, E.J.; Stephens, J. C.; Bedke, D. K.
J. Org. Chem. 2006, 71, 8651–8654
(9) Uchida, R.; Shiomi, K.; Inokoshi, J.; Masuma, R.; Kawakubo, T.;
Tanaka, H.; Iwai, Y.; Omura, S. J. Antibiot. 1996, 49, 932–934
(10) Uchida, R.; Shiomi, K.; Inokoshi, J.; Nishizawa, A.; Hirose, T.;
Tanaka, H.; Iwai, Y.; Omura, S. J. Antibiot. 1996, 49, 886–889
.
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.
(11) Powell, E.; Huang, S.-X.; Xu, Y.; Rajski, S. R.; Wang, Y.; Peters,
N.; Guo, S.; Xu, H. E.; Hoffmann, F. M.; Shen, B.; Xu, W. Biochem.
Pharmacol. 2010, In press.
3526
Org. Lett., Vol. 12, No. 15, 2010

assay confirmed that natural and synthetic 1 displayed
identical (within standard error) abilities to induce ERR/ꢀ
heterodimerization.11
Finally, the absolute stereochemistry of 1 was confirmed
by comparative chiral HPLC and optical rotation analyses.
Both the natural and synthetic 1 were found to be identical
upon chiral HPLC analyses, possessing the same retention
time (Figure 2B) and identical UV-vis spectra, and to have
almost identical optical rotations: +20.9 (c 0.15, CH3OH)
for natural 1 and +23.5 (c 0.28, CH3OH) for synthetic 1
(the slight difference in optical rotations is attributed to
different concentrations). On the basis of these data, we
conclude that 1 from A. rutilus YIM45725 has the S-
configuration at C8; this is consistent with the stereochem-
istries found in the kurasoins and, as unveiled here, 4-hy-
droxysattabacin.6,8
Selective ER modulators are well established and a number
of them are used clinically such as tamoxifen and ralox-
ifene.12,13 Of particular significance during the development
of such agents has been the realization that diols or other
agents, able to form H-bonding networks with the ligand
binding domains of ER spaced a distance of ∼12 Å apart,
can effectively compete for ER binding with E2 and thereby
elicit clinically significant activities. On this basis, we
postulate that the phenol moiety of 1 is likely critical to ER
heterodimerization activity; an understanding of ER-E2
interactions in which the phenol moiety of E2 is involved in
H-bonding supports this hypothesis.13 The small size of 1
yet the pronounced ability to modulate ER activity, and the
relatedness to already established natural products suggests
that further study of this class of natural products is clearly
warranted. Our ability to produce 1 biosynthetically and via
total synthesis provides an outstanding platform to more
rigorously elucidate ER heterodimerization by new analogues
bearing this privileged molecular scaffold.
Figure 2. Synthesis and chiral HPLC analysis of natural vs synthetic
1: (A) reagents and conditions for total synthesis of 1 from the
optically pure starting material 4 [(a) MeOH, HCl, rt, quantative;
(b) TBSCl, imidazole, DMF, rt, 90%; (c) HN(OMe)CH3-HCl,
(CH3)2CHMgCl, THF, 0 °C, 81%; (d) (CH3)2CHCH2MgCl, THF,
0 °C-rt, 67%; (e) TBAF, THF, rt, 86%; (f) Ac2O, pyridine, rt,
72%; (g) pyrrolidine, 1 min, rt, 66%] and (B) chiral HPLC analysis
of racemic 1, 1 from A rutilus (blue), synthetic 1 (red): b, 1; and
1, the R-enantiomer of 1.
rabutylammonium fluoride (TBAF) afforded the correspond-
ing diol 9 in 81% yield. Notably, production of 9 from 4
constitutes a total synthesis of the natural product 4-hy-
droxysattabacin from Sorangium cellulosum (Figure 1C), for
which absolute stereochemistry has been unclear.6 The
optical rotation of 9 was found to be +18.3 (c 0.21, CH3OH),
which compares favorably to the number previously pub-
lished for 4-hydroxysattabacin [+14 (0.3, CHCl3)],6 assigning
4-hydroxysattabacin as the S-enantiomer. Diacetylation of
9 was accomplished in 72% yield upon subjection of the
diol to excess acetic anhydride and pyridine. Selective
deacetylation of the phenol moiety was accomplished by
using neat pyrrolidine at room temperature, affording finally
the synthetic 1 as the S-enantiomer. 1H and 13C NMR spectral
data for synthetic 1 were identical with those previously
obtained for natural 1 from A. rutilus YIM45725. BRET
Acknowledgment. We thank the Analytical Instrumenta-
tion Center of the School of Pharmacy, UW-Madison for
support in obtaining MS and NMR data. This work is
supported in part by NIH grants CA113297 (B.S.), CA125387
(W.X.), MH089442 (W.X.), and T32CA009135 (E.P.) and
the Chinese Ministry of Education 111 Project B08034
(Y.D.).
Supporting Information Available: Full experimental
details for strain cultivation, extraction, compound isolation,
spectral data, and assignment of all natural products and
synthetic intermediates. This material is available free of
charge via the Internet at http://pubs.acs.org.
(12) Jordan, V. C. J. Med. Chem. 2003, 46, 883–908.
(13) Jordan, V. C. J. Med. Chem. 2003, 46, 1081–1111.
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