Syntheses and biological evaluation of b-ring-modified analogues of dafachronic acid A

Article abstract of DOI:10.1021/ol801425v

(Chemical Equation Presented) Synthesis and testing of dafachronic acid A (1) and its derivatives 2 and 3 have revealed that 1, and not a further oxidation product, is the natural ligand for the DAF-12 receptor of Caenorhabditis elegans.

Full text of DOI:10.1021/ol801425v

ORGANIC
LETTERS
2008
Vol. 10, No. 16
3643-3645
Syntheses and Biological Evaluation of
B-Ring-Modified Analogues of
Dafachronic Acid A
Simon Giroux,† Axel Bethke,‡ Nicole Fielenbach,‡ Adam Antebi,‡ and
E. J. Corey*,†
Department of Chemistry and Chemical Biology, HarVard UniVersity, 12 Oxford
Street, Cambridge, Massachusetts 02138, and Huffington Center on Aging, Department
of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
corey@chemistry.harVard.edu
Received June 24, 2008
ABSTRACT
Synthesis and testing of dafachronic acid A (1) and its derivatives 2 and 3 have revealed that 1, and not a further oxidation product, is the
natural ligand for the DAF-12 receptor of Caenorhabditis elegans.
Remarkably, the life span of the nematode Caenorhabditis
elegans can be increased significantly by loss of function of
a handful of genes that affect endocrine function. Among
them, the daf-9 gene encodes a cytochrome P450 enzyme
which is responsible for the biosynthesis of the bile acid-
like steroid, dafachronic acid A (1). Based on various
analytical techniques, it has been recently proposed by
Mangelsdorf and Antebi that 1 is the major ligand for the
nuclear receptor DAF-12, which in its ligand bound form
regulates genes that prevent entry into the dauer stage, a long-
lived quiescent mode.1 However, synthesis of the proposed
ligand remained elusive until a later work, in which the 25-
(S) structure of 1 and its 25-(R)-diastereomer were made.2,3
In this research, we address the question of whether
Figure 1. Structure of glycinoeclepin A.
dafachronic acid A is the true ligand for the nuclear hormone
receptor DAF-12 or just a precursor of a further biooxidation
product which is the actual ligand. We were intrigued by
the fact that dafachronic acid A, with its ∆7-olefinic linkage,
might be further oxidized biologically to a seco acid structure
resembling that of glycinoeclepin A,4,5 a potent hatching
factor for the eggs of the nematode Heterodera glycines
† Harvard University.
‡ Baylor College of Medicine.
(1) (a) Motola, D. L.; Cummins, C. L.; Rottiers, V.; Sharma, K. K.; Li,
T.; Li, Y.; Suino-Powell, K.; Xu, H. E.; Auchus, R. J.; Antebi, A.;
Mangelsdorf, D. J. Cell 2006, 124, 1209–1223. (b) Gerisch, B.; Rottiers,
V.; Li, D.; Motola, D. L.; Cummins, C. L.; Lehrach, H.; Mangelsdorf, D. J.;
Antebi, A. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 5014–5019. (c) Rottiers,
V.; Motola, D. L.; Gerisch, B.; Cummins, C. L.; Nishiwaki, K.; Mangelsdorf,
D. J.; Antebi, A. DeVelopmental Cell 2006, 10, 473–482. (d) (c) For an
online resource on C. elegans, see: http://www.wormbook.org.
(2) Giroux, S.; Corey, E. J. J. Am. Chem. Soc. 2007, 129, 9866–9867.
(3) Giroux, S.; Corey, E. J. Org. Lett. 2008, 10, 801–802.
(4) Glycinoeclepin A, a natural product that is released into soil from
the roots of the soybean plant, is active at 10-12 g/mL as a hatching factor
for H. glycines; see: (a) Fukuzawa, A.; Furusaki, A.; Ikura, M.; Masamune,
T J. Chem. Soc. Chem. Commun. 1985, 221-222, 748. (b) Masamune, T.;
Anetai, M.; Takasugi, M.; Katsui, N. Nature 1982, 297, 495–496
.
10.1021/ol801425v CCC: $40.75
Published on Web 07/19/2008
2008 American Chemical Society

Scheme 1. Synthesis of Analogues 2 and 3 from ꢀ-Stigmasterol
(Figure 1). Consequently, we became interested in exploring
the biological activity of the ꢀ-seco dafachronic acid A
derivative 2, as an analogue of glycinoeclepin A, which might
even be a more active metabolite of 1. In this paper, we
describe the synthesis and biological evaluation of 2. For
comparison, we have also synthesized the 7,8-dihydro
derivative of dafachronic acid A, 3, which would be expected
to be devoid of activity if the seco acid 2 were the real ligand
for DAF-12, rather than dafachronic acid A (1).
The synthesis of the diketo diacid 2 started with the
previously reported 6-keto steroid 4.2 Baeyer-Villiger
oxidation of 4 with trifluoroperacetic acid ((CF3CO)2O, H2O2,
0 °C, CHCl3) afforded the desired 7-membered lactone 5 in
94% yield and as a sole regioisomer. Lactone 5 was cleaved
to a ketoacid intermediate by treatment with Jones’ reagent
(2 equiv, 23 °C, acetone) which was esterified by diaz-
omethane (CH2N2, Et2O) to give ketoester 6 in essentially
quantitative yield over two steps. Saponification of the 3ꢀ-
acetate, oxidation of the resulting alcohol to the ketone, and
hydrolysis gave the diketo diacid 2 in 52% overall yield
(three steps, Scheme 1). Our initial strategy for the synthesis
of 2 involved the oxidation of the ∆7-olefinic linkage in 1
by various methods. Surprisingly, all attempts to directly
oxidize the ∆7 bond to the diketo diacid 2 using O3 then
H2O2, KMnO4, NBu4MnO4, and RuCl3-NaIO4 were unsuc-
cessful.
To synthesize the 7,8-dihydro analogue 3, we have also
used an intermediate from our synthesis of 1.2 Thus, the ∆5-
double bond in 7 was reduced (H2, 1 atm, Pd-C, EtOAc) to
give the fully saturated steroid, and the same three steps as
above were performed to give analogue 3 in 33% overall
yield for the four steps. It should also be mentioned that the
hydrogenation of 1 to 3 failed under several conditions.6
Next, samples of the synthetic dafachronic acid A 1, the
seco-diacid 2, and 7,8-dihydrodafachronic acid A 3 were
evaluated for their bioactivity. First, the ability of synthetic
ligands to rescue daf-9 hormone biosynthetic mutants from
the dauer state was measured. Consistent with 1 being a
natural ligand for DAF-12, dafachronic acid A rescued dauer
formation in the nanomolar range, with half-maximal activity
of 18.5 nM (Figure 2). Similarly, the 7,8-dihydrodafachronic
acid A also gave substantial rescue with half-maximal rescue
at 292 nM. By contrast, the seco-diacid 2 was found not to
rescue C. elegans from the dauer state, indicating that it is
not a ligand. Second, the ability of synthetic ligands to
activate DAF-12 in transcriptional assays on a target gene,
(5) For the syntheses of glycinoeclepin A, see: (a) Murai, A; Tanimoto,
N.; Sakamoto, N.; Masamune, T. J. Am. Chem. Soc. 1988, 110, 1985–
1986.
(6) To the best of our knowledge, no successful hydrogenation of isolated
∆7 double bonds has been reported in the literature. (b) Mori, K.; Watanabe,
H. Pure Appl. Chem. 1989, 61, 543–546. (c) Corey, E. J.; Houpis, I. N.
J. Am. Chem. Soc. 1990, 112, 8997–8998.
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Org. Lett., Vol. 10, No. 16, 2008

Figure 2
.
In vivo activity of sterols 1-3 measured as the percentage
Figure 3. Transcriptional activation of DAF-12 by 1-3 on
lit-1::ptk-luciferase reporter constructs, measuring relative luciferase
units with and without ligand (RLU) vs concentration.
of rescue of daf-9(dh6) null worms from dauer to wild-type gravid
adults.
lit-1, was measured. To do this, plasmid constructs containing
the daf-12 gene and the lit-1 gene fused to a luciferase
reporter were cotransfected into human embryonic kidney
cells (HEK293T) and treated with various doses of the
compounds and luciferase induction measured by light
emission.1 In accord with the dauer rescue results, 2 showed
no actiVity even at 100 µM concentration (Figure 3), whereas
7,8-dihydrodafachronic acid A (3) showed activity similar
to that of dafachronic acid A (1). Specifically, measurement
of the dose-response revealed EC50 values for daf-12
activation to be 114 nM for 7,8-dihydrodafachronic acid A
and 26 nM for dafachronic acid A.
case, ring B oxidative cleavage products are not the active
agents for gene activation of C. elegans DAF-12, and (3)
the ∆7,8 double bond is not essential for dafachronic acid
activity on C. elegans.
Acknowledgment. S.G. is grateful to NSERC (Canada)
for a postdoctoral fellowship. A.A. is grateful for support
from NIH and the Ellison Foundation. We thank Dongling
Li (Baylor College of Medicine) for technical assistance.
Supporting Information Available: Experimental pro-
tocols, characterization for all new compounds, and methods
for performing the dauer assays. This material is available
free of charge via the Internet at http://pubs.acs.org.
These results taken together allow the following conclu-
sions: (1) dafachronic acid A is a natural ligand for DAF-12
nuclear receptor, (2) in contrast to the soybean nematode
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