Published on Web 07/21/2007
Stereocontrolled Synthesis of Dafachronic Acid A, the Ligand for the DAF-12
Nuclear Receptor of Caenorhabditis elegans
Simon Giroux and E. J. Corey*
Department of Chemistry and Chemical Biology, HarVard UniVersity, Cambridge, Massachusetts 02138
The nematode C. elegans has become a valuable engine for
biological discovery since the early studies of Sydney Brenner,1
especially for the investigation of developmental and metabolic
processes. (The developmental course of each of the 1000 or so
somatic cells has already been ascertained.) The progression of C.
elegans through the various life stages depends on the availability
of nutrients. When deprived of food, its metabolism slows and it
enters a “dauer” or diapausal state that prolongs its life. Recently,
it has been discovered that the loss of function of two genes, daf-2
and daf-9, can extend the life span from 2 weeks to ca. 12 weeks,
a finding that attracted even more attention than C. elegans’ survival
tion of the 7-keto group of 8 and dehydration of the resulting 7R-
alcohol gave the ∆ -unsaturated methyl ester 9, from which
7
dafachronic acid A (1) was obtained by the sequence: (1)
deacetylation, (2) oxidation of the hydroxyl at C(3), and (3) ester
saponification.
We have also developed a second pathway for the stereocon-
trolled elaboration of the dafachronic A side chain starting from
the aldehyde 2 (Scheme 2). Diastereoselective addition (Felkin
mode) of vinylmagnesium bromide to 1 in THF at -78 °C followed
by trapping of the intermediate alkoxide by propionic anhydride,
3
Et N, and 4-dimethylaminopyridine afforded selectively the allylic
2
propionate ester 10. Reaction of 10 with lithium diisopropylamide
in THF-HMPA at -78 °C produced an enol silyl ether which
without isolation was heated at reflux to effect highly stereoselective
Claisen rearrangement.11 Hydrogenation of the resulting (E)-â,γ-
of the crash of the Space Shuttle Columbia in 2003. Intensified
interest in these genes has led to the discovery that daf-9 codes for
the protein (DAF-9) which is a cytochrome P450 enzyme respon-
sible for the biosynthesis of a small molecule that activates another
gene, daf-12. Subsequently, Mangelsdorf, Antebi, and their col-
leagues have deduced a structure for the DAF-12 ligand starting
with the hypothesis that it is a sterol that is biosynthesized by DAF-
unsaturated acid 11 (H
2
, Pd-C, 1 atm, EtOAc) provided the acid
6
, identical in all respects to the product obtained by the route
outlined in Scheme 1. Although we have not yet optimized the
yields of 6 by the Claisen route via 10 and 11, it clearly provides
a second, viable and completely stereocontrolled route to dafach-
ronic acid A.
Synthetic dafachronic acid A is currently being subjected to
detailed biological studies by Drs. Adam Antebi, David Mangels-
dorf, and their colleagues. The results of the Antebi laboratory to
date show that synthetic 1 can rescue daf-9 mutants at subnanomolar
concentrations and is equipotent with the natural DAF-12-ligand.
Unfortunately, insufficient natural material is available for spec-
troscopic comparison at this stage.
3
.4
9
-mediated oxidation of a precursor sterol. Since the natural
DAF-12 ligand was only available in trace amounts, insufficient
for structural characterization, these workers carried out a
series of bioassays of many test sterol derivatives in comparison
with the natural ligand. Their findings led them to conclude
7
that a 3-keto group, a ∆ -olefinic linkage, and a 27-carboxylic
function correlated with increased DAF-12 potency and to assign
structure 1 to the natural ligand, which they named dafachronic
acid.3 We undertook the synthesis of 1 in order to obtain de-
finitive evidence of structure and to make the DAF-12 ligand
available for biological investigations, including the study of the
genes affected by DAF-12 activation. We describe herein the first
synthesis of 1, for which we propose the slightly modified name
dafachronic acid A, since additional members of this hormonal
series may emerge.
,4
The synthesis of 1 reported herein is easily scalable and capable
of providing large amounts of this rare nuclear receptor ligand for
detailed study since an overall yield of 37% of dafachronic acid
from the aldehyde 2 has been reproducibly obtained.
The role of dafachronic acid A in regulating the development
of C. elegans is reminiscent of the action of the natural pro-
duct glycinoeclepin A (12) on the nematode Heterodera glycines,
The readily available plant sterol, â-stigmasterol, was transformed
into the known 3,5-cyclosteroid aldehyde 25 by the three-step
sequence shown in Scheme 1. Reaction of 2 with the lithium salt
1
2,13
a predator of the soybean plant (and various other beans).
At concentrations as low as 10
-
12
g/mL, glycinoeclepin A,
6
of the methyl ester 3 in THF afforded the (E,E)-diene ester 4
which is produced in and released from the roots of the soybean
plant, stimulates the hatching of otherwise dormant eggs of
H. glycines.
(
5
>20:1 E:Z) in excellent yield. The (E)-R,â-unsaturated acid
was prepared from 4 by selective hydrogenation of the ∆ -
2
2
olefinic linkage followed by saponification (88% from 4). Further
hydrogenation of 5 using H and achiral catalysts proceeded
2
non-diastereoselectively to form an inseparable 1:1 mixture of
7
2
5-S and 25-R saturated carboxylic acids. However, homogen-
2 8
eous hydrogenation with 4 mol % of Ru(OAc) [(S)-H -BINAP] and
H
2
(1 atm) in MeOH at 50 °C afforded the desired 25-(S)-acid 6
8,9
with 8:1 diastereoselectivity. Recrystallization of this mixture from
diisopropyl ether furnished pure 6 (>10:1 by 13C NMR analysis).7
Esterification of 6 followed by acetolysis provided the 3â-acetoxy-
It is interesting that glycinoeclepin A, a highly oxidized
transformation product of the plant triterpene cycloartenol, and
dafachronic acid A link nematode development to environmental
5
5
10
∆
-steroidal ester 7. Allylic oxidation of 7 to the ∆ -7-ketone and
catalytic hydrogenation produced the saturated 7-ketone 8. Reduc-
9866
9
J. AM. CHEM. SOC. 2007, 129, 9866-9867
10.1021/ja074306i CCC: $37.00 © 2007 American Chemical Society