Stereoselective synthesis of (25r)-dafachronic acids and (25R)-cholestenoic acid as potential ligands for the DAF-12 receptor in Caenorhabditis elegans

Article abstract of DOI:10.1055/s-2008-1077958

Commercially available diosgenin has been used as starting material for a highly efficient synthesis of (25R)-dafachronic acids and (25R)-cholestenoic acid, potential ligands for the receptor DAF-12 in the nematode Caenorhabditis elegans.

Full text of DOI:10.1055/s-2008-1077958

LETTER
1965
Stereoselective Synthesis of (25R)-Dafachronic Acids and (25R)-Cholestenoic
Acid as Potential Ligands for the DAF-12 Receptor in Caenorhabditis elegans
S
ynthesis of (25
R
e
)-Dafachro
n
nic Acids
a
n
é
d(25
R
)-Choleste
M
A
cid artin,a Arndt W. Schmidt,a Gabriele Theumer,a Teymuras V. Kurzchalia,b Hans-Joachim Knölker*a
a
Department Chemie, Technische Universität Dresden, Bergstraße 66, 01069 Dresden, Germany
Fax +49(351)46337030; E-mail: hans-joachim.knoelker@tu-dresden.de
b
Max-Planck Institut für Molekulare Zellbiologie und Genetik, Pfotenhauerstraße 108, 01307 Dresden, Germany
Received 14 May 2008
bited no activity at all. In an independent synthetic study,
Khripach et al. reported the syntheses of (25R)- and (25S)-
D4-dafachronic acid as well as (25R)- and (25S)-choles-
tenoic acid via a multistep elaboration of the steroid side
chain.4 In 2007, Corey et al. described the synthesis of
(25S)-D7-dafachronic acid via a diastereoselective ruthe-
nium-catalyzed hydrogenation.5 More recently, they re-
ported a synthesis of (25R)-D7-dafachronic acid (1) from
b-ergosterol (10 steps and 13% overall yield).6
Abstract: Commercially available diosgenin has been used as start-
ing material for a highly efficient synthesis of (25R)-dafachronic
acids and (25R)-cholestenoic acid, potential ligands for the receptor
DAF-12 in the nematode Caenorhabditis elegans.
Key words: carboxylic acids, oxidations, protecting groups, stereo-
selective synthesis, steroids
Under unfavorable conditions, such as scarcity of food or
overcrowding, the nematode Caenorhabditis elegans en-
ters diapause and forms so-called dauer larvae. The hor-
monal receptor DAF-12 appears to play a key role in this
process.1 Recently, Mangelsdorf et al. reported novel ste-
roidal metabolites from C. elegans identified as 3-keto-
cholesten-26-oic acids. These steroids function as ligands
for the orphan receptor DAF-12 and have been described
as (25R)-D7-dafachronic acid (1) and (25R)-D4-dafachro-
nic acid (2) as well as their 25S-diastereomers (Figure 1).2
The synthesis of both diastereomers of D4-dafachronic
acid starting from the noncommercial (25R)- and (25S)-
26-hydroxycholesterol was described by Mangelsdorf et
al.2 It was found that (25R)-D4-dafachronic acid (2) was
significantly less active than its 25S-isomer. Gill et al.
have shown that (25S)-cholestenoic acid represents anoth-
er ligand for DAF-12.3 However, the 25R-isomer 3 exhi-
In the course of our project directed towards the synthesis
of hormonally active cholesterol derivatives,1,7 we be-
came interested in a diastereoselective route to the (25R)-
cholesten-26-oic acids 1–3. We recognized commercially
available diosgenin (4) as perfect starting material since it
provides the 25R-configuration present in the target com-
pounds 1–3.
Using a modification of the procedure reported by Will-
iams, the Clemmensen reduction of diosgenin afforded
the triol 5 in 85% yield on large scale (Scheme 1).8 Selec-
tive protection of the C-3b and C-26 hydroxy groups us-
ing TBSCl and DBU to 6 followed by removal of the C-16
hydroxy group led to the disilyl ether 7 in 76% overall
yield.8 Several methods are known for allylic oxidations
at C-7 of steroids (Table 1).9–11 We found that Chan-
O
O
OH
OH
H
H
H
H
H
O
O
H
(25R)-Δ4-dafachronic acid (2)
(25R)-Δ7-dafachronic acid (1)
25
H
O
H
O
O
OH
H
H
H
H
H
H
HO
HO
(25R)-cholestenoic acid (3)
diosgenin (4)
Figure 1 (25R)-Cholesten-26-oic acids (1–3) and diosgenin (4)
SYNLETT 2008, No. 13, pp 1965–1968
x
x
.x
x
.2
0
0
8
Advanced online publication: 15.07.2008
DOI: 10.1055/s-2008-1077958; Art ID: G15808ST
© Georg Thieme Verlag Stuttgart · New York

1966
R. Martin et al.
LETTER
Table 1 Allylic Oxidation of 7 to the Cholest-5-en-7-one 8
Grignard reduction using isopropylmagnesium chloride in
diethyl ether afforded diastereoselectively the 7a-alcohol
10 in 70% yield. Elimination of the 7a-alcohol 10 with
thionyl chloride in pyridine led quantitatively to the
cholest-7-ene 11.12 Finally, desilylation with TBAF to the
diol 12 and subsequent Jones oxidation provided (25R)-
D7-dafachronic acid (1).13
A stereoselective route to (25R)-D4-dafachronic acid (2)
proved to be more difficult. Desilylation of 7 provided
(25R)-26-hydroxycholesterol in 89% yield (Scheme 2).
However, Jones oxidation of (25R)-26-hydroxycholester-
Reaction conditions
Yield
(%)
PDC (4.0 equiv), t-BuOOH (4.0 equiv), C6H6, 0 °C to r.t., 28 h9 57
Mn(OAc)3 (10 mol%), t-BuOOH (5.1 equiv), EtOAc, r.t., 2 d10 47
CrO3 (18 equiv), DMP (18 equiv),a CH2Cl2, –20 °C to –10 °C, 56
1 h11
a DMP = 3,5-dimethyl-1H-pyrazole
drasekaran’s procedure9 provides the best results for ally- ol did not provide the desired (25R)-D4-dafachronic acid
lic oxidation of 7 to the cholest-5-en-7-one 8.
(2) but the (25R)-3,6-diketocholest-4-en-26-oic acid (13)
in 56% yield. Using PDC in N,N-dimethylformamide as
oxidizing agent14 provided the undesired compound 13 in
74% yield.13 Oxidation of cholesterols was known to af-
ford cholest-4-en-3,6-diones using Jones reagent,15
PCC,16 or TPAP/NMO.17 Therefore, we decided to oxi-
dize the C-3 and C-26 hydroxy groups in two different
Transfer hydrogenation of 8 provided the ketone 9 in high
yield. In order to establish the D7-double bond, we re-
quired only the 7a-alcohol 10 for stereoelectronic reasons.
However, reduction with lithium aluminum hydride at
low temperature provided the 7a-alcohol 10 in 59% yield
along with the corresponding 7b-alcohol in 31% yield.
R
H
R
O
O
OTBS
OH
H
OH
H
OH
H
b
a
H
H
H
H
H
H
HO
TBSO
HO
4
5
6
OTBS
OTBS
H
H
e
c, d
H
H
H
H
O
TBSO
TBSO
7
8
OTBS
OTBS
H
H
H
f
h
j
g
H
H
O
H
TBSO
TBSO
OH
H
H
9
10
OH
OTBS
i
H
H
H
H
HO
TBSO
H
H
11
12
O
OH
H
H
O
(25R)-Δ7-dafachronic acid (1)
H
Scheme 1 Synthesis of (25R)-D7-dafachronic acid (1). Reagents and conditions: a) Zn, 19% HCl, EtOH, reflux, 2 h, 85%; b) TBSCl, DBU,
THF, r.t., 16 h, 85%; c) MsCl, pyridine, 0 °C to r.t., 16 h; d) LiAlH4, Et2O, 0 °C to reflux, 4 h, 89% over 2 steps; e) PDC, t-BuOOH, benzene,
0 °C to r.t., 28 h, 57%; f) Pd/C, ammonium formate, MeOH–EtOAc (3:4), reflux, 8 h, 90%; g) i-PrMgCl, Et2O, r.t., 1 h, 70%; h) SOCl2, pyridine,
0 °C, 1 h, 100%; i) TBAF, THF, reflux, 24 h, 93%; j) CrO3, H2SO4, acetone, 0 °C, 1 h, 74%.
Synlett 2008, No. 13, 1965–1968 © Thieme Stuttgart · New York

LETTER
Synthesis of (25R)-Dafachronic Acids and (25R)-Cholestenoic Acid
1967
O
OTBS
a, b
OH
H
H
H
H
H
O
H
TBSO
O
7
13
Scheme 2 Synthesis of (25R)-3,6-diketocholest-4-en-26-oic acid (13). Reagents and conditions: a) TBAF, THF, reflux, 20 h, 89%; b) PDC,
DMF, r.t., 18 h, 74%.
steps. This strategy required a differentiation between all Compound 16 represents a crucial intermediate, which
three hydroxy groups at the stage of the triol 5.
has been exploited also for the synthesis of (25R)-cholest-
5-en-26-oic acid (3) (Scheme 4). Oxidation of 16 using
PDC in dichloromethane14 to the aldehyde followed by
further oxidation with sodium chlorite afforded the acid
21 in 82% yield over both steps. Direct oxidation of 16 to
21 by using PDC in DMF as solvent gave only 45% yield.
Desilylation of the acid 21 by the usual method (TBAF in
THF at reflux) led only to impure (25R)-cholestenoic acid
(3) (67% yield). Chromatographic purification of 3
proved to be difficult. Therefore, desilylation of 21 was
carried out with catalytic amounts of concentrated sulfuric
acid in methanol at reflux to provide the methyl ester 22
in 84% yield. Purification of 22 by flash chromatography
and subsequent saponification using lithium hydroxide af-
forded pure (25R)-cholestenoic acid (3) in 97% yield.13
Pivaloyl chloride represents an excellent reagent for se-
lective acylations of primary in the presence of secondary
hydroxy groups.18 However, Williams reported a double
pivaloylation of the triol 5 at C-3b and C-26.8c Treatment
of 5 with only 1.1 equivalents of pivaloyl chloride led to a
selective esterification at C-26 to provide 14 in 81% yield
(Scheme 3). Silylation of the C-3b hydroxy group of 14
using TBSCl provided 15 in 79% yield. Thus, sequential
introduction of the pivaloyl and tert-butyldimethylsilyl
protecting groups resulted in a perfect differentiation
between all three hydroxy groups. Mesylation of the hy-
droxy group at C-16 and subsequent reduction removed
also the pivaloyl group at C-26 to afford the 26-hydroxy
derivative 16 in 89% yield. Acylation of 16 to 17 followed
by cleavage of the silyl ether using TBAF provided 18 in In conclusion, we have developed highly efficient and ste-
88% yield. Using the classical Oppenauer oxidation was reoselective syntheses of (25R)-D7-dafachronic acid (1)
found to avoid allylic oxidation at C-6 (see Scheme 2) and (10 steps, 16% overall yield), (25R)-D4-dafachronic acid
afforded the ketone 19 in 70% yield. Saponification of the (2) (10 steps, 22% overall yield), and (25R)-cholestenoic
acetate to 20 followed by Jones oxidation provided (25R)- acid (3) (9 steps, 32% overall yield) starting from com-
D4-dafachronic acid (2).13
mercially available diosgenin (4). These compounds are
much less active than described for the corresponding
OPiv
OPiv
OH
H
OH
H
OH
H
OH
a
c, d
H
H
H
H
b
H
H
HO
HO
TBSO
5
14
15
OH
OAc
HO
OAc
H
H
H
e
f
g
H
H
H
H
H
H
TBSO
TBSO
16
17
18
O
OAc
OH
OH
H
H
H
h
i
H
H
H
H
H
H
O
O
O
(25R)-Δ4-dafachronic acid (2)
19
20
Scheme 3 Synthesis of (25R)-D4-dafachronic acid (2). Reagents and conditions: a) PivCl, Et3N, cat. DMAP, THF, r.t., 20 h, 81%; b) TBSCl,
DBU, THF, r.t., 17 h, 79%; c) MsCl, pyridine, 0 °C to r.t., 18 h; d) LiAlH4, Et2O, 0 °C to reflux, 4 h, 89% over 2 steps; e) Ac2O, Et3N, cat.
DMAP, THF, r.t., 16 h, 100%; f) TBAF, THF, reflux, 17 h, 88%; g) Al(Oi-Pr)3, acetone–toluene (1:9), reflux, 15 h, 70%; h) cat. K2CO3, MeOH,
r.t., 40 h, 93%; i) CrO3, H2SO4, acetone, 0 °C, 1 h, 79%.
Synlett 2008, No. 13, 1965–1968 © Thieme Stuttgart · New York

1968
R. Martin et al.
LETTER
O
OH
OH
H
H
c
a, b
H
H
H
H
TBSO
TBSO
16
21
O
O
OMe
OH
H
H
H
H
H
H
d
HO
HO
(25R)-cholestenoic acid (3)
22
Scheme 4 Synthesis of (25R)-cholestenoic acid (3). Reagents and conditions: a) PDC, CH2Cl2, r.t., 3.5 h, 89%; b) NaClO2, KH2PO4, 2-meth-
yl-2-butene, THF–H2O (3:1), r.t., 16 h, 92%; c) cat. H2SO4, MeOH, reflux, 16 h, 84%; d) LiOH, THF–MeOH–H2O (2:1:1), r.t., 15 h, 97%.
25S-diastereoisomers.2 However, they could be used for
the clarification of basic principles of dauer larva forma-
tion and biological studies in this direction are underway.
By use of the present methodology, yamogenin, the 25S-
diastereoisomer of diosgenin (4), provides a direct and
stereoselective access to the (25S)-cholesten-26-oic acids.
116.99 (CH), 139.46 (C), 182.51 (C=O), 212.19 (C=O).
Anal. Calcd (%) for C27H42O3: C, 78.21; H, 10.21. Found: C,
78.42; H, 10.41.
(25R)-3,6-Diketocholest-4-en-26-oic acid (13): colorless
solid; mp 105–110 °C. 13C NMR and DEPT (125 MHz,
CDCl3): d = 11.87 (CH3), 16.76 (CH3), 17.49 (CH3), 18.55
(CH3), 20.84 (CH2), 23.59 (CH2), 23.93 (CH2), 27.97 (CH2),
33.83 (CH2), 33.94 (CH2), 34.16 (CH), 35.50 (CH, CH2),
35.60 (CH2), 39.08 (CH2), 39.21 (CH), 39.78 (C), 42.52 (C),
46.77 (CH2), 50.91 (CH), 55.83 (CH), 56.48 (CH), 125.45
(CH), 161.04 (C), 182.27 (C=O), 199.55 (C=O), 202.34
(C=O). HRMS: m/z calcd for C27H40O4 [M+]: 428.2927;
found: 428.2914.
References and Notes
(1) Matyash, V.; Entchev, E. V.; Mende, F.; Wilsch-Bräuninger,
M.; Thiele, C.; Schmidt, A. W.; Knölker, H.-J.; Ward, S.;
Kurzchalia, T. V. PLoS Biol. 2004, 2, 1561.
(25R)-D4-Dafachronic acid (2): colorless solid; mp 148–
150 °C. 13C NMR and DEPT (125 MHz, CDCl3): d = 11.93
(CH3), 16.74 (CH3), 17.35 (CH3), 18.55 (CH3), 20.99 (CH2),
23.60 (CH2), 24.14 (CH2), 28.15 (CH2), 32.00 (CH2), 32.93
(CH2), 33.86 (CH2), 33.94 (CH2), 35.57 (2 CH), 35.64 (2
CH2), 38.58 (C), 39.26 (CH), 39.57 (CH2), 42.37 (C), 53.75
(CH), 55.81 (CH), 55.99 (CH), 123.71 (CH), 171.85 (C),
182.48 (C=O), 199.83 (C=O). Anal. Calcd (%) for C27H42O3:
C, 78.21; H, 10.21. Found: C, 78.03; H, 10.46.
(2) 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.
(3) Held, J. M.; White, M. P.; Fisher, A. L.; Gibson, B. W.;
Lithgow, G. J.; Gill, M. S. Aging Cell 2006, 5, 283.
(4) Khripach, V. A.; Zhabinskii, V. N.; Konstantinova, O. V.;
Khripach, N. B.; Antonchick, A. V.; Antonchick, A. P.;
Schneider, B. Steroids 2005, 70, 551.
(5) Giroux, S.; Corey, E. J. J. Am. Chem. Soc. 2007, 129, 9866.
(6) Giroux, S.; Corey, E. J. Org. Lett. 2008, 10, 801.
(7) Schmidt, A. W.; Doert, T.; Goutal, S.; Gruner, M.; Mende,
F.; Kurzchalia, T. V.; Knölker, H.-J. Eur. J. Org. Chem.
2006, 3687.
(25R)-Cholestenoic acid (3): colorless solid; mp 168–170 °C
(MeCN). 13C NMR and DEPT (125 MHz, CDCl3): d = 11.85
(CH3), 16.74 (CH3), 18.63 (CH3), 19.38 (CH3), 21.05 (CH2),
23.62 (CH2), 24.27 (CH2), 28.21 (CH2), 31.58 (CH2), 31.87
(CH, CH2), 33.91 (CH2), 35.62 (CH), 35.72 (CH2), 36.47
(C), 37.22 (CH2), 39.27 (CH), 39.73 (CH2), 42.21 (CH2),
42.30 (C), 50.07 (CH), 56.05 (CH), 56.70 (CH), 71.82 (CH),
121.71 (CH), 140.69 (C), 182.46 (C=O). Anal. Calcd (%) for
C27H44O3: C, 77.83; H, 10.64. Found: C, 77.26; H, 10.33.
(14) Corey, E. J.; Schmidt, G. Tetrahedron Lett. 1979, 399.
(15) (a) Šolaja, B. A.; Milić, D. R.; Došen-Mićović, L. I. Steroids
1994, 59, 330. (b) Chodounská, H.; Pouzar, V.; Buděšínský,
M.; Slavíková, B.; Kohout, L. Steroids 2004, 69, 605.
(16) Brunel, J. M.; Loncle, C.; Vidal, N.; Dherbomez, M.;
Letourneux, Y. Steroids 2005, 70, 907.
(17) (a) Miranda Moreno, M. J. S.; Sá e Melo, M. L.;
Campos Neves, A. S. Tetrahedron Lett. 1991, 32, 3201.
(b) Acosta, C. K.; Rao, P. N.; Kim, H. K. Steroids 1993, 58,
205.
(18) (a) Nagaoka, H.; Rutsch, W.; Schmid, G.; Iio, H.; Johnson,
M. R.; Kishi, Y. J. Am. Chem. Soc. 1980, 102, 7962.
(b) Boschelli, D.; Takemasa, T.; Nishitani, Y.; Masamune,
S. Tetrahedron Lett. 1985, 26, 5239. (c) Nicolaou, K. C.;
Webber, S. E. Synthesis 1986, 453.
(8) (a) Williams, J. R.; Chai, D.; Wright, D. Steroids 2002, 67,
1041. (b) Williams, J. R.; Chai, D.; Bloxton, J. D.; Gong, H.;
Solvibile, W. R. Tetrahedron 2003, 59, 3183. (c) Gong, H.;
Williams, J. R. Org. Lett. 2006, 8, 2253.
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52, 5048.
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3149.
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(13) Characteristic Spectroscopic Data for the
(25R)-Cholesten-26-oic Acids 1, 13, 2, and 3
(25R)-D7-Dafachronic acid (1): colorless solid; mp 174–
175 °C. 13C NMR and DEPT (125 MHz, CDCl3): d = 11.87
(CH3), 12.44 (CH3), 16.72 (CH3), 18.74 (CH3), 21.66 (CH2),
22.91 (CH2), 23.68 (CH2), 27.90 (CH2), 30.02 (CH2), 33.85
(CH2), 34.35 (C), 35.62 (CH2), 36.01 (CH), 38.09 (CH2),
38.73 (CH2), 39.24 (CH), 39.38 (CH2), 42.82 (CH), 43.33
(C), 44.20 (CH2), 48.78 (CH), 54.87 (CH), 56.01 (CH),
Synlett 2008, No. 13, 1965–1968 © Thieme Stuttgart · New York