Stereoselective synthesis of the hormonally active (25S)- Δ7-dafachronic acid, (25S)-Δ4-dafachronic acid, (25S)-dafachronic acid, and (25S)-cholestenoic acid

Article abstract of DOI:10.1039/b815064h

We report a stereoselective synthesis of the (25S)-cholestenoic-26-acids which are highly efficient ligands for the hormonal receptor DAF-12 in Caenorhabditis elegans.

Full text of DOI:10.1039/b815064h

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Stereoselective synthesis of the hormonally active (25S)-D7-dafachronic acid,
(25S)-D4-dafachronic acid, (25S)-dafachronic acid, and (25S)-cholestenoic
acid†
Rene´ Martin,a Frank Da¨britz,a Eugeni V. Entchev,b Teymuras V. Kurzchaliab and Hans-Joachim Kno¨lker*a
Received 29th August 2008, Accepted 2nd October 2008
First published as an Advance Article on the web 17th October 2008
DOI: 10.1039/b815064h
We report
a
stereoselective synthesis of the (25S)-
cholestanol,1 the saturated (25S)-dafachronic acid 3 appeared to
be also an attractive target molecule. Herein, we report a flexible
synthesis of the (25S)-configurated steroidal acids 1–4.
cholestenoic-26-acids which are highly efficient ligands for
the hormonal receptor DAF-12 in Caenorhabditis elegans.
Recently, Corey and Giroux described the synthesis of both
diastereoisomers of 1, which they called dafachronic acid A.5,6
For the (25S)-diastereoisomer they started from b-stigmasterol
using a diastereoselective ruthenium-catalyzed hydrogenation.5
The (25R)-diastereoisomer was synthesized from b-ergosterol.6
In an independent synthetic study, Khripach and co-workers
reported the synthesis of both diastereoisomers of 2 and 4 using a
diastereoselective elaboration of the steroid side chain.7
The life cycle and longevity of the nematode Caenorhabditis
elegans are regulated by several genes, among them daf -9.1,2
DAF-9 protein, a cytochrome P450, is involved in the biosynthesis
of the dafachronic acids. Mangelsdorf et al. identified these new
steroidal hormones, products of DAF-9 activity, as ligands for the
nuclear hormone receptor DAF-12. In the presence of dafachronic
acids, DAF-12 is inactive and reproductive development occurs,
whereas in the absence of the hormone the dauer larva is
formed. The dauer-pathway genes and concomitant hormones
also influence the life span of worms.2 The steroids were assigned
as (25S)-D7- and (25S)-D4-dafachronic acid (1) and (2) (Fig. 1).2
Starting from commercially available 3b-hydroxychol-5-en-24-
oic acid (5) we have developed a highly stereoselective synthesis
of one crucial intermediate providing access to all three (25S)-
dafachronic acids 1–3 and (25S)-cholestenoic acid (4) as ligands
for DAF-12. Transformation of 5 to the known aldehyde 6 was
achieved by disilylation of 5 with tert-butyldimethylsilyl chloride
followed by reduction of the silyl ester using lithium aluminium
hydride (Scheme 1).8 Swern oxidation of the alcohol provided the
aldehyde 6 in 3 steps and 94% overall yield.9 For introduction of
the stereogenic center at C-25 we applied a stereoselective Evans
aldol reaction using commercially available (S)-(+)-4-isopropyl-
3-propionyl-2-oxazolidinone (7).10 The resulting aldol product 8
was obtained in 95% yield as a single stereoisomer. Reduction
of 8 using lithium borohydride followed by esterification of the
C-26 hydroxy group with pivaloyl chloride provided compound
9 in high yield. For transformation into the natural products,
removal of the hydroxy group at C-24 was required. Mesylation
of 9 and subsequent reduction with lithium aluminium hydride
afforded the desired alcohol along with the C24/C25-olefin as
an inseparable mixture. However, Barton deoxygenation of 9
provided 10 in excellent yield.11 It should be emphasized that
radical removal of the C-24 hydroxy group occurred without
detectable epimerization at C-25. The orthogonally diprotected
(25S)-26-hydroxycholesterol 10, which is available in 8 steps and
66% overall yield, represents our key intermediate and has been
converted to all four (25S)-steroidal acids. Allylic oxidation of
10 provided the cholest-5-en-7-one 11 in 75% yield.12 Palladium-
catalyzed hydrogenation of 11 and subsequent reduction with
Fig. 1 (25S)-Cholestenoic-26-acids 1–4.
The (25R)-dafachronic acids are much weaker ligands for
DAF-12 compared with the 25S-isomers 1 and 2.2 Independently,
Gill et al. identified (25S)-cholestenoic acid (4) as a ligand
for DAF-12.3 Recently, we described the total syntheses of the
(25R)-dafachronic acids and (25R)-cholestenoic acid starting from
diosgenin.4 As we found that cholesterol can be replaced by
L-Selectrideꢀ at -78 ◦C diastereoselectively provided the 7a-
R
aDepartment Chemie, Technische Universita¨t Dresden, Bergstrasse 66,
01069 Dresden, Germany. E-mail: hans-joachim.knoelker@tu-dresden.de;
Fax: +49 35-463-37030
alcohol 12 in excellent yield. Elimination of 12 using thionyl
chloride in pyridine13 followed by removal of both protecting
groups afforded the diol 13 in 71% overall yield. As the (25S)-
26-hydroxy-D7-cholesterol 13 is unprecedented in this series, we
confirmed the configuration at C-25 by an X-ray crystal structure
determination (Fig. 2).‡ Finally, Jones oxidation of 13 led to (25S)-
D7-dafachronic acid (1) in 89% yield.§
bMax Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer-
strasse 108, 01307 Dresden, Germany
† Electronic supplementary information (ESI) available: Copies of the 1H
and 13C NMR spectra for all compounds described. CCDC reference
number 697683. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/b815064h
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Scheme 1 Synthesis of the orthogonally diprotected diol 10 as the crucial intermediate and transformation into (25S)-D7-dafachronic acid (1).
Scheme 2 Transformation of the crucial intermediate 10 into (25S)-D4-dafachronic acid (2), (25S)-dafachronic acid (3), and (25S)-cholestenoic acid (4).
For the transformation of compound 10 into 2, removal of the
silyl protecting group and Oppenauer oxidation of the C-3 hydroxy
group afforded the cholest-4-en-3-one 14. Subsequent cleavage of
the pivaloyl protecting group and Jones oxidation of the C-26
hydroxy group provided (25S)-D4-dafachronic acid (2).¶
Sequential removal of both protecting groups by desilylation
of 10 with TBAF followed by treatment with lithium alu-
minium hydride led to the known (25S)-26-hydroxycholesterol
(15) (Scheme 2).14 Catalytic hydrogenation of the diol 15 and
subsequent Jones oxidation provided (25S)-dafachronic acid (3)
in 81% yield over 4 steps.ꢀ
Fig. 2 Molecular structure of the diol 13 in the crystal form.
4294 | Org. Biomol. Chem., 2008, 6, 4293–4295
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=
=
For the synthesis of 4, the pivaloyl protecting group of 10
was removed using lithium aluminium hydride. Subsequent Swern
oxidation9 of the C-26 hydroxy group and further oxidation with
sodium chlorite led to the C-3 silyl-protected acid 16 in 81% yield
over 3 steps. In the 25R-series, we found that chromatographic
separation of cholestenoic acid proved to be very difficult.4
Therefore, compound 16 was converted to the 3-hydroxy 26-methyl
ester by treatment with catalytic amounts of concentrated sulfuric
acid in methanol at reflux. After chromatographic purification,
cleavage of the methyl ester with lithium hydroxide provided pure
(25S)-cholestenoic acid (4) in 99% yield.**
In conclusion, we have developed a highly efficient and stereo-
selective synthesis of the crucial intermediate 10 which serves as
precursor for the hormonally active steroids 1–4 in excellent yields
(1: 15 steps, 27% overall yield; 2: 12 steps, 19% overall yield;
3: 12 steps, 53% overall yield; 4: 13 steps, 46% overall yield).
The Evans aldol reaction of aldehyde 6 as the key-step of our
synthesis proceeded with complete stereoselectivity and provided
compound 8 as a single stereoisomer, also on a large scale. Our
approach is superior with respect to efficiency and overall yields
as compared with the previously reported syntheses. Moreover,
the present route is highly flexible as it provides all 4 steroidal
acids in amounts of up to 1 g via a common intermediate and
paves the way for detailed biological studies. Thus, it opened up
a simple route to the non-natural (25S)-dafachronic acid (3). In
a preliminary biological assay, the hormonal activity of 3 was
comparable to that of the other (25S)-diastereoisomers 1, 2, and
4. Further investigations in this direction are currently underway.
(CH), 117.00 (CH), 139.48 (C), 182.55 (C O), 212.17 (C O); anal. calc.
for C27H42O3: C 78.21, H 10.21; found: C 78.21, H 10.31%.
¶ Characteristic spectroscopic data for (25S)-D4-dafachronic acid (2):
colorless solid, mp 173–174 ◦C; 13C NMR and DEPT (125 MHz, CDCl3):
d = 11.93 (CH3), 17.01 (CH3), 17.35 (CH3), 18.54 (CH3), 20.99 (CH2), 23.69
(CH2), 24.14 (CH2), 28.15 (CH2), 32.00 (CH2), 32.93 (CH2), 33.94 (CH2),
34.00 (CH2), 35.58 (2 CH), 35.64 (CH2), 35.68 (CH2), 38.58 (C), 39.34
(CH), 39.58 (CH2), 42.37 (C), 53.75 (CH), 55.82 (CH), 55.98 (CH), 123.71
=
=
(CH), 171.86 (C), 182.35 (C O), 199.84 (C O); anal. calc. for C27H42O3: C
78.21, H 10.21; found: C 78.21, H 10.12%.
ꢀ Characteristic spectroscopic data for (25S)-dafachronic acid (3): light
yellow solid, mp 123–126 ◦C; 13C NMR and DEPT (125 MHz, CDCl3):
d = 11.45 (CH3), 12.05 (CH3), 16.99 (CH3), 18.56 (CH3), 21.42 (CH2),
23.71 (CH2), 24.19 (CH2), 28.21 (CH2), 28.94 (CH2), 31.68 (CH2), 34.00
(CH2), 35.36 (CH), 35.61 (C, CH), 35.71 (CH2), 38.18 (CH2), 38.53 (CH2),
39.37 (CH), 39.86 (CH2), 42.57 (C), 44.71 (CH2), 46.67 (CH), 53.74 (CH),
=
=
56.15 (CH), 56.23 (CH), 182.70 (C O), 212.41 (C O); HRMS: m/z calc.
for C27H44O3 [M+]: 416.3290; found: 416.3291.
** Characteristic spectroscopic data for (25S)-cholestenoic acid (4): color-
less solid, mp 172–175 ◦C; 13C NMR and DEPT (125 MHz, CDCl3): d =
11.84 (CH3), 17.02 (CH3), 18.62 (CH3), 19.38 (CH3), 21.05 (CH2), 23.70
(CH2), 24.26 (CH2), 28.21 (CH2), 31.61 (CH2), 31.87 (CH, CH2), 34.05
(CH2), 35.63 (CH), 35.75 (CH2), 36.48 (C), 37.22 (CH2), 39.21 (CH), 39.73
(CH2), 42.24 (CH2), 42.30 (C), 50.07 (CH), 56.03 (CH), 56.71 (CH), 71.81
=
(CH), 121.71 (CH), 140.71 (C), 181.58 (C O); anal. calc. for C27H44O3: C
77.83, H 10.64; found: C 77.99, H 10.77%.
1 (a) V. Matyash, E. V. Entchev, F. Mende, M. Wilsch-Bra¨uninger, C.
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Gruner, F. Mende, T. V. Kurzchalia and H.-J. Kno¨lker, Eur. J. Org.
Chem., 2006, 3687.
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Acknowledgements
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Kno¨lker, Synlett, 2008, 1965.
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experiments.
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Notes and references
‡ Crystallographic data for the diol 13: C27H46O2, M = 402.64 g mol-1,
crystal size: 0.27 ¥ 0.12 ¥ 0.10 mm3, orthorhombic, space group P212121,
3
˚
˚
a = 34.899(7), b = 9.3865(19), c = 7.5000(15) A, V = 2456.8(9) A , Z = 4,
-3
-1
˚
rcalcd = 1.089 g cm , m = 0.066 mm , l = 0.71073 A, T = 223(2) K, q
9 A. J. Mancuso, S.-L. Huang and D. Swern, J. Org. Chem., 1978, 43,
2480.
range = 3.19–25.40◦, reflections collected: 35264, independent: 2611 (Rint
=
0.0748), 266 parameters. The structure was solved by direct methods and
10 (a) D. A. Evans, J. Bartroli and T. L. Shih, J. Am. Chem. Soc., 1981,
103, 2127; (b) D. A. Evans, J. V. Nelson, E. Vogel and T. R. Taber,
J. Am. Chem. Soc., 1981, 103, 3099; (c) D. A. Evans, J. M. Takacs, L. R.
McGee, M. D. Ennis, D. J. Mathre and J. Bartroli, Pure Appl. Chem.,
1981, 53, 1109.
refined by full-matrix least-squares on F2; final R indices [I > 2s(I)]: R1 =
-3
˚
0.0468; wR2 = 0.1065; maximal residual electron density: 0.242 e A .
CCDC-697683 contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge from The Cambridge
crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
§ Characteristic spectroscopic data for (25S)-D7-dafachronic acid (1):
colorless solid, mp 139–143 ◦C; 13C NMR and DEPT (125 MHz, CDCl3):
d = 11.89 (CH3), 12.45 (CH3), 17.01 (CH3), 18.74 (CH3), 21.68 (CH2),
22.92 (CH2), 23.79 (CH2), 27.92 (CH2), 30.04 (CH2), 34.01 (CH2), 34.38
(C), 35.68 (CH2), 36.04 (CH), 38.11 (CH2), 38.75 (CH2), 39.36 (CH), 39.40
(CH2), 42.83 (CH), 43.35 (C), 44.22 (CH2), 48.81 (CH), 54.90 (CH), 56.02
11 D. H. R. Barton and S. W. McCombie, J. Chem. Soc., Perkin Trans. 1,
1975, 1574.
12 N. Chidambaram and S. Chandrasekaran, J. Org. Chem., 1987, 52,
5048.
13 H.-J. Kno¨lker, A. Ecker, P. Struwe, A. Steinmeyer, G. Mu¨ller and G.
Neef, Tetrahedron, 1997, 53, 91.
14 C.-Y. Byon, M. Gut and V. Toome, J. Org. Chem., 1981, 46, 3901.
This journal is
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Org. Biomol. Chem., 2008, 6, 4293–4295 | 4295
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