Synthesis and hormonal activity of the (25s)-cholesten-26-oic acids -potent ligands for the daf-12 receptor in caenorhabditis elegans

Article abstract of DOI:10.1002/ejoc.200900443

Using a highly stereoselective Evans aldol reaction for the introduction of the stereogenic center at C-25, we describe an efficient synthesis of the orthogonally diprotected (25S)-26-hydroxycholesterol 11. In a few synthetic steps, this crucial intermediate 11 has been converted into the four (25S)-cholesten-26-oic acids 1-4, which have been obtained in 12-15 steps and 19-53% overall yield based on commercially available 3p-hydroxychol-5-en-24-oic acid (5). Our biological studies of the compounds 1-4 reveal that (25S)-Δ⁷-dafachronic acid (1) represents the most active steroidal ligand for the hormonal receptor DAF-12 in Caenorhabditis elegans. Moreover, the saturated (25S)-dafachronic acid (3) represents a new ligand for this receptor and the (25S)-steroidal acids are more active as compared to their corresponding (25R)-counterparts.

Full text of DOI:10.1002/ejoc.200900443

FULL PAPER
DOI: 10.1002/ejoc.200900443
Synthesis and Hormonal Activity of the (25S)-Cholesten-26-oic Acids –
Potent Ligands for the DAF-12 Receptor in Caenorhabditis elegans
René Martin,[a] Eugeni V. Entchev,[b] Frank Däbritz,[a] Teymuras V. Kurzchalia,[b] and
Hans-Joachim Knölker*[a]
Keywords: Diastereoselectivity / Hormones / Oxidation / Protecting groups / Steroids
Using a highly stereoselective Evans aldol reaction for the
introduction of the stereogenic center at C-25, we describe
an efficient synthesis of the orthogonally diprotected (25S)-
26-hydroxycholesterol 11. In a few synthetic steps, this cru-
cial intermediate 11 has been converted into the four (25S)-
cholesten-26-oic acids 1–4, which have been obtained in 12–
15 steps and 19–53% overall yield based on commercially
available 3β-hydroxychol-5-en-24-oic acid (5). Our biological
studies of the compounds 1–4 reveal that (25S)-∆7-dafach-
ronic acid (1) represents the most active steroidal ligand for
the hormonal receptor DAF-12 in Caenorhabditis elegans.
Moreover, the saturated (25S)-dafachronic acid (3) represents
a new ligand for this receptor and the (25S)-steroidal acids
are more active as compared to their corresponding (25R)-
counterparts.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
Introduction
Reproductive development of nematodes such as Caeno-
rhabditis elegans and Pristionchus pacificus is controlled by
steroidal ligands, called dafachronic acids (Figure 1).[1,2] In
C. elegans, the biosynthesis of these steroids requires ac-
tivity of the cytochrome P450 DAF-9.[3] Dafachronic acids
are ligands which inactivate the nuclear hormone receptor
DAF-12 and thus, lead to reproductive development of
worms. In daf-9 mutant worms, incapable of dafachronic
acid biosynthesis, DAF-12 is active and worms enter the
diapause state generating dauer larvae. Another ligand
known to bind at DAF-12 is (25S)-cholestenoic acid (4).[4]
Mangelsdorf and colleagues prepared (25S)-∆4-dafach-
ronic acid (2) and its C-25 epimer from the corresponding
26-hydroxycholesterols.[1a] In 2007, the structure of the
other ligand, (25S)-∆7-dafachronic acid (1), has been con-
firmed by a synthesis from Corey and Giroux.[5] Moreover,
it has been shown that (25S)-∆7-dafachronic acid (1) repre-
sents the most active ligand known so far.[1a,5] Interestingly,
the synthesis of both C-25 epimers of 2 and 4 was described
previously by Khripach et al.[6] Our investigations on the
synthesis and biological activity of cholesterol derivatives,[7]
led us to an elegant and concise synthesis of the 25R-dia-
stereoisomers of 1, 2 and 4 starting from commercially
Figure 1. Hormonally active steroidal acids 1–4.
available diosgenin.[8,9] Also in the 25R-series, the ∆7-
dafachronic acid exhibited the highest hormonal activity.[9]
Since yamogenin, the C-25 epimer of diosgenin, was not
available from commercial sources, we devised a novel
stereoselective construction of the side chain for the synthe-
sis of all three (25S)-cholesten-26-oic acids 1, 2 and 4, as
well as the saturated (25S)-dafachronic acid (3).[10] The syn-
thesis of (25S)-dafachronic acid (3) was also reported by
Corey and co-workers.[11]
[a] Department Chemie, Technische Universität Dresden,
Bergstraße 66, 01069 Dresden, Germany
Results and Discussion
Fax: +49-351-463-37030
The Evans aldol reaction represents a powerful synthetic
method for the enantioselective construction of stereogenic
carbon centers.[12,13] However, applications to stereoselec-
tive synthesis of steroid side chains are rare.[14] For our pur-
E-mail: hans-joachim.knoelker@tu-dresden.de
[b] Max-Planck-Institut für Molekulare Zellbiologie und Genetik,
Pfotenhauerstraße 108, 01307 Dresden, Germany
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200900443.
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Scheme 1. Stereoselective synthesis of the crucial intermediate 11. Reagents and conditions: (a) 3.2 equiv. TBSCl, 8.1 equiv. imidazole,
2.2 equiv. DMAP, DMF, 25 °C, 18 h; (b) 4.0 equiv. LiAlH4, THF, 25 °C, 17 h, 96% for 2 steps; (c) 4.0 equiv. DMSO, 2.0 equiv. (COCl)2,
CH2Cl2, –78 °C, then 6, 20 min, 5.0 equiv. Et3N, to 25 °C, 98%; (d) 1.3 equiv. (S)-(+)-4-isopropyl-3-propionyl-2-oxazolidinone, 1.43 equiv.
Bu2BOTf, 1.69 equiv. Et3N, CH2Cl2, 0 °C, 1 h, then –78 °C, 7, 30 min, then 0 °C, 80 min, 95%; (e) 1.1 equiv. LiBH4, 1.0 equiv. H2O,
Et2O, 0 °C, 2 h; (f) 1.3 equiv. PivCl, pyridine/CH2Cl2 (1:1), 0 °C, 2 h, 81% for 2 steps; (g) 1.0 equiv. NaHMDS, 20.0 equiv. CS2, THF,
–78 °C to 0 °C, then 2.0 equiv. MeI, 30 min, 98%; (h) 10 mol-% AIBN, 15.0 equiv. Bu3SnH, reflux, 5 min, 93%.
poses, commercially available 3β-hydroxychol-5-en-24-oic commended. Using the strategy described above, the or-
acid (5) appeared to be the ideal starting material thogonally diprotected (25S)-26-hydroxycholesterol 11 is
(Scheme 1).[15] Treatment of 5 with tert-butylchlorodime- readily available in 8 steps and 66% overall yield. Com-
thylsilane (TBSCl) in the presence of imidazole and DMAP pound 11 represents the central intermediate of our synthe-
led to the intermediate silyl ester which on subsequent re- sis and has been converted into the four (25S)-steroidal ac-
duction using lithium aluminium hydride provided almost ids 1–4.
quantitatively the 24-hydroxy derivative 6. Oxidation of the
alcohol 6 with PDC afforded the aldehyde 7 in 96% yield.
For an access to (25S)-∆7-dafachronic acid (1), a double
bond shift was required. Allylic oxidation following Chand-
Moreover, Swern oxidation of 6 provided the aldehyde 7 rasekaran’s procedure afforded the enone 12 in 75% yield
in 98% yield even on large scale.[16] By using the standard (Scheme 2).[18] Palladium-catalyzed hydrogenation using
conditions reported by Evans,[12] (1.3 equiv. of commercial ethyl acetate as solvent led to the ketone 13.
(S)-(+)-4-isopropyl-3-propionyl-2-oxazolidinone, triethyl-
The reduction of cyclohexanones using sterically hin-
amine and dibutylboron triflate), the aldol product 8 was dered reducing agents under kinetic conditions provides the
available on large scale as a single stereoisomer in 95% axial alcohols.[19] Thus, treatment of ketone 13 with -Se-
yield. Reduction of 8 with lithium borohydride followed by lectride® afforded stereoselectively the 7α-alcohol 14 in 90%
selective pivaloylation of the primary hydroxy group af- yield. Using the commercially available Burgess reagent in
forded compound 9 in 81% yield over both steps. In the benzene under reflux, the cholest-7-ene 15 could be isolated
next step, the hydroxy group at C-24 had to be removed. in 78% yield (Table 1).[20] In the 25R-series, elimination of a
Mesylation of the hydroxy group followed by treatment structurally related alcohol with thionyl chloride in pyridine
with lithium aluminium hydride afforded the corresponding provided quantitatively the corresponding ∆7-olefin.[8,9]
C-26 hydroxy compound along with the C-24/C-25 olefin. Treatment of 14 with 3.0 equiv. of thionyl chloride in pyr-
Both compounds were not separable by flash chromatog- idine provided the olefin 15 in only 72% yield. However,
raphy on silica gel. The reaction of xanthates with tributyl- increasing the amount of thionyl chloride to 5.0 equiv. af-
stannane in the presence of AIBN provides deoxygenated forded 15 in 87% yield. Removal of the silyl and pivaloyl
products in high yields and thus, represents a promising al- protecting groups was achieved by treatment with first
ternative.[17] For the synthesis of the corresponding xan- TBAF and then lithium aluminium hydride to afford the
thate, alcohol 9 was treated with NaHMDS and carbon di- diol 16 in 82% yield. Changing the sequence of the removal
sulfide at low temperature. Addition of iodomethane pro- of the protecting groups resulted in only 69% yield of 16.
vided the xanthate 10 almost quantitatively. After a reac- At this stage of our synthesis, the 25S-configuration of the
tion time of only 5 min, Barton deoxygenation of 10 af- 26-hydroxycholest-7-en-3β-ol (16) has been unambiguously
forded the deoxygenated compound 11 in 93% yield. In or- confirmed by an X-ray crystal structure determination (Fig-
der to achieve good results in this transformation, ure 2).[10] Finally, Jones oxidation of the diol 16 provided
recrystallisation of commercial AIBN from methanol is re- (25S)-∆7-dafachronic acid (1) in 89% yield.
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Synthesis and Hormonal Activity of (25S)-Cholesten-26-oic Acids
Scheme 2. Synthesis of (25S)-∆7-dafachronic acid (1) from the intermediate 11. Reagents and conditions: (a) 4.0 equiv. PDC, 8.0 equiv.
tBuOOH, Celite®, benzene, 0 °C to 25 °C, 41 h, 75%; (b) 10% Pd/C, H2, EtOAc, 25 °C, 16 h, 95%; (c) 1.3 equiv. -Selectride®, THF,
–78 °C, 1.5 h, 90%; (d) 5.0 equiv. SOCl2, pyridine, 0 °C, 40 min, 87%; (e) 1.5 equiv. TBAF, THF, reflux, 16 h; (f) 4.0 equiv. LiAlH4, THF,
0 °C to 25 °C, 17 h, 82% for 2 steps; (g) 5.0 equiv. Jones reagent, acetone, 0 °C, 90 min, 89%.
Table 1. Elimination of the 7α-hydroxy group from 14.
cholest-4-en-3-one 18 in 86% yield. Cleavage of the pivalate
proved to be difficult (Table 2). Reaction of the pivalate 18
with bis[tributyltin(IV)] oxide in benzene under reflux re-
sulted in 100% recovery of starting material 18.[21] Saponifi-
cation of 18 with lithium hydroxide afforded only 29% of
the desired 26-hydroxy derivative 19. Using one equivalent
of potassium carbonate in methanol at room temperature
provided after 6 d the alcohol 19 in 39% yield along with
20% of starting material 18. The best result was obtained
by treatment of 18 with sodium methoxide in methanol at
room temperature and afforded the alcohol 19 in 56% yield
along with 15% of starting material. Completion of the
synthesis was achieved by Jones oxidation of the alcohol 19
to provide (25S)-∆4-dafachronic acid (2) in 65% yield.
Reaction conditions
% Yield of 15
2.0 equiv. Burgess reagent, benzene, reflux, 2 h
3.0 equiv. SOCl2, pyridine, 0 °C, 30 min
5.0 equiv. SOCl2, pyridine, 0 °C, 40 min
78
72
87
Figure 2. X-ray crystal structure of the diol 16 (orthorhombic,
P212121).
Table 2. Removal of the pivaloyl protecting group from 18.
Reaction conditions
% Yield of 19
Transformation of intermediate 11 to (25S)-∆4-dafach-
ronic acid (2) required only a few steps (Scheme 3). As we
have described previously for the 25R series,[8,9] treatment
of 26-hydroxycholesterol with Jones reagent leads by con-
comitant allylic oxidation to the undesired 3,6-diketochol-
est-4-en-26-oic acid. Therefore, we decided to achieve a se-
quential oxidation of the two hydroxy groups at C-3 and C-
3.0 equiv. LiOH, MeOH/H2O (10:1), 50 °C, 18 h
1.0 equiv. K2CO3, MeOH, 25 °C, 6 d
2.0 equiv. NaOMe, MeOH, 25 °C, 5 d
29
39[a]
56[b]
[a] 20% of starting material recovered. [b] 15% of starting material
recovered.
For the synthesis of (25S)-dafachronic acid (3), both pro-
26. Selective removal of the silyl ether with TBAF in THF tecting groups had to be removed from our crucial interme-
under reflux afforded the 3β-alcohol 17. Oppenauer oxi- diate 11 (Scheme 4). Removal of the pivalate using lithium
dation of the hydroxy group at C-3 occurred with concomi- aluminium hydride and subsequent desilylation by treat-
tant isomerisation of the double bond and afforded the ment with TBAF afforded the known (25S)-26-hydroxycho-
Scheme 3. Synthesis of (25S)-∆4-dafachronic acid (2). Reagents and conditions: (a) 1.5 equiv. TBAF, THF, reflux, 17 h, 92%; (b) 1.5 equiv.
Al(OiPr)3, acetone/toluene (1:9), 100 °C, 5 h, 86%; (c) 2.0 equiv. NaOMe, MeOH, 25 °C, 5 d, 56 % of 19, 15% of 18; (d) 5.0 equiv. Jones
reagent, 0 °C, 90 min, 65%.
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Scheme 4. Synthesis of (25S)-dafachronic acid (3). Reagents and conditions: (a) 1.5 equiv. TBAF, THF, reflux, 17 h; (b) 4.0 equiv. LiAlH4,
THF, 25 °C, 16 h, 93% for 2 steps; (c) 10% Pd/C, H2, MeOH/CH2Cl2 (1:1), 25 °C, 24 h, 99%; (d) 5.0 equiv. Jones reagent, acetone, 0 °C,
60 min, 88%.
lesterol (20) in only 69% yield.[22] However, as described
For the conversion of compound 11 to (25S)-choles-
above for the synthesis of the diol 16, a reversal of the se- tenoic acid (4), the pivaloyl protecting group had to be re-
quence by first removing the silyl and then the pivaloyl pro- moved selectively. Treatment of 11 with lithium aluminium
tecting group gave a much better result and provided (25S)- hydride afforded the alcohol 22 in 91% yield (Scheme 5).
26-hydroxycholesterol (20) in 93% yield over both steps. Swern oxidation of 22 followed by oxidation of the crude
Palladium-catalyzed hydrogenation of 20 led to (25S)-5α- aldehyde with sodium chlorite provided the silyl-protected
cholestan-3β,26-diol (21). Finally, Jones oxidation of 21 af- (25S)-cholestenoic acid 23 in 89% yield over two steps. In
forded (25S)-dafachronic acid (3) in 88% yield.[10,11]
our studies of the 25R-series, we found that chromato-
Scheme 5. Transformation of 11 into (25S)-cholestenoic acid (4). Reagents and conditions: (a) 4.0 equiv. LiAlH4, THF, 25 °C, 17 h, 91%;
(b) 4.0 equiv. DMSO, 2.0 equiv. (COCl)2, CH2Cl2, –78 °C, then 22, 20 min, 5.0 equiv. Et3N, to 25 °C; (c) 2.0 equiv. NaClO2, 10.0 equiv.
2-methyl-2-butene, KH2PO4, THF/H2O (3:1), 25 °C, 24 h, 89% for 2 steps; (d) cat. H2SO4, MeOH, reflux, 16 h, 87%; (e) 3.0 equiv. LiOH,
THF/MeOH/H2O (1:1:1), 25 °C, 24 h, 99%.
Figure 3. Bioactivity of (25S)-dafachronic acids 1–3 and (25S)-cholestenoic acid (4). A: rescue of diapause in daf-9(dh6) mutant worms
by feeding with the indicated (25S)-cholesten-26-oic acids. B: without addition of (25S)-cholesten-26-oic acids, daf-9(dh6) mutant worms
(no fluorescence) arrest as dauer like larvae (white arrow). C–E: 250 nM (25S)-dafachronic acids 1–3 rescue daf-9(dh6) mutant worms
to adults (Λ). F: 250 nM (25S)-cholestenoic acid (4) rescues only partially daf-9(dh6) mutant worms forming arrested larvae, often with
molting defects (*). White triangles in all images indicate fluorescent daf-9(dh6);dhEx24 mutant worms which develop to adults without
requirement of exogenous (25S)-dafachronic or (25S)-cholestenoic acid.
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Synthesis and Hormonal Activity of (25S)-Cholesten-26-oic Acids
graphic purification of cholestenoic acid is difficult.[8,9] idal acids: (25S)-∆7-dafachronic acid (1) (15 steps, 27%
Thus, esterification was carried out with concomitant cleav- overall yield), (25S)-∆4-dafachronic acid (2) (12 steps, 19%
age of the silyl ether using catalytic amounts of concen- overall yield), (25S)-dafachronic acid (3) (12 steps, 53%
trated sulfuric acid in methanol under reflux. The methyl overall yield) and (25S)-cholestenoic acid (4) (13 steps, 46%
ester 24 was purified by flash chromatography on silica gel overall yield). Our present synthesis of the (25S)-cholesten-
and isolated in 87% yield. A subsequent saponification of 26-oic acids 1–4 is clearly superior with respect to overall
the ester with lithium hydroxide provided almost quantita- yields and efficiency as compared to the previous ap-
tively pure (25S)-cholestenoic acid (4).
proaches to these compounds.[5,6,11] The present methodol-
The bioactivity of 25S-steroidal acids 1–4 was investi- ogy for the assembly of the side chain can be applied to the
gated by rescue of daf-9 mutant worms from dauer arrest synthesis of other (25S)-steroids.
(Figure 3). The daf-9(dh6) mutant worms lacking DAF-9
Our efficient access to the (25S)-cholesten-26-oic acids
protein activity cannot generate dafachronic acids.[1a,3b] In 1–4 also set the stage for detailed biological studies towards
consequence, these mutant worms arrest as dauer-like lar- their hormonal activity in controlling the life cycle of C.
vae (Figure 3, A,B). In the experimental setup, some daf- elegans. Our study of the biological activity of (25S)-
9(dh6) mutant worms are obtained as progeny from the dafachronic acids emphasizes the importance of the 3-hy-
strain daf-9(dh6);dhEx24.[3b] The daf-9(dh6) mutant droxy group and suggests that worms can not efficiently oxi-
worms are identified by the absence of green fluorescence, dise the 3-hydroxy group of (25S)-cholestenoic acid (4). The
which is carried by the extrachromosomal array dhEx24. In fact that (25S)-∆4-dafachronic acid (2) and (25S)-dafach-
the parental strain daf-9(dh6);dhEx24, the extrachromo- ronic acid (3) show a similar activity indicates that either
somal array dhEx24 rescues the daf-9(dh6) mutation (Fig- the double bond at C-4 is not important for the activity or,
ure 3, B).[3b] Normal reproductive development of daf- that the worms very efficiently convert 3 into 2. The latter
9(dh6) mutant worms is only possible by exogenous supply reaction might be carried out by a steroid dehydrogenase.[23]
of dafachronic acid (Figure 3, A–E).
The comparison of the biological activities of the (25S)-
If the supplied amount of dafachronic acid is not suf- steroidal acids described in the present publication with
ficient for complete rescue, arrested larvae, often with molt- those of the (25R)-dafachronic acids reported in our pre-
ing defects, are observed (Figure 3 A, F). Our experiments vious paper[9] emphasizes that the (25S)-steroidal acids are
show that the rescue of daf-9(dh6) mutant worms from more active than their corresponding (25R)-diastereoiso-
dauer arrest is dependent on the concentration of steroidal mers. Moreover, it is shown that (25S)-∆7-dafachronic acid
acids. The rescue of daf-9(dh6) mutant worms from dauer (1) has the highest biological activity among all these steroi-
arrest by feeding with the 25S-steroidal acids 1–4 demon- dal acids. The bioactivity of (25R)-∆7-dafachronic acid (1)
strates the difference in activity for these ligands (Figure 3, is in the range of the bioactivity observed for (25S)-∆4-
A). Our results emphasize that (25S)-∆7-dafachronic acid dafachronic acid (2) and (25S)-dafachronic acid (3).
(1) is the most active ligand. Moreover, it became obvious Whereas (25R)-∆4-dafachronic acid is almost as active as
that (25S)-∆4-dafachronic acid (2) and (25S)-dafachronic (25S)-cholestenoic acid (4). Thus, by our biological studies
acid (3) have a moderate activity, which is about one order the functional groups of the steroids and their stereochem-
of magnitude lower than the activity of 1. The least active istry required for efficacious signaling can be concluded.
compound in this series is (25S)-cholestenoic acid (4) with
an activity about one order of magnitude lower than found
for the ligands 2 and 3. We have compared the activities
of the (25S)-steroidal acids 1–4 with those of the (25R)-
Experimental Section
General: All reactions were carried out in dry solvents and oven-
dried glassware under argon atmosphere. Tetrahydrofuran, ethyl
acetate, dichloromethane, and diethyl ether were dried in a solvent
purification system (MBraun-SPS). Acetone was distilled from
phosphorus pentoxide and stored over molecular sieves (3 Å). Ben-
zene was dried with sodium. Toluene was purchased from Acros
Organics (water content less than 50 ppm). Dry methanol was pur-
chased from VWR Prolabo (water content less than 20 ppm). Pyr-
idine was obtained from Fluka (water content less than 50 ppm).
Triethylamine was heated under reflux with calcium hydride for
48 h and stored over 3 Å molecular sieves. Commercial AIBN was
recrystallised from methanol. Dibutylboron triflate was obtained
from Acros Organics as a 1  solution in dichloromethane. All
other chemicals were used as received from commercial sources.
Flash chromatography was performed using silica gel from Acros
Organics (0.063–0.200 mm). Thin layer chromatography was per-
formed with TLC plates from Merck (60 F254) using anisaldehyde
solution for visualization. Melting points were measured on an
Electrothermal IA9100 melting point apparatus. Specific rotation
dafachronic acids, which have been reported in our previous
publication.[9] As a result, the activity of (25R)-∆7-dafach-
ronic acid is in the same range as observed for (25S)-∆4-
dafachronic acid (2) and (25S)-dafachronic acid (3). While
(25R)-∆4-dafachronic acid has an activity comparable to
that of (25S)-cholestenoic acid (4).
Conclusions
We have developed a highly efficient synthetic route to
all four of the (25S)-cholesten-26-oic acids 1–4 by using a
completely stereoselective Evans aldol reaction as key-step.
Thus, compound 8 was obtained as a single stereoisomer
even on a multigram scale and our crucial intermediate 11
became available in 8 steps and 66% overall yield. Interme-
diate 11 has been exploited to prepare all four (25S)-stero- values were obtained from a Perkin–Elmer 341 polarimeter. Infra-
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red spectra were recorded on a Thermo Nicolet Avatar 360 FT-IR
spectrometer using the ATR method (Attenuated Total Reflec-
tance). NMR spectra were recorded on a Bruker DRX 500 NMR
quenched by addition of a saturated aqueous solution of ammo-
nium chloride (20 mL). The layers were separated and the aqueous
layer was extracted with dichloromethane (2ϫ50 mL). The com-
bined organic layers were dried with magnesium sulfate and the
solvent was evaporated. Purification of the residue by flash
chromatography on silica gel (petroleum ether/diethyl ether, 10:1)
afforded the aldehyde 7, yield 1.081 g (98%). Colourless solid; m.p.
137–139 °C (ref.[15] 132–137 °C). 1H NMR (500 MHz, CDCl3): δ =
0.04 (s, 6 H), 0.66 (s, 3 H), 0.87 (s, 9 H), 0.91 (d, J = 6.5 Hz, 3 H),
0.95–1.18 (m, 6 H), 0.98 (s, 3 H), 1.24–1.34 (m, 3 H), 1.40–1.60 (m,
6 H), 1.68–1.86 (m, 4 H), 1.93–2.00 (m, 2 H), 2.15 (ddd, J = 13.3,
4.9, 2.2 Hz, 1 H), 2.25 (m, 1 H), 2.35 (m, 1 H), 2.44 (m, 1 H), 3.47
(m, 1 H), 5.30 (m, 1 H), 9.76 (t, J = 1.9 Hz, 1 H) ppm. 13C NMR
and DEPT (125 MHz, CDCl3): δ = –4.60 (2 CH3), 11.85 (CH3),
18.26 (C), 18.40 (CH3), 19.41 (CH3), 21.02 (CH2), 24.23 (CH2),
25.93 (3 CH3), 27.94 (CH2), 28.17 (CH2), 31.87 (CH, CH2), 32.05
(CH2), 35.32 (CH), 36.55 (C), 37.35 (CH2), 39.72 (CH2), 40.92
(CH2), 42.37 (C), 42.78 (CH2), 50.11 (CH), 55.76 (CH), 56.73
(CH), 72.60 (CH), 121.09 (CH), 141.54 (C), 203.25 (CHO) ppm.
C30H52O2Si (472.82): C 76.21, H 11.09, found: C 76.27, H 11.14%.
For further spectroscopic data see ref.[15].
1
spectrometer. Complete assignment of the H and 13C signals was
achieved by HSQC experiments. Chemical shifts δ are reported in
ppm with the deuterated solvent as internal standard. The follow-
ing abbreviations have been used: s: singlet, d: doublet, dd: doublet
of doublets, dt: doublet of triplets, t: triplet, q: quartet, sext: sextet,
sept: septet, m: multiplet, br: broad. Mass spectra were recorded
on a Finnigan MAT-95 spectrometer (electron impact, 70 eV) or by
GC/MS-coupling using an Agilent Technologies 6890N GC System
equipped with a 5973 Mass Selective Detector (electron impact EI,
70 eV). ESI-MS spectra were recorded on an Esquire LC with an
ion trap detector from Bruker. Positive and negative ions were de-
tected. Elemental analyses were measured on an EuroVector
EuroEA3000 elemental analyser. X-ray analyses: Bruker–Nonius
Kappa CCD equipped with a 700 series Cryostream low tempera-
ture device from Oxford Cryosystems and STOE IPDS 2 image
plate. Software: Collect (Nonius BV, 1999), Dirax/lsq (Duisenberg,
1992), SHELXS-97 (G. M. Sheldrick, 1990), EvalCCD (Duisen-
berg et al., 2003), SADABS version 2.10 (G. M. Sheldrick, Bruker
AXS Inc., 2002), SHELXL-97 (G. M. Sheldrick, 1997), Schakal-
99 (E. Keller, 1999).
(4S,24ЈR,25ЈS)-3-[3Јβ-(tert-Butyldimethylsilyloxy)-24Ј-hydroxychol-
est-5Ј-en-26Ј-oyl]-4-isopropyloxazolidin-2-one (8): A 1.0  solution
of dibutylboron triflate (3.28 mL, 3.28 mmol) was added to a solu-
tion of (S)-(+)-4-isopropyl-3-propionyl-2-oxazolidinone (505 µL,
2.98 mmol) in dichloromethane (10 mL) at 0 °C. After 5 min, tri-
ethylamine (539 µL, 3.87 mmol) was added dropwise and the re-
sulting mixture was stirred at 0 °C for 1 h. The reaction mixture
was cooled to –78 °C and a solution of the aldehyde 7 (1.081 g,
2.29 mmol) in dichloromethane (10 mL) was added dropwise. Stir-
ring was continued at –78 °C for 30 min. Then, the mixture was
warmed to 0 °C and stirring was continued for additional 80 min.
Methanol (10 mL) and 30% aqueous H2O2 (10 mL) were added
and the resulting mixture was stirred at 0 °C for 30 min. Water
(100 mL) was added and the layers were separated. The aqueous
layer was extracted with dichloromethane (2ϫ50 mL). The com-
bined organic layers were dried with magnesium sulfate and the
solvent was evaporated. Purification of the residue by flash
chromatography on silica gel (petroleum ether/diethyl ether, 4:1 to
2:1) afforded the aldol product 8 as a single stereoisomer, yield
3β-(tert-Butyldimethylsilyloxy)chol-5-en-24-ol (6): tert-Butylchloro-
dimethylsilane
(3.78 g,
25.09 mmol),
imidazole
(4.32 g,
63.50 mmol) and DMAP (2.2 g, 17.25 mmol) were added to a solu-
tion of 3β-hydroxychol-5-en-24-oic acid (5) (2.936 g, 7.84 mmol) in
DMF (50 mL). Additional DMF (50 mL) was added and the solu-
tion was stirred at room temperature for 18 h. After addition of
water (250 mL), the resulting mixture was extracted with diethyl
ether (3ϫ50 mL). The combined organic layers were washed with
water (2ϫ100 mL), brine (100 mL) and then dried with magnesium
sulfate. Evaporation of the solvent gave the crude product which
was dissolved in THF (50 mL). Lithium aluminium hydride (1.19 g,
31.36 mmol) was added in portions to this solution at 0 °C and the
resulting mixture was stirred at room temperature for 17 h. Water
(50 mL) was slowly added and the mixture was extracted with di-
ethyl ether (3ϫ50 mL). The combined organic layers were dried
with magnesium sulfate and the solvent was removed. Purification
of the residue by flash chromatography on silica gel (petroleum
ether/diethyl ether, 4:1) provided the alcohol 6, yield 3.561 g (96%).
1.428 g (95%). Colourless solid; m.p. 172–174 °C. IR (ATR): ν =
˜
1
Colourless solid; m.p. 171–173 °C (ref.[15] 146.5–152 °C). H NMR
3533, 2928, 2858, 1773, 1701, 1680, 1458, 1386, 1367, 1302, 1236,
1207, 1143, 1078, 1057, 1017, 959, 886, 870, 835, 807, 774, 719
cm–1. 1H NMR (500 MHz, CDCl3): δ = 0.04 (s, 6 H), 0.66 (s, 3 H),
0.87 (d, J = 6.5 Hz, 3 H), 0.87 (s, 9 H), 0.89–1.28 (m, 9 H), 0.91
(d, J = 7.1 Hz, 6 H), 0.98 (s, 3 H), 1.24 (d, J = 7.1 Hz, 3 H), 1.38–
1.60 (m, 9 H), 1.70 (m, 1 H), 1.79 (dt, J = 13.3, 3.3 Hz, 1 H), 1.82–
1.85 (m, 1 H), 1.93–2.00 (m, 2 H), 2.15 (ddd, J = 13.3, 4.8, 2.1 Hz,
1 H), 2.25 (m, 1 H), 2.33 (dsept, J = 4.0, 7.0 Hz, 1 H), 2.91 (br. d,
J = 2.1 Hz, 1 H), 3.46 (m, 1 H), 3.76 (dq, J = 2.6, 7.1 Hz, 1 H),
3.87 (m, 1 H), 4.21 (dd, J = 8.6, 3.3 Hz, 1 H), 4.27 (t, J = 8.6 Hz,
(500 MHz, CDCl3): δ = 0.04 (s, 6 H), 0.67 (s, 3 H), 0.88 (s, 9 H),
0.93 (d, J = 6.6 Hz, 3 H), 0.95–1.17 (m, 6 H), 0.98 (s, 3 H), 1.21–
1.27 (m, 2 H), 1.39–1.72 (m, 11 H), 1.79 (dt, J = 13.3, 3.5 Hz, 1
H), 1.82–1.84 (m, 1 H), 1.93–2.01 (m, 2 H), 2.15 (ddd, J = 13.3,
4.9, 2.2 Hz, 1 H), 2.25 (m, 1 H), 3.47 (m, 1 H), 3.60 (m, 2 H), 5.30
(m, 1 H) ppm. 13C NMR and DEPT (125 MHz, CDCl3): δ = –4.60
(2 CH3), 11.84 (CH3), 18.27 (C), 18.66 (CH3), 19.42 (CH3), 21.03
(CH2), 24.25 (CH2), 25.93 (3 CH3), 28.22 (CH2), 29.37 (CH2), 31.82
(CH2), 31.87 (CH), 31.90 (CH2), 32.06 (CH2), 35.55 (CH), 36.56
(C), 37.36 (CH2), 39.76 (CH2), 42.31 (C), 42.79 (CH2), 50.15 (CH),
55.95 (CH), 56.76 (CH), 63.60 (CH2), 72.63 (CH), 121.13 (CH),
141.54 (C) ppm. C30H54O2Si (474.83): C 75.88, H 11.46, found: C
75.94, H 11.34%. For further spectroscopic data see ref.[15]
1 H), 4.46 (ddd, J = 8.6, 4.0, 3.3 Hz, 1 H), 5.30 (m, 1 H) ppm. 13
C
NMR and DEPT (125 MHz, CDCl3): δ = –4.61 (2 CH3), 10.78
(CH3), 11.84 (CH3), 14.66 (CH3), 17.90 (CH3), 18.25 (C), 18.66
(CH3), 19.41 (CH3), 21.02 (CH2), 24.25 (CH2), 25.92 (3 CH3), 28.18
(CH2), 28.30 (CH), 30.07 (CH2), 31.87 (CH), 31.89 (CH2), 32.03
(CH2), 32.06 (CH2), 35.45 (CH), 36.55 (C), 37.34 (CH2), 39.73
(CH2), 42.08 (CH), 42.30 (C), 42.79 (CH2), 50.13 (CH), 55.79
(CH), 56.73 (CH), 58.18 (CH), 63.29 (CH2), 71.54 (CH), 72.61
(CH), 121.14 (CH), 141.52 (C), 153.50 (C=O), 177.93 (C=O) ppm.
ESI-MS: m/z = 658.5 [(M + H)+], 640.4, 508.4. C39H67NO5Si
(658.04): C 71.18, H 10.26, N 2.13, found: C 71.43, H 10.34, N
2.25%.
3β-(tert-Butyldimethylsilyloxy)chol-5-en-24-al (7): Oxalyl chloride
(396 µL, 4.68 mmol) was added slowly to a solution of DMSO
(665 µL, 9.36 mmol) in dichloromethane (10 mL) at –78 °C. After
5 min, a solution of the alcohol 6 (1.109 g, 2.34 mmol) in dichloro-
methane (20 mL) was added and stirring was continued at –78 °C
for 20 min. Then, triethylamine (1.63 mL, 11.70 mmol) was added,
the solution was warmed to room temperature and stirring was
continued for additional 10 min. The reaction mixture was
3708
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 3703–3714

Synthesis and Hormonal Activity of (25S)-Cholesten-26-oic Acids
(24R,25R)-3β-(tert-Butyldimethylsilyloxy)-26-(pivaloyloxy)cholest-5- (CH2), 72.61 (CH), 84.56 (CH), 121.12 (CH), 141.55 (C), 178.41
en-24-ol (9): A 2.0  solution of lithium borohydride in THF
(908 µL, 1.815 mmol) and water (30 µL, 1.65 mmol) were added
to a solution of the aldol product 8 (1.085 g, 1.65 mmol) in diethyl
ether (25 mL) at 0 °C. After stirring for 2 h at 0 °C, water (20 mL)
and 2  NaOH (20 mL) were slowly added to the reaction mix-
ture, which was subsequently extracted with dichloromethane
(2ϫ50 mL), diethyl ether (2ϫ50 mL) and ethyl acetate
(2ϫ50 mL). The combined organic layers were dried with magne-
sium sulfate and the solvent was evaporated. The crude diol was
dissolved in pyridine (15 mL) and dichloromethane (15 mL). Af-
ter addition of pivaloyl chloride (264 µL, 2.145 mmol) at 0 °C, the
resulting mixture was stirred at 0 °C for 2 h and then water
(50 mL) was added. The mixture was extracted with dichloro-
methane (3ϫ50 mL) and the combined organic layers were dried
with magnesium sulfate. Evaporation of the solvent and purifica-
tion of the residue by flash chromatography on silica gel (petro-
leum ether/diethyl ether, 6:1) provided the pivalate 9, yield 818 mg
(C=O), 215.94 (C=S) ppm.
(25S)-3β-(tert-Butyldimethylsilyloxy)-26-(pivaloyloxy)cholest-5-ene
(11): AIBN (3.5 mg, 21.1 µmol) was added to a solution of the
xanthate 10 (149 mg, 211 µmol) in toluene (5 mL). The flask con-
taining this mixture was put in an oil bath, which was already
heated at 110 °C.
A solution of tributylstannane (851 µL,
3.165 mmol) in toluene (2 mL) was slowly added to the hot reaction
mixture. After 5 min, the yellow solution became colourless, indi-
cating complete conversion. After cooling to room temperature, a
saturated aqueous solution of sodium hydrogen carbonate (50 mL)
was added and the mixture was extracted with diethyl ether
(3ϫ50 mL). The combined organic layers were dried with magne-
sium sulfate and the solvent was evaporated. Purification of the
residue by flash chromatography on silica gel (petroleum ether/di-
ethyl ether, 100:1 to 70:1) afforded compound 11, yield 118 mg
(93%). Colourless solid; m.p. 116–118 °C. IR (ATR): ν = 2929,
˜
2895, 2856, 1728, 1471, 1397, 1368, 1282, 1252, 1152, 1090, 1033,
989, 959, 939, 890, 870, 835, 804, 771, 666 cm–1. 1H NMR
(500 MHz, CDCl3): δ = 0.04 (s, 6 H), 0.66 (s, 3 H), 0.86–1.28 (m,
10 H), 0.88 (s, 9 H), 0.90 (d, J = 6.6 Hz, 3 H), 0.92 (d, J = 6.8 Hz,
3 H), 0.98 (s, 3 H), 1.19 (s, 9 H), 1.30–1.59 (m, 10 H), 1.69–1.83
(m, 4 H), 1.94 (m, 1 H), 1.99 (dt, J = 13.0, 3.3 Hz, 1 H), 2.15 (ddd,
J = 13.3, 4.9, 2.1 Hz, 1 H), 2.26 (m, 1 H), 3.47 (m, 1 H), 3.82 (dd,
J = 10.7, 6.7 Hz, 1 H), 3.93 (dd, J = 10.7, 5.6 Hz, 1 H), 5.30 (m, 1
H) ppm. 13C NMR and DEPT (125 MHz, CDCl3): δ = –4.60 (2
CH3), 11.82 (CH3), 17.07 (CH3), 18.27 (C), 18.70 (CH3), 19.42
(CH3), 21.04 (CH2), 23.20 (CH2), 24.27 (CH2), 25.93 (3 CH3), 27.22
(3 CH3), 28.22 (CH2), 31.88 (CH), 31.92 (CH2), 32.07 (CH2), 32.70
(CH), 33.86 (CH2), 35.67 (CH), 36.14 (CH2), 36.57 (C), 37.36
(CH2), 38.84 (C), 39.77 (CH2), 42.30 (C), 42.80 (CH2), 50.17 (CH),
56.01 (CH), 56.77 (CH), 69.11 (CH2), 72.63 (CH), 121.14 (CH),
141.56 (C), 178.64 (C=O) ppm. MS (70 eV): m/z (%) = 600 (1)
[M+], 585 (3), 543 (73) [(M – tBu)+], 441 (12), 367 (100), 295 (11),
255 (14), 161 (20), 159 (79). HRMS: m/z calcd. for C34H59O3Si
[(M – tBu)+]: 543.4233, found: 543.4246.
(81%). Colourless solid; m.p. 133–136 °C. IR (ATR): ν = 3525,
˜
2958, 2926, 2898, 2851, 1710, 1471, 1459, 1382, 1287, 1247, 1174,
1131, 1076, 1029, 1010, 975, 940, 892, 873, 834, 805, 777 cm–1
.
1H NMR (500 MHz, CDCl3): δ = 0.04 (s, 6 H), 0.66 (s, 3 H), 0.88
(s, 9 H), 0.90–1.33 (m, 8 H), 0.911 (d, J = 7.0 Hz, 3 H), 0.915 (d,
J = 6.4 Hz, 3 H), 0.98 (s, 3 H), 1.20 (s, 9 H), 1.37–1.58 (m, 10 H),
1.69–1.74 (m, 1 H), 1.79 (dt, J = 13.3, 3.3 Hz, 1 H), 1.82–1.87 (m,
2 H), 1.94 (m, 1 H), 1.99 (dt, J = 12.4, 3.3 Hz, 1 H), 2.15 (ddd, J
= 13.3, 4.9, 2.2 Hz, 1 H), 2.25 (m, 1 H), 3.47 (m, 1 H), 3.53 (m,
1 H), 3.92 (dd, J = 11.0, 5.8 Hz, 1 H), 4.19 (dd, J = 11.0, 7.6 Hz,
1 H), 5.30 (m, 1 H) ppm. 13C NMR and DEPT (125 MHz,
CDCl3): δ = –4.60 (2 CH3), 10.27 (CH3), 11.84 (CH3), 18.26 (C),
18.67 (CH3), 19.41 (CH3), 21.03 (CH2), 24.25 (CH2), 25.93 (3
CH3), 27.21 (3 CH3), 28.26 (CH2), 30.60 (CH2), 31.87 (CH), 31.90
(CH2), 32.06 (CH2), 32.28 (CH2), 35.50 (CH), 36.56 (C), 37.36
(CH2), 37.95 (CH), 38.83 (C), 39.75 (CH2), 42.31 (C), 42.79
(CH2), 50.14 (CH), 55.83 (CH), 56.75 (CH), 66.83 (CH2), 71.88
(CH), 72.62 (CH), 121.13 (CH), 141.54 (C), 178.89 (C=O) ppm.
GC-MS (70 eV): m/z (%) = 559 (29) [(M – tBu)+], 383 (18), 365
(16), 341 (27), 331 (11), 255 (15), 211 (20), 175 (11), 171 (11), 161
(33), 159 (100). C38H68O4Si (617.03): C 73.97, H 11.11, found: C
74.04, H 11.19%.
(25S)-3β-(tert-Butyldimethylsilyloxy)-26-(pivaloyloxy)cholest-5-en-
7-one (12): A 5.5  solution of tert-butyl hydroperoxide in decane
(629 µL, 3.46 mmol), PDC (1.30 g, 3.46 mmol) and Celite® (1.03 g)
were added to a solution of compound 11 (520 mg, 0.865 mmol) in
O-(24R,25R)-3β-(tert-Butyldimethylsilyloxy)-26-(pivaloyloxy)chol- benzene (30 mL) at 0 °C. The resulting mixture was stirred at room
est-5-en-24-yl S-Methyl Xanthate (10):
A
1.0  solution of
temperature for 24 h. A second portion of tert-butyl hydroperoxide
(629 µL, 3.46 mmol) was added and stirring was continued for 17 h.
The reaction mixture was filtered through a short pad of silica gel
with diethyl ether (500 mL) and the solvent was removed. Purifica-
tion of the residue by flash chromatography on silica gel (petroleum
ether/diethyl ether, 20:1) provided the enone 12, yield 398 mg
NaHMDS in THF (136 µL, 136 µmol) and carbon disulfide
(164 µL, 2.72 mmol) were added to a solution of the alcohol 9
(84 mg, 136 µmol) in THF at –78 °C. After stirring for 30 min at
–78 °C, the solution was warmed to 0 °C and iodomethane (17 µL,
272 µmol) was added. Stirring was continued for additional 30 min,
water (50 mL) was added and the solution was extracted with di-
ethyl ether (3ϫ50 mL). The combined organic layers were dried
with magnesium sulfate and the solvent was removed to provide
the xanthate 10, yield 94 mg (98%). Yellow oil (which crystallised
(75%). Colourless crystals; m.p. 153–155 °C. IR (ATR): ν = 2934,
˜
2858, 1730, 1666, 1626, 1461, 1375, 1282, 1253, 1153, 1091, 1033,
955, 937, 892, 877, 835, 804, 771 cm–1. 1H NMR (500 MHz,
CDCl3): δ = 0.05 (s, 6 H), 0.66 (s, 3 H), 0.88 (s, 9 H), 0.90 (d, J =
6.4 Hz, 3 H), 0.91 (d, J = 6.7 Hz, 3 H), 1.00–1.66 (m, 17 H), 1.17
(s, 3 H), 1.19 (s, 9 H), 1.74–1.82 (m, 2 H), 1.85–1.91 (m, 2 H), 2.01
(dt, J = 12.7, 3.3 Hz, 1 H), 2.22 (dd, J = 12.3, 10.9 Hz, 1 H), 2.33–
2.43 (m, 3 H), 3.59 (m, 1 H), 3.83 (dd, J = 10.7, 6.7 Hz, 1 H), 3.93
(dd, J = 10.7, 5.6 Hz, 1 H), 5.65 (d, J = 1.4 Hz, 1 H) ppm. 13C
NMR and DEPT (125 MHz, CDCl3): δ = –4.70 (CH3), –4.67
(CH3), 11.93 (CH3), 17.07 (CH3), 17.28 (CH3), 18.15 (C), 18.85
(CH3), 21.16 (CH2), 23.26 (CH2), 25.82 (3 CH3), 26.28 (CH2), 27.22
1
on standing). H NMR (500 MHz, CDCl3): δ = 0.04 (s, 6 H), 0.65
(s, 3 H), 0.85–0.93 (m, 1 H), 0.88 (s, 9 H), 0.91 (d, J = 6.5 Hz, 3
H), 0.95–1.26 (m, 7 H), 0.98 (s, 3 H), 1.02 (d, J = 7.0 Hz, 3 H),
1.20 (s, 9 H), 1.36–1.57 (m, 8 H), 1.70 (m, 1 H), 1.77–1.83 (m, 3
H), 1.93–1.98 (m, 2 H), 2.12–2.27 (m, 4 H), 2.54 (s, 3 H), 3.47 (m,
1 H), 3.97 (d, J = 6.5 Hz, 2 H), 5.30 (m, 1 H), 5.78 (m, 1 H) ppm.
13C NMR and DEPT (125 MHz, CDCl3): δ = –4.60 (2 CH3), 11.85
(2 CH3), 18.27 (C), 18.57 (CH3), 18.87 (CH3), 19.41 (CH3), 21.03
(CH2), 24.23 (CH2), 25.93 (3 CH3), 27.05 (CH2), 27.20 (3 CH3), (3 CH3), 28.54 (CH2), 31.71 (CH2), 32.70 (CH), 33.83 (CH2), 35.61
28.02 (CH2), 31.31 (CH2), 31.88 (CH, CH2), 32.06 (CH2), 35.17 (CH), 36.15 (CH2), 36.39 (CH2), 38.34 (C), 38.68 (CH2), 38.85 (C),
(CH), 36.08 (CH), 36.56 (C), 37.36 (CH2), 38.78 (C), 39.74 (CH2), 42.51 (CH2), 43.05 (C), 45.38 (CH), 49.90 (CH), 49.95 (CH), 54.66
42.31 (C), 42.79 (CH2), 50.14 (CH), 55.49 (CH), 56.73 (CH), 65.56 (CH), 69.10 (CH2), 71.31 (CH), 125.78 (CH), 165.89 (C), 178.65
Eur. J. Org. Chem. 2009, 3703–3714
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3709

H.-J. Knölker et al.
FULL PAPER
(C=O), 202.47 (C=O) ppm. GC-MS (70 eV): m/z (%) = 614 (2)
367 (17), 345 (14), 343 (10), 331 (76), 281 (24), 273 (16), 269 (32),
[M+], 557 (41), 381 (100), 269 (25). C38H66O4Si (615.01): C 74.21, 259 (14), 257 (22), 255 (13), 213 (14), 211 (23), 75 (100). C38H70O4Si
H 10.82, found: C 74.24, H 10.92%.
(619.05): C 73.73, H 11.40, found: C 73.84, H 11.48%.
(25S)-3β-(tert-Butyldimethylsilyloxy)-26-(pivaloyloxy)-5α-cholest-7-
ene (15): Thionyl chloride (41 µL, 565 µmol) was added to a solu-
tion of the 7α-alcohol 14 (70 mg, 113 µmol) in pyridine (5 mL) at
0 °C. The resulting mixture was stirred at 0 °C for 40 min and the
solvent was evaporated. Purification of the residue by flash
chromatography on silica gel (petroleum ether/diethyl ether, 50:1)
afforded the cholest-7-ene 15, yield 59 mg (87%). Colourless solid;
(25S)-3β-(tert-Butyldimethylsilyloxy)-26-(pivaloyloxy)cholest-5α-an-
7-one (13): A solution of the enone 12 (100 mg, 163 µmol) in ethyl
acetate (10 mL) was added to a Schlenk flask, loaded with Pd/C
(10%, 17.3 mg, 16.3 µmol Pd). The resulting mixture was stirred at
room temperature for 16 h under an hydrogen atmosphere. Fil-
tration of the mixture over a short pad of Celite® with ethyl acetate,
evaporation of the solvent and purification of the crude product
by flash chromatography on silica gel (petroleum ether/diethyl
ether, 10:1) afforded the ketone 13, yield 95 mg (95%). Colourless
m.p. 79–80 °C. IR (ATR): ν = 2930, 2854, 1728, 1471, 1398, 1377,
˜
1282, 1252, 1161, 1102, 1084, 1006, 982, 940, 871, 835, 815, 795,
1
772 cm–1. H NMR (500 MHz, CDCl3): δ = 0.04 (s, 6 H), 0.51 (s,
solid; m.p. 116–117 °C. IR (ATR): ν = 2929, 2857, 1726, 1704,
˜
3 H), 0.77 (s, 3 H), 0.87 (s, 9 H), 0.90 (d, J = 6.4 Hz, 3 H), 0.92 (d,
J = 6.7 Hz, 3 H), 0.99–1.80 (m, 26 H), 1.19 (s, 9 H), 1.83–1.87 (m,
1 H), 2.00 (dt, J = 12.3, 3.2 Hz, 1 H), 3.53 (m, 1 H), 3.83 (dd, J =
10.7, 6.8 Hz, 1 H), 3.93 (dd, J = 10.7, 5.6 Hz, 1 H), 5.14 (m, 1 H)
ppm. 13C NMR and DEPT (125 MHz, CDCl3): δ = –4.58 (2 CH3),
11.80 (CH3), 13.07 (CH3), 17.06 (CH3), 18.28 (C), 18.83 (CH3),
21.49 (CH2), 22.93 (CH2), 23.28 (CH2), 25.94 (3 CH3), 27.21 (3
CH3), 27.94 (CH2), 29.69 (CH2), 31.86 (CH2), 32.69 (CH), 33.84
(CH2), 34.21 (C), 36.08 (CH, CH2), 37.31 (CH2), 38.43 (CH2),
38.84 (C), 39.56 (CH2), 40.37 (CH), 43.37 (C), 49.50 (CH), 55.02
(CH), 56.02 (CH), 69.11 (CH2), 71.89 (CH), 117.53 (CH), 139.53
(C), 178.65 (C=O) ppm. GC-MS (70 eV): m/z (%) = 600 (6) [M+],
585 (7), 545 (9), 543 (74), 468 (21), 467 (20), 441 (22), 367 (15), 255
(10), 161 (19), 159 (69), 75 (100). C38H68O3Si (601.03): C 75.94, H
11.40, found: C 75.82, H 11.40%.
1471, 1375, 1284, 1250, 1162, 1099, 1054, 1007, 986, 943, 873, 835,
797, 772 cm–1. 1H NMR (500 MHz, CDCl3): δ = 0.025 (s, 3 H),
0.026 (s, 3 H), 0.63 (s, 3 H), 0.86 (s, 9 H), 0.89 (d, J = 6.7 Hz, 3
H), 0.91 (d, J = 6.8 Hz, 3 H), 0.93–1.56 (m, 20 H), 1.06 (s, 3 H),
1.19 (s, 9 H), 1.69–1.78 (m, 3 H), 1.85–1.89 (m, 1 H), 1.94–2.00 (m,
2 H), 2.16 (m, 1 H), 2.33 (m, 2 H), 3.53 (m, 1 H), 3.82 (dd, J =
10.7, 6.7 Hz, 1 H), 3.92 (dd, J = 10.7, 5.6 Hz, 1 H) ppm. 13C NMR
and DEPT (125 MHz, CDCl3): δ = –4.66 (2 CH3), 11.85 (CH3),
12.02 (CH3), 17.05 (CH3), 18.20 (C), 18.75 (CH3), 21.83 (CH2),
23.23 (CH2), 24.92 (CH2), 25.87 (3 CH3), 27.21 (3 CH3), 28.40
(CH2), 31.52 (CH2), 32.71 (CH), 33.83 (CH2), 35.55 (CH), 36.01
(C), 36.12 (CH2), 36.21 (CH2), 38.44 (CH2), 38.74 (CH2), 38.84
(C), 42.47 (C), 46.20 (CH2), 47.13 (CH), 48.83 (CH), 49.99 (CH),
54.92 (CH), 55.44 (CH), 69.10 (CH2), 71.50 (CH), 178.64 (C=O),
212.44 (C=O) ppm. GC-MS (70 eV): m/z (%) = 601 (3) [(M –
Me)+], 561 (8), 559 (59), 457 (28), 383 (29), 365 (25), 271 (31), 253
(11), 177 (11), 161 (35), 159 (89), 75 (100), 57 (73). C38H68O4Si
(617.03): C 73.97, H 11.11, found: C 74.06, H 11.22%.
(25S)-26-Hydroxy-5α-cholest-7-en-3β-ol (16): A 1.0  solution of
tetrabutylammonium fluoride in THF (0.53 mL, 530 µmol) was
added to a solution of the cholest-7-ene 15 (214 mg, 356 µmol) in
THF (15 mL). The mixture was heated under reflux for 16 h. After
cooling to room temperature, water was added (50 mL) and the
resulting mixture was extracted with diethyl ether (3ϫ50 mL). The
combined organic layers were dried with magnesium sulfate and
the solvent was evaporated. The crude product was dissolved in
THF (10 mL) and lithium aluminium hydride (54 mg, 1.424 mmol)
was added at 0 °C. The resulting mixture was stirred at room tem-
perature for 17 h. Then, 10% sulfuric acid (10 mL) and water
(50 mL) were added. The mixture was extracted with ethyl acetate
(3ϫ50 mL) and the combined organic layers were dried with mag-
nesium sulfate. Evaporation of the solvent and purification of the
residue by flash chromatography on silica gel (petroleum ether/
ethyl acetate, 3:1) provided the diol 16, which has been recrystal-
lised from ethyl acetate, yield 118 mg (82%). Colourless crystals;
(25S)-3β-(tert-Butyldimethylsilyloxy)-26-(pivaloyloxy)cholest-5α-an-
7α-ol (14): A 1.0  solution of -Selectride® in THF (754 µL,
754 µmol) was added to a solution of the ketone 13 (358 mg,
580 µmol) in THF (20 mL) at –78 °C. Stirring was continued at the
same temperature for 1.5 h. The reaction mixture was quenched
by addition of a saturated aqueous solution of sodium hydrogen
carbonate (10 mL), methanol (5 mL) and 30% aqueous H2O2
(5 mL), and was warmed to 0 °C. After stirring for 30 min, water
(50 mL) and diethyl ether (50 mL) were added and the layers were
separated. The aqueous layer was extracted with diethyl ether
(2ϫ50 mL) and the combined organic layers were dried with mag-
nesium sulfate. Evaporation of the solvent and purification of the
residue by flash chromatography on silica gel (petroleum ether/di-
ethyl ether, 10:1) provided the 7α-alcohol 14 as a single stereoiso-
mer, yield 324 mg (90%). Colourless solid; m.p. 90–92 °C. IR
m.p. 165–167 °C. IR (ATR): ν = 3303, 2914, 2865, 1464, 1447,
˜
1381, 1366, 1347, 1100, 1052, 1032, 1019, 976, 941, 848, 832, 729
cm–1. 1H NMR (500 MHz, CDCl3): δ = 0.52 (s, 3 H), 0.78 (s, 3 H),
0.91 (d, J = 6.7 Hz, 6 H), 0.98–1.30 (m, 9 H), 1.33–1.64 (m, 12 H),
1.69–1.90 (m, 6 H), 2.01 (dt, J = 12.7, 3.4 Hz, 1 H), 3.41 (dd, J =
10.5, 6.5 Hz, 1 H), 3.50 (dd, J = 10.5, 5.7 Hz, 1 H), 3.58 (m, 1 H),
5.14 (dd, J = 4.7, 2.2 Hz, 1 H) ppm. 13C NMR and DEPT
(125 MHz, CDCl3): δ = 11.84 (CH3), 13.04 (CH3), 16.72 (CH3),
18.85 (CH3), 21.52 (CH2), 22.93 (CH2), 23.51 (CH2), 27.95 (CH2),
29.62 (CH2), 31.45 (CH2), 33.63 (CH2), 34.18 (C), 35.81 (CH),
36.17 (CH, CH2), 37.11 (CH2), 37.95 (CH2), 39.53 (CH2), 40.21
(CH), 43.36 (C), 49.40 (CH), 55.01 (CH), 56.07 (CH), 68.34 (CH2),
71.04 (CH), 117.44 (CH), 139.56 (C) ppm. MS (70 eV): m/z (%) =
402 (100) [M+], 387 (30), 273 (15), 255 (46), 231 (16), 229 (11), 213
(15), 161 (11). HRMS: m/z calcd. for C27H46O2 [M+]: 402.3498,
found: 402.3490.
(ATR): ν = 3501, 2929, 2855, 1728, 1708, 1471, 1398, 1380, 1285,
˜
1250, 1162, 1101, 1075, 1032, 1006, 975, 947, 871, 835, 814, 772
cm–1. 1H NMR (500 MHz, CDCl3): δ = 0.03 (s, 6 H), 0.63 (s, 3 H),
0.78 (s, 3 H), 0.86 (s, 9 H), 0.89 (d, J = 6.5 Hz, 3 H), 0.91 (d, J =
6.8 Hz, 3 H), 0.98–1.05 (m, 2 H), 1.06–1.16 (m, 6 H), 1.19 (s, 9 H),
1.21–1.68 (m, 17 H), 1.56 (dt, J = 12.6, 3.1 Hz, 1 H), 1.74–1.86 (m,
2 H), 1.92 (dt, J = 12.6, 3.2 Hz, 1 H), 3.57 (m, 1 H), 3.81 (m, 1 H),
3.82 (dd, J = 10.7, 6.7 Hz, 1 H), 3.93 (dd, J = 10.7, 5.6 Hz, 1 H)
ppm. 13C NMR and DEPT (125 MHz, CDCl3): δ = –4.60 (CH3),
–4.58 (CH3), 11.26 (CH3), 11.80 (CH3), 17.05 (CH3), 18.25 (C),
18.63 (CH3), 20.95 (CH2), 23.17 (CH2), 23.63 (CH2), 25.94 (3 CH3),
27.21 (3 CH3), 28.19 (CH2), 31.85 (CH2), 32.71 (CH), 33.85 (CH2),
35.57 (C), 35.66 (CH), 36.10 (CH2), 36.29 (CH2), 36.90 (CH2),
37.20 (CH), 38.17 (CH2), 38.84 (C), 39.50 (CH2), 39.53 (CH), 42.65
(C), 45.91 (CH), 50.57 (CH), 55.99 (CH), 68.11 (CH), 69.11 (CH2),
71.96 (CH), 178.64 (C=O) ppm. GC-MS (70 eV): m/z (%) = 603
(2) [(M – Me)+], 561 (13), 485 (18), 451 (12), 383 (13), 378 (15),
Crystallographic Data for Compound 16: C27H46O2, M = 402.64
gmol–1, crystal size: 0.27ϫ0.12ϫ0.10 mm3, orthorhombic, space
3710
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 3703–3714

Synthesis and Hormonal Activity of (25S)-Cholesten-26-oic Acids
group P212121, a = 34.899(7), b = 9.3865(19), c = 7.5000(15) Å, V
= 2456.8(9) Å3, Z = 4, ρcalcd. = 1.089 gcm–3, µ = 0.066 mm–1, λ =
0.71073 Å, T = 223(2) K, θ range = 3.19–25.40°, reflections col-
lected: 35264, independent: 2611 (Rint = 0.0748), data/restraints/
parameters: 1947/0/266. The structure was solved by direct meth-
ods and refined by full-matrix least-squares on F2; final R indices
[IϾ2σ(I)]: R1 = 0.0468; wR2 = 0.1065; maximal residual electron
density: 0.242 eÅ–3. 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.
ESI-MS: m/z = 509.4 [(M + Na)+]. C32H54O3 (486.77): C 78.96, H
11.18, found: C 79.02, H 10.95%.
(25S)-26-(Pivaloyloxy)cholest-4-en-3-one (18): Aluminium isopro-
poxide (152 mg, 746 µmol) was added to a solution of the alcohol
17 (242 mg, 497 µmol) in acetone (1 mL) and toluene (9 mL). The
resulting mixture was stirred at 100 °C for 5 h. After cooling to
room temperature, water (50 mL) and diethyl ether (50 mL) were
added, and the layers were separated. The aqueous layer was ex-
tracted with diethyl ether (2ϫ50 mL) and the combined organic
layers were dried with magnesium sulfate. Evaporation of the sol-
vent and purification of the residue by flash chromatography on
silica gel (petroleum ether/diethyl ether, 4:1) provided the enone 18,
(25S)-∆7-Dafachronic Acid [(25S)-3-Keto-5α-cholest-7-en-26-oic
Acid] (1): A freshly prepared solution of Jones reagent (CrO3:
156 mg, 1.565 mmol; concd. H2SO4: 137 µL, 2.46 mmol) in water
(1 mL) was added to a solution of the diol 16 (126 mg, 313 µmol)
in acetone (12 mL) at 0 °C. The resulting mixture was stirred at
0 °C for 90 min, then 2-propanol (5 mL) and water (50 mL) were
added. The resulting dark green mixture was extracted with ethyl
acetate (3ϫ50 mL). The combined organic layers were dried with
magnesium sulfate and the solvent was evaporated. The residue was
purified by flash chromatography on silica gel (petroleum ether/
ethyl acetate, 3:1 + 1% acetic acid) to provide (25S)-∆7-dafachronic
acid (1), yield 116 mg (89%). Colourless solid; m.p. 139–143 °C
yield 207 mg (86%). Pale yellow solid; m.p. 63–64 °C. IR (ATR): ν
˜
= 2933, 2868, 2851, 1727, 1671, 1616, 1478, 1466, 1433, 1397, 1377,
1332, 1282, 1227, 1157, 1031, 977, 959, 933, 860, 770, 684 cm–1. 1H
NMR (500 MHz, CDCl3): δ = 0.69 (s, 3 H), 0.85–0.92 (m, 1 H),
0.89 (d, J = 6.6 Hz, 3 H), 0.91 (d, J = 6.7 Hz, 3 H), 0.93–1.62 (m,
17 H), 1.16 (s, 3 H), 1.19 (s, 9 H), 1.68 (dt, J = 4.8, 14.0 Hz, 1 H),
1.74–1.84 (m, 3 H), 1.98–2.03 (m, 2 H), 2.25 (ddd, J = 14.5, 4.0,
2.3 Hz, 1 H), 2.32 (dt, J = 16.7, 3.8 Hz, 1 H), 2.36–2.44 (m, 2 H),
3.82 (dd, J = 10.7, 6.8 Hz, 1 H), 3.93 (dd, J = 10.7, 5.6 Hz, 1 H),
5.71 (s, 1 H) ppm. 13C NMR and DEPT (125 MHz, CDCl3): δ =
11.92 (CH3), 17.06 (CH3), 17.35 (CH3), 18.61 (CH3), 20.98 (CH2),
23.21 (CH2), 24.14 (CH2), 27.21 (3 CH3), 28.15 (CH2), 32.00 (CH2),
32.69 (CH), 32.92 (CH2), 33.83 (CH2), 33.96 (CH2), 35.57 (CH),
35.64 (CH, CH2), 36.07 (CH2), 38.57 (C), 38.83 (C), 39.57 (CH2),
42.36 (C), 53.76 (CH), 55.82 (CH), 55.96 (CH), 69.08 (CH2), 123.72
(CH), 171.72 (C), 178.64 (C=O), 199.69 (C=O) ppm. GC-MS
(70 eV): m/z (%) = 484 (88) [M+], 469 (10), 442 (17), 382 (10), 361
(23), 271 (25), 245 (12), 244 (13), 229 (45), 124 (100). C32H52O3
(484.75): C 79.29, H 10.81, found: C 79.23, H 10.79%.
(ref.[5] 143 °C). [α]2D0 = +33.9 (c = 0.49, CHCl ). IR (ATR): ν =
˜
3
2939, 2872, 2804, 1724, 1705, 1439, 1420, 1383, 1254, 1233, 1208,
1184, 1159, 1143, 1122, 1013, 974, 938, 844, 832, 793, 749, 729
1
cm–1. H NMR (500 MHz, CDCl3): δ = 0.54 (s, 3 H), 0.91 (d, J =
6.5 Hz, 3 H), 1.00 (s, 3 H), 1.02–1.06 (m, 1 H), 1.17 (d, J = 7.0 Hz,
3 H), 1.19–1.89 (m, 20 H), 2.03 (dt, J = 12.6, 3.4 Hz, 1 H), 2.12
(ddd, J = 13.4, 6.0, 2.5 Hz, 1 H), 2.20–2.28 (m, 3 H), 2.40 (dd, J =
14.6, 6.0 Hz, 1 H), 2.45 (m, 1 H), 5.17 (d, J = 2.3 Hz, 1 H), 11.12
(br. s, 1 H) ppm. 13C NMR and DEPT (125 MHz, CDCl3): δ =
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 (CH), 117.00 (CH), 139.48 (C), 182.55
(C=O), 212.17 (C=O) ppm. ESI-MS: m/z = 415.3 [(M + H)+].
C27H42O3 (414.62): C 78.21, H 10.21, found: C 78.21, H 10.31%.
(25S)-26-Hydroxycholest-4-en-3-one (19): Sodium methoxide
(43 mg, 792 µmol) was added to a solution of compound 18
(192 mg, 396 µmol) in methanol (3 mL). The mixture was stirred at
room temperature for 5 d, then 2  HCl (20 mL) and ethyl acetate
(50 mL) were added. The layers were separated and the aqueous
layer was extracted with ethyl acetate (2ϫ50 mL). The combined
organic layers were dried with magnesium sulfate and the solvent
was evaporated. Purification of the residue by flash chromatog-
raphy on silica gel (petroleum ether/ethyl acetate, 5:1 to 2:1) af-
forded recovered pivalate 18, yield 29 mg (15%) and the more polar
alcohol 19, yield 89 mg (56%). Colourless solid; m.p. 126–127 °C.
(25S)-26-(Pivaloyloxy)cholest-5-en-3β-ol (17): A 1.0  solution of
tetrabutylammonium fluoride in THF (1.25 mL, 1.25 mmol) was
added to a solution of the silyl ether 11 (500 mg, 0.832 mmol) in
THF (20 mL). The mixture was heated under reflux for 17 h. After
cooling to room temperature, water (50 mL) was added and the
mixture was extracted with diethyl ether (3ϫ50 mL). The com-
bined organic layers were dried with magnesium sulfate and the
solvent was evaporated. Purification of the residue by flash
chromatography on silica gel (petroleum ether/ethyl acetate, 6:1)
provided the alcohol 17, yield 373 mg (92%). Colourless solid; m.p.
IR (ATR): ν = 3414, 2932, 2866, 2850, 1659, 1612, 1462, 1446,
˜
1376, 1332, 1273, 1231, 1190, 1043, 956, 933, 864, 686 cm–1. 1H
NMR (500 MHz, CDCl3): δ = 0.69 (s, 3 H), 0.86–0.96 (m, 1 H),
0.896 (d, J = 6.5 Hz, 3 H), 0.903 (d, J = 6.7 Hz, 3 H), 0.97–1.14
(m, 8 H), 1.16 (s, 3 H), 1.22–1.29 (m, 1 H), 1.32–1.63 (m, 9 H),
1.67 (dt, J = 4.8, 14.0 Hz, 1 H), 1.78–1.86 (m, 2 H), 1.98–2.06 (m,
2 H), 2.25 (ddd, J = 14.6, 4.1, 2.4 Hz, 1 H), 2.32 (dt, J = 17.0,
3.8 Hz, 1 H), 2.34–2.44 (m, 2 H), 3.40 (dd, J = 10.5, 6.5 Hz, 1 H),
3.49 (dd, J = 10.5, 5.7 Hz, 1 H), 5.71 (s, 1 H) ppm. 13C NMR and
DEPT (125 MHz, CDCl3): δ = 11.92 (CH3), 16.70 (CH3), 17.34
(CH3), 18.63 (CH3), 20.98 (CH2), 23.41 (CH2), 24.13 (CH2), 28.16
(CH2), 32.00 (CH2), 32.92 (CH2), 33.61 (CH2), 33.95 (CH2), 35.56
(CH), 35.64 (CH2), 35.71 (CH), 35.79 (CH), 36.15 (CH2), 38.57
(C), 39.58 (CH2), 42.35 (C), 53.76 (CH), 55.82 (CH), 56.01 (CH),
68.29 (CH2), 123.70 (CH), 171.78 (C), 199.74 (C=O) ppm. MS
(70 eV): m/z (%) = 400 (100) [M+], 385 (12), 366 (12), 358 (31), 277
(25), 276 (11), 271 (13), 229 (59), 124 (91). HRMS: m/z calcd. for
C27H44O2 [M+]: 400.3341, found: 400.3329.
101–103 °C. IR (ATR): ν = 3416, 2962, 2934, 2903, 2865, 1729,
˜
1479, 1461, 1397, 1376, 1365, 1283, 1160, 1057, 1023, 984, 957,
926, 841, 799, 770, 742 cm–1. 1H NMR (500 MHz, CDCl3): δ =
0.66 (s, 3 H), 0.90 (d, J = 7.6 Hz, 3 H), 0.91 (d, J = 6.9 Hz, 3 H),
0.93–1.30 (m, 10 H), 0.99 (s, 3 H), 1.19 (s, 9 H), 1.31–1.59 (m, 10
H), 1.75–1.85 (m, 4 H), 1.94–2.00 (m, 2 H), 2.20–2.30 (m, 2 H),
3.51 (m, 1 H), 3.82 (dd, J = 10.7, 6.7 Hz, 1 H), 3.93 (dd, J = 10.7,
5.6 Hz, 1 H), 5.34 (m, 1 H) ppm. 13C NMR and DEPT (125 MHz,
CDCl3): δ = 11.83 (CH3) 17.07 (CH3), 18.69 (CH3), 19.38 (CH3),
21.05 (CH2), 23.21 (CH2), 24.26 (CH2), 27.21 (3 CH3), 28.22 (CH2),
31.63 (CH2), 31.87 (CH, CH2), 32.69 (CH), 33.85 (CH2), 35.67
(CH), 36.14 (CH2), 36.48 (C), 37.22 (CH2), 38.84 (C), 39.73 (CH2),
42.27 (CH2), 42.29 (C), 50.08 (CH), 56.01 (CH), 56.72 (CH), 69.11 (25S)-∆4-Dafachronic Acid [(25S)-3-Ketocholest-4-en-26-oic Acid]
(CH2), 71.77 (CH), 121.67 (CH), 140.75 (C), 178.66 (C=O) ppm. (2): A freshly prepared solution of Jones reagent (CrO3: 111 mg,
Eur. J. Org. Chem. 2009, 3703–3714
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3711

H.-J. Knölker et al.
FULL PAPER
1.11 mmol; concd. H2SO4: 97 µL, 1.743 mmol) in water (0.5 mL)
was added to a solution of the alcohol 19 (89 mg, 222 µmol) in
acetone (12 mL) at 0 °C. The resulting mixture was stirred at 0 °C
for 90 min, then 2-propanol (5 mL) and water (50 mL) were added.
The resulting dark green mixture was extracted with ethyl acetate
(3ϫ50 mL). The combined organic layers were dried with magne-
sium sulfate and the solvent was evaporated. The residue was puri-
fied by flash chromatography on silica gel (petroleum ether/ethyl
acetate, 2:1) to provide (25S)-∆4-dafachronic acid (2), yield 60 mg
(65%). Colourless solid; m.p. 173–174 °C (ref.[6] 172–175 °C). [α]2D0
ture of Pd/C (10%, 33.5 mg, 31.5 µmol Pd) in methanol (5 mL).
The reaction mixture was stirred under an hydrogen atmosphere at
room temperature for 24 h and then filtered with ethyl acetate over
a short pad of Celite®. The solvent was evaporated to provide the
pure saturated diol 21, yield 126 mg (99%). Colourless solid; m.p.
169–171 °C. IR (ATR): ν = 3240, 2969, 2932, 2848, 1463, 1449,
˜
1385, 1369, 1358, 1332, 1320, 1256, 1236, 1171, 1127, 1080, 1049,
1030, 1005, 987, 956, 917, 797, 736, 719 cm–1. 1H NMR (500 MHz,
CDCl3): δ = 0.58–0.61 (m, 1 H), 0.63 (s, 3 H), 0.79 (s, 3 H), 0.80–
0.93 (m, 1 H), 0.89 (d, J = 6.6 Hz, 3 H), 0.90 (d, J = 6.7 Hz, 3 H),
= +61.9 (c = 0.47, CHCl ). IR (ATR): ν = 2929, 2848, 1725, 1649, 0.94–1.15 (m, 9 H), 1.17–1.49 (m, 12 H), 1.51–1.66 (m, 4 H), 1.69
˜
3
1610, 1467, 1450, 1413, 1375, 1362, 1333, 1306, 1282, 1237, 1199,
1168, 1117, 1030, 947, 932, 901, 871, 843, 781 cm–1. 1H NMR
(500 MHz, CDCl3): δ = 0.69 (s, 3 H), 0.82–0.93 (m, 1 H), 0.89 (d,
J = 6.5 Hz, 3 H), 0.96–1.29 (m, 8 H), 1.16 (s, 3 H), 1.17 (d, J =
6.9 Hz, 3 H), 1.31–1.69 (m, 9 H), 1.68 (dt, J = 4.7, 13.9 Hz, 1 H),
1.78–1.86 (m, 2 H), 1.98–2.03 (m, 2 H), 2.25 (ddd, J = 14.6, 4.0,
2.4 Hz, 1 H), 2.32 (dt, J = 17.3, 4.0 Hz, 1 H), 2.36–2.44 (m, 2 H),
2.45 (sext, J = 6.9 Hz, 1 H), 5.71 (s, 1 H) ppm. 13C NMR and
DEPT (125 MHz, CDCl3): δ = 11.93 (CH3), 17.01 (CH3), 17.35
(dt, J = 13.2, 3.6 Hz, 1 H), 1.75–1.81 (m, 2 H), 1.94 (dt, J = 12.6,
3.4 Hz, 1 H), 3.40 (dd, J = 10.5, 6.5 Hz, 1 H), 3.50 (dd, J = 10.5,
5.7 Hz, 1 H), 3.57 (m, 1 H) ppm. 13C NMR and DEPT (125 MHz,
CDCl3): δ = 12.06 (CH3), 12.31 (CH3), 16.71 (CH3), 18.67 (CH3),
21.23 (CH2), 23.43 (CH2), 24.19 (CH2), 28.25 (CH2), 28.71 (CH2),
31.50 (CH2), 32.07 (CH2), 33.64 (CH2), 35.44 (C), 35.48 (CH),
35.77 (CH), 35.81 (CH), 36.22 (CH2), 36.98 (CH2), 38.19 (CH2),
40.01 (CH2), 42.57 (C), 44.83 (CH), 54.32 (CH), 56.21 (CH), 56.46
(CH), 68.35 (CH2), 71.37 (CH) ppm. MS (70 eV): m/z (%) = 404
(CH3), 18.54 (CH3), 20.99 (CH2), 23.69 (CH2), 24.14 (CH2), 28.15 (100) [M+], 389 (22), 388 (14), 386 (16), 371 (25), 278 (11), 248 (22),
(CH2), 32.00 (CH2), 32.93 (CH2), 33.94 (CH2), 34.00 (CH2), 35.58 234 (40), 233 (90), 217 (52), 215 (78). HRMS: m/z calcd. for
(2 CH), 35.64 (CH2), 35.68 (CH2), 38.58 (C), 39.34 (CH), 39.58
C27H48O2 [M+]: 404.3654, found: 404.3652. C27H48O2 (404.67): C
(CH2), 42.37 (C), 53.75 (CH), 55.82 (CH), 55.98 (CH), 123.71 80.14, H 11.96, found: C 79.53, H 11.66%.
(CH), 171.86 (C), 182.35 (C=O), 199.84 (C=O) ppm. ESI-MS m/z
= 415.3 [(M + H)+]. C27H42O3 (414.62): C 78.21, H 10.21, found:
C 78.21, H 10.12%.
(25S)-Dafachronic Acid [(25S)-3-Keto-5α-cholestan-26-oic Acid]
(3): A freshly prepared solution of Jones reagent (CrO3: 124 mg,
1.24 mmol; concd. H2SO4: 111 µL, 1.99 mmol) in water (0.7 mL)
was added to a solution of the diol 21 (100 mg, 247 µmol) in ace-
tone (12 mL) at 0 °C. The resulting mixture was stirred at 0 °C
for 60 min, then 2-propanol (5 mL) and water (50 mL) were
added. The resulting dark green mixture was extracted with ethyl
acetate (3ϫ50 mL). The combined organic layers were dried with
magnesium sulfate and the solvent was evaporated. The residue
was purified by flash chromatography on silica gel (petroleum
ether/ethyl acetate, 3:1 + 1% acetic acid) to provide (25S)-dafach-
ronic acid (3), yield 91 mg (88%). Light yellow solid; m.p. 123–
(25S)-26-Hydroxycholesterol [(25S)-Cholest-5-en-3β,26-diol] (20): A
1.0  solution of tetrabutylammonium fluoride in THF (249 µL,
249 µmol) was added to a solution of compound 11 (100 mg,
166 µmol) in THF (10 mL). The mixture was heated under reflux
for 17 h. After cooling to room temperature, water (50 mL) was
added and the resulting mixture was extracted with diethyl ether
(3ϫ50 mL). The combined organic layers were dried with magne-
sium sulfate and the solvent was evaporated. The crude product
was dissolved in THF (15 mL) and lithium aluminium hydride
(25 mg, 664 µmol) was added. The mixture was stirred at room
temperature for 16 h, then water (10 mL) and 10% HCl were added
(10 mL). The mixture was extracted with ethyl acetate (3ϫ50 mL)
and the combined organic layers were dried with magnesium sul-
fate. Evaporation of the solvent and purification of the residue by
flash chromatography on silica gel (petroleum ether/ethyl acetate,
3:1) provided the diol 20, yield 62 mg (93%). Colourless solid; m.p.
126 °C. [α]D20 = +44.9 (c = 1.0, CHCl ). IR (ATR): ν = 2931, 2863,
˜
3
1702, 1443, 1416, 1379, 1292, 1232, 1173, 1030, 955, 804, 732
cm–1 1H NMR (500 MHz, CDCl3): δ = 0.66 (s, 3 H), 0.68–0.73
.
(m, 1 H), 0.84–0.92 (m, 1 H), 0.88 (d, J = 6.5 Hz, 3 H), 0.97–1.41
(m, 16 H), 0.99 (s, 3 H), 1.17 (d, J = 7.0 Hz, 3 H), 1.47–1.58 (m,
3 H), 1.63–1.70 (m, 2 H), 1.76–1.83 (m, 1 H), 1.95–2.02 (m, 2 H),
2.07 (ddd, J = 14.7, 3.8, 2.2 Hz, 1 H), 2.24 (d, J = 14.7 Hz, 1 H),
2.26–2.30 (m, 1 H), 2.35 (dd, J = 13.9, 6.5 Hz, 1 H), 2.45 (sext, J
= 7.0 Hz, 1 H) ppm. 13C NMR and DEPT (125 MHz, CDCl3): δ
= 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) ppm. MS (70 eV): m/z (%) = 416
(33) [M+], 402 (19), 398 (14), 387 (13), 370 (18), 246 (23), 233 (13),
232 (42), 231 (100), 218 (19), 217 (53). HRMS: m/z calcd. for
C27H44O3 [M+]: 416.3290, found: 416.3291.
173–175 °C (ref.[22] 171–174 °C). IR (ATR): ν = 3318, 2931, 2864,
˜
1464, 1376, 1231, 1193, 1132, 1107, 1053, 1039, 1022, 987, 954,
1
926, 839, 799, 736 cm–1. H NMR (500 MHz, CDCl3): δ = 0.66 (s,
3 H), 0.88–1.17 (m, 9 H), 0.90 (d, J = 6.8 Hz, 6 H), 0.99 (s, 3 H),
1.20–1.27 (m, 1 H), 1.33–1.63 (m, 11 H), 1.77–1.85 (m, 3 H), 1.93–
2.01 (m, 2 H), 2.23 (m, 1 H), 2.28 (ddd, J = 13.0, 5.0, 1.9 Hz, 1
H), 3.40 (dd, J = 10.5, 6.5 Hz, 1 H), 3.48–3.54 (m, 1 H), 3.50 (dd,
J = 10.5, 5.7 Hz, 1 H), 5.33 (m, 1 H) ppm. 13C NMR and DEPT
(125 MHz, CDCl3): δ = 11.84 (CH3), 16.70 (CH3), 18.71 (CH3),
19.38 (CH3), 21.04 (CH2), 23.42 (CH2), 24.26 (CH2), 28.22 (CH2),
31.61 (CH2), 31.86 (CH), 31.87 (CH2), 33.63 (CH2), 35.75 (CH),
35.80 (CH), 36.22 (CH2), 36.47 (C), 37.21 (CH2), 39.73 (CH2),
42.26 (CH2), 42.28 (C), 50.07 (CH), 56.06 (CH), 56.72 (CH), 68.32
(CH2), 71.76 (CH), 121.68 (CH), 140.73 (C) ppm. MS (70 eV): m/z
(%) = 402 (100) [M+], 387 (38), 384 (70), 369 (43), 317 (47), 291
(76), 273 (24), 255 (32), 231 (20), 213 (42). HRMS: m/z calcd. for
C27H46O2 [M+]: 402.3498, found: 402.3494. C27H46O2 (402.65): C
80.54, H 11.51, found: C 80.71, H 11.59%.
(25S)-3β-(tert-Butyldimethylsilyloxy)cholest-5-en-26-ol (22): Lith-
ium aluminium hydride (63 mg, 1.664 mmol) was slowly added to
a solution of the pivalate 11 (250 mg, 0.416 mmol) in THF (10 mL)
at 0 °C. The reaction mixture was stirred at room temperature for
17 h, then water (10 mL) and 10% sulfuric acid were added. The
mixture was extracted with diethyl ether (3ϫ100 mL) and the com-
bined organic layers were dried with magnesium sulfate. Removal
of the solvent and purification of the residue by flash chromatog-
(25S)-5α-Cholestan-3β,26-diol (21): A solution of the diol 20
(127 mg, 315 µmol) in dichloromethane (5 mL) was added to a mix-
3712
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Synthesis and Hormonal Activity of (25S)-Cholesten-26-oic Acids
raphy on silica gel (petroleum ether/diethyl ether, 5:1) provided the
alcohol 22, yield 195 mg (91%). Colourless solid; m.p. 165–166 °C.
Methyl (25S)-3β-Hydroxycholesten-5-en-26-oate (24): A catalytic
amount of concentrated sulfuric acid was added to a solution of
the acid 23 (227 mg, 0.428 mmol) in methanol (10 mL) and the
mixture was heated under reflux for 16 h. After cooling to room
temperature, a saturated aqueous solution of sodium hydrogen
IR (ATR): ν = 3302, 2929, 2857, 1462, 1381, 1367, 1250, 1196,
˜
1093, 1024, 1006, 989, 958, 886, 869, 835, 803, 775, 732, 669 cm–1.
1H NMR (500 MHz, CDCl3): δ = 0.04 (s, 6 H), 0.66 (s, 3 H), 0.87–
0.94 (m, 1 H), 0.88 (s, 9 H), 0.90 (d, J = 6.6 Hz, 3 H), 0.91 (d, J = carbonate (25 mL) and ethyl acetate (50 mL) were added. The lay-
6.7 Hz, 3 H), 0.95–1.17 (m, 8 H), 0.98 (s, 3 H), 1.24 (m, 1 H), 1.33–
1.62 (m, 11 H), 1.68–1.72 (m, 1 H), 1.77–1.85 (m, 1 H), 1.79 (dt, J
ers were separated and the aqueous layer was extracted with ethyl
acetate (2ϫ50 mL). The combined organic layers were dried with
= 13.4, 3.6 Hz, 1 H), 1.92–2.01 (m, 2 H), 2.15 (ddd, J = 13.3, 4.9, magnesium sulfate and the solvent was evaporated. The residue
2.2 Hz, 1 H), 2.26 (m, 1 H), 3.41 (dd, J = 10.5, 6.6 Hz, 1 H), 3.47 was purified by flash chromatography on silica gel (petroleum
(m, 1 H), 3.50 (dd, J = 10.5, 5.7 Hz, 1 H), 5.30 (m, 1 H) ppm. 13C ether/ethyl acetate, 6:1) to provide the methyl ester 24, yield
NMR and DEPT (125 MHz, CDCl3): δ = –4.60 (2 CH3), 11.84 161 mg (87%). Colourless crystals; m.p. 118–119 °C. IR (ATR): ν
˜
(CH3), 16.72 (CH3), 18.27 (C), 18.72 (CH3), 19.42 (CH3), 21.04
(CH2), 23.41 (CH2), 24.27 (CH2), 25.93 (3 CH3), 28.24 (CH2), 31.88 1222, 1192, 1164, 1140, 1107, 1054, 1041, 1025, 1011, 987, 959,
(CH), 31.92 (CH2), 32.06 (CH2), 33.65 (CH2), 35.76 (CH), 35.82 841, 804, 765, 741 cm–1 1H NMR (500 MHz, CDCl3): δ = 0.66
= 3443, 2931, 2901, 2886, 2866, 1732, 1460, 1378, 1363, 1312,
.
(CH), 36.24 (CH2), 36.56 (C), 37.36 (CH2), 39.78 (CH2), 42.30 (C),
42.79 (CH2), 50.17 (CH), 56.06 (CH), 56.77 (CH), 68.35 (CH2),
(s, 3 H), 0.87–1.16 (m, 8 H), 0.89 (d, J = 6.5 Hz, 3 H), 0.99 (s, 3
H), 1.13 (d, J = 7.0 Hz, 3 H), 1.19–1.66 (m, 12 H), 1.76–1.85 (m,
72.63 (CH), 121.15 (CH), 141.55 (C) ppm. GC-MS (70 eV): m/z 3 H), 1.93–1.99 (m, 1 H), 1.99 (dt, J = 12.6, 3.5 Hz, 1 H), 2.22–
(%) = 459 (100) [(M – tBu)+], 389 (17), 377 (54), 343 (10), 331 (19), 2.24 (m, 1 H), 2.28 (ddd, J = 13.0, 5.1, 2.0 Hz, 1 H), 2.42 (sext, J
281 (21), 273 (12), 269 (14). C33H60O2Si (516.91): C 76.68, H 11.70,
found: C 76.82, H 11.88%.
= 7.0 Hz, 1 H), 3.51 (m, 1 H), 3.66 (s, 3 H), 5.34 (m, 1 H) ppm.
13C NMR and DEPT (125 MHz, CDCl3): δ = 11.84 (CH3), 17.23
(CH3), 18.60 (CH3), 19.38 (CH3), 21.05 (CH2), 23.76 (CH2), 24.26
(CH2), 28.20 (CH2), 31.62 (CH2), 31.86 (CH), 31.87 (CH2), 34.31
(CH2), 35.61 (CH), 35.75 (CH2), 36.47 (C), 37.22 (CH2), 39.51
(CH), 39.73 (CH2), 42.27 (CH2), 42.29 (C), 50.07 (CH), 51.45
(CH3), 56.02 (CH), 56.72 (CH), 71.77 (CH), 121.68 (CH), 140.74
(C), 177.45 (C=O) ppm. C28H46O3 (430.66): C 78.09, H 10.77,
found: C 78.06, H 10.88%.
(25S)-3β-(tert-Butyldimethylsilyloxy)cholest-5-en-26-oic Acid (23):
Oxalyl chloride (84 µL, 0.994 mmol) was added to a solution of
DMSO (141 µL, 1.988 mmol) in dichloromethane (5 mL) at
–78 °C. After 5 min, a solution of the alcohol 22 (257 mg,
0.497 mmol) in dichloromethane (10 mL) was added and the reac-
tion mixture was stirred at –78 °C for 20 min. Then, triethylamine
(346 µL, 2.485 mmol) was added dropwise, the solution was
warmed to room temperature and stirring was continued for
10 min. A saturated aqueous solution of ammonium chloride
(50 mL) was added and the layers were separated. The aqueous
layer was extracted with dichloromethane (2ϫ50 mL). The com-
bined organic layers were dried with magnesium sulfate and the
solvent was evaporated to afford the aldehyde. 2-Methyl-2-butene
(527 µL, 4.97 mmol) and KH2PO4 (200 mg) were added to the solu-
tion of the aldehyde in THF (6 mL) and water (1 mL). After ad-
dition of a solution of sodium chlorite (90 mg, 0.994 mmol) in
water (1 mL), the mixture was stirred at room temperature for 24 h.
Then, 10% HCl (25 mL) and diethyl ether (50 mL) were added,
and the layers were separated. The aqueous layer was extracted
with diethyl ether (2ϫ50 mL) and the combined organic layers
were dried with magnesium sulfate. Removal of the solvent and
purification of the residue by flash chromatography on silica gel
(petroleum ether/diethyl ether, 3:1) provided the acid 23, yield
(25S)-Cholestenoic Acid [(25S)-3β-Hydroxycholest-5-en-26-oic Acid]
(4): Lithium hydroxide (11.5 mg, 480 µmol) was added to a solution
of the methyl ester 24 (69 mg, 160 µmol) in THF/methanol/water
(1:1:1, 6 mL) and the mixture was stirred at room temperature for
24 h. Methanol and THF were removed in vacuo and the residue
was thoroughly extracted with dichloromethane (3ϫ30 mL) to re-
move traces of unreacted starting material. The combined dichloro-
methane layers were discarded. Then, 10% hydrochloric acid was
added to the aqueous residue (pH Ͻ 4) and the mixture was ex-
tracted with ethyl acetate (3ϫ40 mL). The combined organic layers
were dried with magnesium sulfate and the solvent was evaporated
to provide pure (25S)-cholestenoic acid (4), yield 67 mg (99%). An
analytically pure sample was obtained by recrystallization from
acetonitrile. Colourless solid; m.p. 172–175 °C (MeCN) (ref.[6] 157–
160 °C). [α]2D0 = –22.9 (c = 0.14, CHCl ). IR (ATR): ν = 3262, 2934,
˜
3
2922, 2890, 2862, 1674, 1457, 1434, 1421, 1374, 1277, 1225, 1137,
1113, 1088, 1052, 1020, 987, 953, 927, 890, 841, 819, 797, 737, 658
cm–1. 1H NMR (500 MHz, CDCl3): δ = 0.66 (s, 3 H), 0.87–1.27
(m, 9 H), 0.90 (d, J = 6.5 Hz, 3 H), 0.99 (s, 3 H), 1.17 (d, J =
7.0 Hz, 3 H), 1.31–1.59 (m, 10 H), 1.63–1.69 (m, 1 H), 1.77–1.85
(m, 3 H), 1.93–2.01 (m, 2 H), 2.22–2.30 (m, 2 H), 2.46 (sext, J =
7.0 Hz, 1 H), 3.52 (m, 1 H), 5.34 (m, 1 H) ppm. 13C NMR and
DEPT (125 MHz, CDCl3): δ = 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) ppm. ESI-MS: m/z
= 399.4 [(M + H – H2O)+]. C27H44O3 (416.64): C 77.83, H 10.64,
found: C 77.99, H 10.77%.
235 mg (89%). Colourless solid; m.p. 183–185 °C. IR (ATR): ν =
˜
2931, 2880, 2854, 1704, 1464, 1418, 1380, 1294, 1248, 1226, 1198,
1
1083, 988, 957, 888, 870, 835, 803, 774, 733, 669 cm–1. H NMR
(500 MHz, CDCl3): δ = 0.05 (s, 6 H) 0.65 (s, 3 H), 0.86–0.94 (m, 1
H), 0.88 (s, 9 H), 0.90 (d, J = 6.5 Hz, 3 H), 0.95–1.28 (m, 8 H),
0.98 (s, 3 H), 1.17 (d, J = 7.0 Hz, 3 H), 1.31–1.59 (m, 10 H), 1.63–
1.72 (m, 2 H), 1.76–1.83 (m, 2 H), 1.92–1.98 (m, 1 H), 1.98 (dt, J
= 12.6, 3.2 Hz, 1 H), 2.15 (ddd, J = 13.3, 4.9, 2.1 Hz, 1 H), 2.26
(m, 1 H), 2.45 (sext, J = 7.0 Hz, 1 H), 3.47 (m, 1 H), 5.30 (m, 1 H),
11.08 (br. s, 1 H) ppm. 13C NMR and DEPT (125 MHz, CDCl3): δ
= –4.60 (2 CH3), 11.84 (CH3), 16.99 (CH3), 18.27 (C), 18.62 (CH3),
19.41 (CH3), 21.04 (CH2), 23.71 (CH2), 24.27 (CH2), 25.93 (3 CH3),
28.22 (CH2), 31.88 (CH), 31.91 (CH2), 32.05 (CH2), 34.03 (CH2),
35.63 (CH), 35.76 (CH2), 36.56 (C), 37.36 (CH2), 39.35 (CH), 39.76
(CH2), 42.31 (C), 42.79 (CH2), 50.16 (CH), 56.04 (CH), 56.76
(CH), 72.64 (CH), 121.14 (CH), 141.55 (C), 182.60 (C=O) ppm.
Supporting Information (see also the footnote on the first page of
this article): Copies of the 1H and 13C NMR spectra for compound
ESI-MS: m/z = 553.4 [(M + Na)+]. C33H58O3Si (530.90): C 74.66, 10 and copies of the HSQC spectra for the (25S)-steroidal acids
H 11.01, found: C 74.75, H 10.94%.
1–4.
Eur. J. Org. Chem. 2009, 3703–3714
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3713

H.-J. Knölker et al.
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We thank Dr. Margit Gruner for 2D NMR experiments and Prof.
Adam Antebi for the daf-9(dh6);dhEx24 strain. We are grateful to
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Received: April 21, 2009
Published Online: June 10, 2009
3714
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 3703–3714