Stereoselective synthesis and hormonal activity of novel dafachronic acids and naturally occurring steroids isolated from corals

Article abstract of DOI:10.1039/c2ob25394a

A stereoselective synthesis of (25S)-Δ¹-, (25S)-Δ^1,4-, (25S)-Δ^1,7-, (25S)- Δ⁸⁽¹⁴⁾-, (25S)-Δ^4,6,8(14)-dafachronic acid, methyl (25S)-Δ^1,4-dafachronate and (25S)-5α-hydroxy-3,6- dioxocholest-7-en-26-oic acid is described. (25S)-Δ^1,4- Dafachronic acid and its methyl ester are natural products isolated from corals and have been obtained by synthesis for the first time. (25S)-5α-Hydroxy- 3,6-dioxocholest-7-en-26-oic acid represents a promising synthetic precursor for cytotoxic marine steroids.

Full text of DOI:10.1039/c2ob25394a

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Volume 10 | Number 21 | 7 June 2012 | Pages 4137–4300
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Hans-Joachim Knölker et al.
Stereoselective synthesis and hormonal activity of novel dafachronic acids
and naturally occurring steroids isolated from corals
1477-0520(2012)10:21;1-G

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Organic &
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COMMUNICATION
Stereoselective synthesis and hormonal activity of novel dafachronic acids and
naturally occurring steroids isolated from corals†
Ratni Saini,a Sebastian Boland,b Olga Kataeva,a Arndt W. Schmidt,a Teymuras V. Kurzchalia*b and
Hans-Joachim Knölker*a
Received 23rd February 2012, Accepted 8th March 2012
DOI: 10.1039/c2ob25394a
A stereoselective synthesis of (25S)-Δ1-, (25S)-Δ1,4-, (25S)-
been prepared from 3β-hydroxychol-5-en-24-oic acid (3) in 8
Δ1,7-, (25S)-Δ8(14)-, (25S)-Δ4,6,8(14)-dafachronic acid, methyl
steps and 66% overall yield using an Evans aldol reaction as key
step (Scheme 1).6 With 4 as relay compound both hormonally
active dafachronic acids became readily accessible. Desilylation
of 4, Oppenauer oxidation with isomerization of the double
(25S)-Δ1,4-dafachronate and (25S)-5α-hydroxy-3,6-dioxochol-
est-7-en-26-oic acid is described. (25S)-Δ1,4-Dafachronic acid
and its methyl ester are natural products isolated from corals
and have been obtained by synthesis for the first time. (25S)-
5α-Hydroxy-3,6-dioxocholest-7-en-26-oic acid represents a
promising synthetic precursor for cytotoxic marine steroids.
The genes daf-9 and daf-12 play a key role in controlling the life
cycle and longevity of the nematode Caenorhabditis elegans.1,2
It was found that daf-9 encodes a cytochrome P450 oxidase
which completes the synthesis of dafachronic acids 1 and 2
(Fig. 1). These steroidal acids are ligands for the hormone recep-
tor DAF-12.3 By binding of dafachronic acids, DAF-12 is inacti-
vated and the nematodes undergo normal reproductive
development. In the absence of these ligands, DAF-12 is acti-
vated and dauer larvae are generated.2
Due to their hormonal activity and the importance for investi-
gations in developmental biology, several synthetic routes have
been reported for the dafachronic acids.4–10 We have developed a
highly efficient synthetic route to (25S)-Δ4-dafachronic acid (1)
and (25S)-Δ7-dafachronic acid (2).6,10 A crucial intermediate for
our approach is the orthogonally diprotected diol 4 which has
Scheme 1 Synthesis of (25S)-Δ4- and (25S)-Δ7-dafachronic acid (1)
and (2) and (25S)-dafachronic acid (5) using the orthogonally dipro-
tected diol 4 as relay compound. 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, rt, 5 d,
87%; (d) 5.0 equiv. Jones reagent, 0 °C, 2 h, 85%; (e) 1.5 equiv. TBAF,
THF, reflux, 17 h; (f) 4.0 equiv. LiAlH4, THF, rt, 16 h, 93% for two
steps; (g) 10% Pd/C, H2, MeOH–CH2Cl2 (1 : 1), rt, 24 h, 99%; (h) 5.0
equiv. Jones reagent, acetone, 0 °C, 60 min, 88%; (i) 4.0 equiv. PDC,
8.0 equiv. tBuOOH, Celite®, benzene, 0 °C to rt, 41 h, 75%; ( j) 10%
Pd/C, H2, EtOAc, rt, 16 h, 95%; (k) 1.3 equiv. L-Selectride®, THF,
−78 °C, 1.5 h, 90%; (l) 5.0 equiv. SOCl2, pyridine, 0 °C, 40 min, 87%;
(m) 1.5 equiv. TBAF, THF, reflux, 16 h; (n) 4.0 equiv. LiAlH4, THF,
0 °C to rt, 17 h, 82% for two steps; (o) 5.0 equiv. Jones reagent,
acetone, 0 °C, 90 min, 89%.
Fig. 1 Steroidal ligands for the hormonal DAF-12 receptor of C.
elegans.
aDepartment Chemie, Technische Universität Dresden, Bergstrasse 66,
01069 Dresden, Germany. E-mail: hans-joachim.knoelker@tu-dresden.
de; Fax: +49 351-463-37030
bMax Planck Institute of Molecular Cell Biology and Genetics,
Pfotenhauerstrasse 108, 01307 Dresden, Germany
†Electronic supplementary information (ESI) available: 1H and 13C
NMR spectra of compounds 9–11, 13, 16, 20 and 23. 2D NMR spectra
(COSY, HSQC, HMBC and NOESY) of compound 20. CCDC 867379
and 867380. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c2ob25394a
This journal is © The Royal Society of Chemistry 2012
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Scheme 3 Synthesis of 13 and (25S)-Δ1,7-dafachronic acid (10).
Reagents and conditions: (a) cat. H2SO4, MeOH, reflux, 7 h, 96%; (b)
1.3 equiv. DDQ, 5 mol% TBSCl, dioxane, rt, 24 h, 65% (20% 12); (c)
3.0 equiv. LiOH, THF–MeOH–H2O (1 : 1 : 1), rt, 24 h, 88%.
Scheme 2 Synthesis of (25S)-Δ1-, (25S)-Δ1,4- and (25S)-Δ1,7-dafachro-
nic acid (9), (10) and (11). Reagents and conditions: (a) 10 mol%
Pd(OCOCF3)2, 10 mol% DMSO, O2, HOAc, 80 °C, 24 h, 93% 9, 75%
10, 86% 11.
Scheme 4 Synthesis of (25S)-Δ8(14)-dafachronic acid (16). Reagents
and conditions: (a) PtO2, H2, EtOAc–HOAc (10 : 1), rt, 2 d, 94%; (b)
1.5 equiv. TBAF, THF, rt, 24 h; (c) 2.0 equiv. LiAlH4, THF, 0 °C to rt,
8 h, 93% for two steps; (d) 5.0 equiv. Jones reagent, acetone, 0 °C, 2 h,
78%.
bond, cleavage of the pivalate and Jones oxidation led to (25S)-
Δ4-dafachronic acid (1) in an improved 39% overall yield (12
steps). Alternatively, sequential removal of both protecting
groups of compound 4 by desilylation with TBAF and sub-
sequent cleavage of the pivalate via reduction using lithium alu-
minium hydride, catalytic hydrogenation of the double bond and
Jones oxidation provided the unnatural (25S)-dafachronic acid
(5) (12 steps, 53% overall yield). For the synthesis of (25S)-Δ7-
dafachronic acid (2), the double bond of the orthogonally dipro-
tected diol 4 was moved from the 5,6- to the 7,8-position via the
following four-step sequence. Allylic oxidation at C-7 to the
enone 6, hydrogenation of the 5,6-double bond, stereoselective
reduction to the 7α-alcohol 7 and finally, elimination of water
afforded compound 8. Removal of both protecting groups of 8
(first of the silyl and then of the pivaloyl group) followed by
Jones oxidation provided (25S)-Δ7-dafachronic acid (2) in 15
steps and 27% overall yield.
Herein, we describe an efficient synthesis of novel dafachronic
acids and of naturally occurring steroidal acids which have been
isolated from corals. A first investigation of their hormonal
activity is also presented. Using the palladium(II)-catalyzed
process reported by Stahl et al. 3-oxosteroids can be regioselec-
tively dehydrogenated at the 1,2-position.11 Thus, (25S)-dafa-
chronic acid (5), (25S)-Δ4-dafachronic acid (1) and (25S)-Δ7-
dafachronic acid (2) have been transformed directly into (25S)-
Δ1-dafachronic acid (9), (25S)-Δ1,4-dafachronic acid (10) and
(25S)-Δ1,7-dafachronic acid (11) in yields ranging from 75 to
93% (Scheme 2).‡11 In this context, it is noteworthy that com-
pound 10 was recently described as a natural product. Namikoshi
et al. isolated (25S)-3-oxocholesta-1,4-dien-26-oic acid (10)
from the Indonesian soft coral Minabea sp.12 Thus, our present
route also constitutes the first synthesis of this natural product.
In an alternative approach to (25S)-Δ1,4-dafachronic acid (10),
(25S)-Δ4-dafachronic acid (1) was initially converted to the cor-
responding methyl ester 12 (Scheme 3). Dehydrogenation with
1.3 equiv. 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)
in the presence of tert-butyldimethylsilyl chloride (TBSCl)
afforded the cross-conjugated dienone 13 in 65% yield along
with 20% of starting material.‡13 The course of this reaction is
much more difficult to control as compared to the procedure
described by Stahl et al. Use of larger amounts of DDQ by over-
oxidation led to methyl (25S)-Δ1,4,6-dafachronate. Interestingly,
methyl (25S)-Δ1,4-dafachronate (13) also represents a natural
product. A few years ago, Zubía et al. isolated methyl 3-oxocho-
lesta-1,4-dien-26-oate from the Antarctic octocoral Anthomastus
bathyproctus.14 However, they left the configuration at C-25 of
their natural product undetermined. A comparison of the value
reported by Zubía et al. for the 13C NMR signal of C-27 (δ =
17.0 ppm)14 with the value we have observed for C-27 of com-
pound 13 (δ = 17.23 ppm) led us to assign an S configuration for
C-25 of natural methyl 3-oxocholesta-1,4-dien-26-oate. Our
assignment is based on the distinct difference found between the
13C NMR signals for C-27 of the (25S) and the (25R) series:
This difference is observed for the carboxylic acids (compare
Table 2 in ref. 10) as well as for the esters.5b,6b
Starting from 8, we synthesised (25S)-Δ8(14)-dafachronic acid
(16) (Scheme 4). Isomerisation of the double bond provided
quantitatively the diprotected Δ8(14)-compound 14.15 Cleavage of
first the silyl and then the pivaloyl protecting group led to the
Δ8(14)-steroiddiol 15. The regio- and the stereochemistry of 15
has been confirmed by an X-ray crystal structure determination
(Fig. 2).§ Finally, Jones oxidation afforded (25S)-Δ8(14)-dafa-
chronic acid (16) in 16 steps and 25% overall yield based on 3.‡
In a further study, we investigated the chemistry of a corre-
sponding B-ring diene 3β,26-steroid diol resembling vitamin
D.16 Starting from enone 6, an additional double bond at pos-
ition 7,8 was introduced via the Shapiro reaction (Scheme 5).17
Conversion of 6 to the tosylhydrazone 17 followed by treatment
with an excess of lithium hydride provided the 5,7-diene 18.
4160 | Org. Biomol. Chem., 2012, 10, 4159–4163
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Fig. 3 Molecular structure of (25S)-cholesta-5,7-diene-3β,26-diol (19)
in the crystal (ORTEP plot at the 50% probability level).
Fig. 2 Molecular structure of (25S)-cholest-8(14)-en-3β,26-diol (15) in
the crystal (ORTEP plot at the 50% probability level).
Fig. 4 NOESY spectrum of compound 20 (600 MHz, CDCl3).
triazoline-3,5-dione (PTAD) afforded the adduct 22.19 Jones
oxidation of 22 proceeded with concomitant elimination of the
heterocycle and provided directly (25S)-Δ4,6,8(14)-dafachronic
acid (23).‡
Scheme 5 Synthesis of (25S)-Δ4,6,8(14)-dafachronic acid (23). Reagents
and conditions: (a) 7.5 equiv. p-toluenesulfonyl hydrazide, THF, reflux,
24 h, 81%; (b) 100 equiv. LiH, toluene, 100 °C, 4 h, 66%; (c) 3.0 equiv.
TBAF, THF, rt, 24 h, 90%; (d) 1.2 equiv. LiAlH4, Et2O–CH2Cl2 (1 : 1),
0 °C to rt, 4 h, 84%; (e) 5.0 equiv. Jones reagent, acetone, 0 °C, 30 min,
50%; (f) 1.5 equiv. LiAlH4, CH2Cl2–Et2O (1 : 1), 0 °C to rt, 3 h, 100%;
(g) 1.3 equiv. 4-phenyl-1,2,4-triazoline-3,5-dione, CH2Cl2, rt, 30 min,
86%; (h) 5.0 equiv. Jones reagent, acetone, 0 °C, 30 min, 75%.
Finally, we have tested the hormonal activity of the novel
(25S)-dafachronic acids in rescuing worms from dauer arrest.
The details of our bioassay protocol have been described pre-
viously.5b,6b Mutant worms daf-9(dh-6) are lacking the DAF-9
activity. Therefore, they cannot produce the ligands for the
DAF-12 receptor and arrest as dauer larvae. Feeding of daf-9(dh-
6) mutant worms with the (25S)-dafachronic acids described
above led to a rescue from dauer arrest and to the development
of adults. The ability to rescue the daf-9(dh-6) mutant worms
from dauer arrest and to induce normal development of adults
varies significantly for the different (25S)-dafachronic acids
(Fig. 5).
Removal of the silyl and then the pivaloyl protecting group
afforded (25S)-cholesta-5,7-diene-3β,26-diol (19). At this stage,
the structural assignment has been additionally confirmed by
X-ray analysis of single crystals of 19 (Fig. 3).¶ Jones oxidation
of compound 19 generated as expected the ketone at C-3 and the
carboxylic acid at C-26. However, an additional dioxygenation
of the 5,6-double bond led to (25S)-5α-hydroxy-3,6-dioxochol-
est-7-en-26-oic acid (20).‡ This structural assignment has been
confirmed by extensive 2D NMR experiments (Fig. 4 and ESI†).
It is noteworthy that Shin et al. have isolated bioactive steroids
from the gorgonian Acalycigorgia inermis with structural fea-
tures very similar to compound 20.18 The synthesis of these
natural products is currently in progress in our laboratories.
Cleavage of the pivalate of the 5,7-diene 18 led to compound
21 which on Diels–Alder cycloaddition with 4-phenyl-1,2,4-
The activity of the (25S)-dafachronic acids is dependent on
the location of the double bond(s). The most active compounds
are (25S)-Δ1,7-dafachronic acid (11) and (25S)-Δ7-dafachronic
acid (2) with a double bond at the 7,8-position as a common
structural feature. They induce normal development of the
worms already at concentrations below 10 nM. (25S)-Δ1-Dafa-
chronic acid (9), (25S)-Δ4-dafachronic acid (1) and (25S)-Δ1,4
-
dafachronic acid (10) belong to the group of second most active
This journal is © The Royal Society of Chemistry 2012
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Spectroscopic data for (25S)-Δ8(14)-dafachronic acid (16): Colourless
crystals, mp 138–140 °C; 13C NMR and DEPT (125 MHz, CDCl3): δ =
11.93 (CH3), 16.99 (CH3), 18.25 (CH3), 18.97 (CH3), 20.05 (CH2),
23.59 (CH2), 25.89 (CH2), 27.01 (CH2), 29.14 (CH2), 29.29 (CH2),
34.04 (CH2), 34.27 (CH), 35.54 (CH2), 36.94 (CH2), 37.11 (C), 38.04
(CH2), 38.24 (CH2), 39.34 (CH), 42.74 (C), 44.66 (CH2), 46.39 (CH),
48.74 (CH), 56.79 (CH), 125.41 (C), 143.45 (C), 182.50 (CvO),
212.35 (CvO); anal. calc. for C27H42O3: C 78.21, H 10.21; found: C
78.30, H 10.28.
Spectroscopic data for (25S)-5α-hydroxy-3,6-dioxocholest-7-en-26-
oic acid (20): Light yellow crystals; 13C NMR and DEPT (150 MHz,
CDCl3): δ = 12.48 (CH3), 15.86 (CH3), 17.04 (CH3), 18.72 (CH3),
22.06 (CH2), 22.51 (CH2), 23.69 (CH2), 27.66 (CH2), 31.97 (CH2),
33.96 (CH2), 35.52 (CH2), 35.81 (CH), 37.35 (CH2), 38.83 (CH2),
39.18 (CH), 40.83 (C), 43.71 (CH), 44.63 (C), 44.83 (CH2), 55.74
(CH), 56.11 (CH), 79.87 (C), 119.53 (CH), 165.89 (C), 181.43 (CvO),
197.03 (CvO), 210.18 (CvO).
Spectroscopic data for (25S)-Δ4,6,8(14)-dafachronic acid (23): Yellow
crystals, mp 147–150 °C; 13C NMR and DEPT (125 MHz, CDCl3): δ =
16.62 (CH3), 17.03 (CH3), 18.74 (CH3), 18.80 (CH3), 18.95 (CH2),
23.58 (CH2), 25.30 (CH2), 27.16 (CH2), 33.98 (CH2), 34.03 (CH2),
34.07 (CH2), 34.40 (CH), 35.43 (CH2), 35.63 (CH2), 36.73 (C), 39.36
(CH), 44.12 (CH), 44.21 (C), 55.54 (CH), 122.90 (CH), 124.38 (CH),
124.45 (C), 134.09 (CH), 156.09 (C), 164.60 (C), 182.42 (CvO),
199.77 (CvO).
§Crystal data for (25S)-cholest-8(14)-en-3β,26-diol (15): C27H46O2,
crystal size: 0.28 × 0.25 × 0.19 mm3, M = 402.64 g mol−1, monoclinic,
space group: P21, λ = 0.71073 Å, a = 11.0257(6), b = 7.5583(4), c =
14.7907(9) Å, β = 95.487(4)°, V = 1226.94(12) Å3, Z = 2, ρc = 1.090 g
cm−3, μ = 0.066 mm−1, T = 198(2) K, θ range = 1.38–29.48°, reflections
collected: 27 556, independent: 6718 (Rint = 0.0672), 274 parameters.
The structure was solved by direct methods and refined by full-matrix
Fig. 5 Hormonal activity of the different dafachronic acids.
compounds which at concentrations of 50 nM effect a
rescue from dauer arrest for about 60–80% of the worms. (25S)-
Δ4,6,8(14)-Dafachronic acid (23) and (25S)-Δ8(14)-dafachronic acid
(16) are the least active compounds in this series. Previously, we
have shown that (25S)-dafachronic acid (5) has about the same
activity as (25S)-Δ4-dafachronic acid (1).6b,10 The present results
emphasise the importance of the double bond at position 7,8 for
the hormonal activity of dafachronic acids.
least-squares on F2; final R indices [I > 2σ(I)]: R1 = 0.0442, wR2
=
We are grateful to the ESF EuroMembrane Network (DFG
grant KN 240/13-1) and the Deutsche Forschungsgemeinschaft
(grant KN 240/16-1) for financial support of our project.
0.0837; maximal residual electron density: 0.133 e Å−3. CCDC 867379.
¶Crystal data for (25S)-cholesta-5,7-diene-3β,26-diol (19): C27H44O2,
crystal size: 0.63 × 0.15 × 0.04 mm3, M = 400.62 g mol−1, monoclinic,
space group: P21, λ = 0.71073 Å, a = 11.658(3), b = 6.105(2), c =
17.568(5) Å, β = 106.10(2)°, V = 1201.3(6) Å3, Z = 2, ρc = 1.108 g
cm−3, μ = 0.067 mm−1, T = 150(2) K, θ range = 1.21–27.00°, reflections
collected: 18 278, independent: 5131 (Rint = 0.1437), 274 parameters.
The structure was solved by direct methods and refined by full-matrix
Notes and references
‡Spectroscopic data for (25S)-Δ1-dafachronic acid (9): Colourless crys-
tals, mp 112–115 °C; 13C NMR and DEPT (125 MHz, CDCl3): δ =
12.17 (CH3), 12.95 (CH3), 17.00 (CH3), 18.56 (CH3), 21.22 (CH2),
23.70 (CH2), 24.08 (CH2), 27.63 (CH2), 28.20 (CH2), 31.28 (CH2),
34.01 (CH2), 35.62 (CH), 35.64 (CH), 35.69 (CH2), 38.96 (C), 39.26
(CH), 39.76 (CH2), 40.98 (CH2), 42.70 (C), 44.28 (CH), 49.91 (CH),
56.10 (CH), 56.33 (CH), 127.32 (CH), 158.68 (CH), 181.95 (CvO),
200.39 (CvO); anal. calc. for C27H42O3: C 78.21, H 10.21; found: C
77.61, H 10.35.
least-squares on F2; final R indices [I > 2σ(I)]: R1 = 0.0534, wR2
=
0.0850; maximal residual electron density: 0.160 e Å−3. CCDC 867380.
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Spectroscopic data for (25S)-Δ1,4-dafachronic acid (10): Light yellow
crystals, mp 129–130 °C; 13C NMR and DEPT (125 MHz, CDCl3): δ =
12.03 (CH3), 17.02 (CH3), 18.49 (CH3), 18.65 (CH3), 22.84 (CH2),
23.69 (CH2), 24.35 (CH2), 28.09 (CH2), 32.92 (CH2), 33.67 (CH2),
33.99 (CH2), 35.49 (CH), 35.56 (CH), 35.64 (CH2), 39.28 (CH), 39.45
(CH2), 42.64 (C), 43.66 (C), 52.34 (CH), 55.41 (CH), 55.94 (CH),
123.71 (CH), 127.37 (CH), 156.20 (CH), 169.69 (C), 181.97 (CvO),
186.56 (CvO); anal. calc. for C27H40O3: C 78.60, H 9.77; found: C
78.70, H 9.85.
Spectroscopic data for (25S)-Δ1,7-dafachronic acid (11): Colourless
crystals, mp 100–102 °C; 13C NMR and DEPT (125 MHz, CDCl3): δ =
11.92 (CH3), 12.60 (CH3), 17.03 (CH3), 18.74 (CH3), 21.55 (CH2),
22.86 (CH2), 23.76 (CH2), 27.87 (CH2), 28.56 (CH2), 34.01 (CH2),
35.64 (CH2), 36.03 (CH), 37.37 (C), 39.27 (CH2 and CH), 39.64 (CH),
40.11 (CH2), 43.54 (C), 45.24 (CH), 55.15 (CH), 55.97 (CH), 117.83
(CH), 127.13 (CH), 138.67 (C), 157.43 (CH), 181.71 (CvO), 199.90
(CvO).
Spectroscopic data for methyl (25S)-Δ1,4-dafachronate (13): Light
yellow crystals, mp 75–80 °C; 13C NMR and DEPT (75 MHz, CDCl3):
δ = 12.03 (CH3), 17.23 (CH3), 18.48 (CH3), 18.67 (CH3), 22.84 (CH2),
23.79 (CH2), 24.35 (CH2), 28.08 (CH2), 32.91 (CH2), 33.67 (CH2),
34.27 (CH2), 35.51 (CH), 35.55 (CH), 35.65 (CH2), 39.47 (CH2), 39.50
(CH), 42.65 (C), 43.62 (C), 51.43 (CH3), 52.36 (CH), 55.43 (CH),
55.98 (CH), 123.75 (CH), 127.42 (CH), 156.00 (CH), 169.44 (C),
177.37 (CvO), 186.44 (CvO); anal. calc. for C28H42O3: C 78.83, H
9.92; found: C 78.94, H 9.77.
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