Synthesis and crystal structure of [26,27-2H6] 24-epi-cathasterone

Article abstract of DOI:10.1039/b203323b

The first synthesis of [26,27-²H₆]24-epi-cathasterone 8 via (20S)-3β-acetoxy-6,6-(ethylenedioxy)-20-formyl-5αpregnane 5 starting from stigmasterol is described. The aldehyde 5 was alkylated with lithium butyldimethyl-(E)-2,3dimethyl[3,3,3,4,4,4-²H₆]butenylalumi nate 6 prepared from 3-[²H₃]methyl[4,4,4-²H₃]but-l-yne. The structure was determined using spectral data and X-ray crystallographic analysis.

Full text of DOI:10.1039/b203323b

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Synthesis and crystal structure of [26,27-2H6] 24-epi-cathasterone
Adelheid Kolbe,a Petra Fuchs,a Andrea Porzel,a Ute Baumeister,b Alfred Kolbe*b and
Günter Adama
1
a Institut für Pflanzenbiochemie, Weinberg 3, D-06120 Halle, Saale, Germany
b Institut für Physikalische Chemie der Martin-Luther-Universität, Mühlpforte 1,
D-06108 Halle, Saale, Germany. E-mail: kolbe@chemie.uni-halle.de
Received (in Cambridge, UK) 4th April 2002, Accepted 5th July 2002
First published as an Advance Article on the web 6th August 2002
The first synthesis of [26,27-2H6]24-epi-cathasterone 8 via (20S)-3β-acetoxy-6,6-(ethylenedioxy)-20-formyl-5α-
pregnane 5 starting from stigmasterol is described. The aldehyde 5 was alkylated with lithium butyldimethyl-(E)-2,3-
dimethyl[3,3,3,4,4,4-2H6]butenylaluminate 6 prepared from 3-[2H3]methyl[4,4,4-2H3]but-1-yne. The structure was
determined using spectral data and X-ray crystallographic analysis.
Table 1 Selected torsion angles τ/Њ for [26,27-2H6]24-epi-cathasterone
Introduction
The brassinosteroids are steroidal phytohormones ubiquitously
occurring in the plant kingdom, exhibiting high growth
promoting and antistress activity.1 Cathasterone ((22S,24R)-
3β,22-dihydroxy-5α-ergostan-6-one) was shown to be a bio-
synthetic precursor of brassinolide in cultured cells of Cathar-
anthus roseus2 by feeding experiments with deuterium-labelled
cathasterone. We synthesised deuterium-labelled 24-epi-catha-
sterone ([26,27-2H6](22S,24S)-3β,22-dihydroxy-5α-ergostan-6-
one) as the corresponding precursor in the analogously natur-
ally occurring 24-epi-brassinosteroid series.3,4 Here we report
the first synthesis of [26,27-2H6]epi-cathasterone 8 via (20S)-3β-
acetoxy-6,6-(ethylenedioxy)-20-formyl-5α-pregnane 5 starting
from stigmasterol. The intermediate aldehyde 5 was alkylated
with lithium butyldimethyl-(E)-2,3-dimethyl[3,3,3,4,4,4-2H6]-
butenylaluminate 6. [26,27-2H6]24-epi-cathasterone was needed
for biosynthetic studies and also for the determining of the
endogenous level of 24-epi-cathasterone in plants.
Atoms
Molecule A
Molecule B
C(13)–C(17)–C(20)–C(21)
C(13)–C(17)–C(20)–C(22)
C(17)–C(20)–C(22)–C(23)
C(17)–C(20)–C(22)–O(3)
C(20)–C(22)–C(23)–C(24)
C(22)–C(23)–C(24)–C(25)
C(22)–C(23)–C(24)–C(28)
C(23)–C(24)–C(25)–C(26)
C(23)–C(24)–C(25)–C(27)
Ϫ54.9(11)
Ϫ179.0(8)
178.5(8)
55.3(9)
169.5(8)
Ϫ50.7(12)
Ϫ177.9(9)
Ϫ163.9(8)
71.4(10)
Ϫ173.8(9)
Ϫ177.1(9)
Ϫ51.4(13)
Ϫ53.6(12)
69.0(13)
151.9(9)
Ϫ81.6(12)
Ϫ69.9(13)
56.6(14)
[26,27-2H6]24-epi-cathasterone was purified by flash chromato-
graphy (SiO2, EtOAc–n-hexane 1 : 1 to 7 : 3 v/v) and preparative
HPLC (octadecylsilane (ODS), MeCN–H2O 95 : 5). 3.5 mg puri-
fied [26,27-2H6]24-epi-cathasterone 8 was obtained, 14.9% from
the [26,27-2H6]3β-acetoxy-6,6-(ethylenedioxy)-22R-hydroxy-24-
methyl-5α-cholest-23-ene 7.
The deuteriation rate of the labelled 24-epi-cathasterone
amounts to 86%. In the NMR-spectra of the deuteriated com-
pounds we therefore found small signals for the positions 26
and 27 due to non-labelled compounds.
Results and discussion
For the synthesis of [26,27-2H6]24-epi-cathasterone 8 stigma-
sterol was used as the starting material. Our synthetic route
is shown in Scheme 1. At first (22E,24S)-3β,5-cyclo-24-
ethylcholest-22-en-6-one 1 was prepared from stigmasterol as
described by Lichtblau.5 Then the cyclopropane ring was
opened with AcOH and H2SO4 to give (22E,24S)-3β-hydroxy-
The deuterium labelled 24-epi-cathasterone is suitable for
biosynthetic studies and for the determining of the endogenous
levels of 24-epi-cathasterone in plants because a sufficient
amount of 6 H-atoms is exchanged.
The structure of the [26,27-2H6]24-epi-cathasterone was
confirmed by X-ray crystallographic analysis (Fig. 1 and 2,
Table 1 and 2) and NMR (Table 3). The spectral data (MS, 1H
NMR and 13C NMR) of the intermediate compounds and of
the [26,27-2H6]24-epi-cathasterone are in agreement with the
given structures.
24-ethyl-5α-cholest-22-en-6-one in
a similar manner as
Aburatani et al.6 had described for the preparation of
(22R,23R,24S)-3β,22,23-triacetoxy-5α-ergostan-6-one. In the
next steps the hydroxy and the keto groups were protected as
acetoxy and ethylenedioxy, respectively.7 Then the double bond
in the side chain was oxidised with OsO4. The obtained 22,23-
diole 4 was cleaved with H5IO6 in presence of a small amount of
pyridine to prevent the extensive loss of the protecting group at
the keto group.8 The desired aldehyde 5 was obtained in 84.2%
yield of the diol. The alkylation of the aldehyde with lithium
butyldimethyl-(E)-2,3-dimethyl[3,3,3,4,4,4-2H6]butenylalumin-
ate 6 gave after purification by flash chromatography on SiO2
and by preparative HPLC 21% of the desired (22R)-allylic
alcohol
7
([26,27-2H6]3β-acetoxy-6,6-(ethylenedioxy)-22R-
This (22R)-allylic
hydroxy-24-methyl-5α-cholest-23-ene).
alcohol was catalytically hydrogenated using 10% Pt on carbon
as a catalyst. In the next two steps the protecting groups were
removed. The dioxolane-type acetal was cleaved at first by trans-
acetalisation9 and then the acetyl-group from the 3β-position
was removed with NaOMe. In the next step the obtained crude
Fig. 1 Structure of the two molecules in the unit cell of [26,27-2H6]24-
epi-cathasterone with atom labelling scheme (H atoms have been
omitted for clarity, O–H ؒ ؒ ؒ O bridges shown as broken lines
connecting the donor and acceptor O atoms).16
2022
J. Chem. Soc., Perkin Trans. 1, 2002, 2022–2027
This journal is © The Royal Society of Chemistry 2002
DOI: 10.1039/b203323b

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Scheme 1 a: AcOH, H2SO4, Py–(Ac)2O; b: 2,2-dimethyl-1,3-dioxolane, pTsOH; c: OsO4; d: H5IO6, NaHCO3; e: 10% Pt/C; f: p-Ts-pyridine.
X-Ray†
[26,27-2H6]24-epi-Cathasterone crystallizes in the triclinic space
group P1 with two steroid and two crystal water molecules per
unit cell, i.e. the two molecules are symmetrically independent
(Fig. 1). Both of them show the expected bond lengths and
angles, which are in good agreement in the range of the
standard uncertainties with one another and also with that
of similar steroid molecules like 3-acetyl-22,24-di-epi-
cathasterone.10 ‡ Besides effects caused by the different consti-
tution (torsion angles including O(2) and absolute configuration
of C(22) of the reference molecule) and by disorder (O(1) in
molecule A, which could not be resolved because of the poor
quality of data), considerable conformational variations are
restricted to the side chain of the steroid molecule (see Table 1).
While these chains (C(20) to C(25)) are essential in the all-trans
form for both molecules A and B of the title compound (all
torsion angles are in the anti-periplanar range of conformation,
cf. Fig. 1), the reference molecule shows different partial con-
formations, readily explainable by packing effects. The confor-
mational differences between the chains of molecules A and B
are also due to their arrangement within the crystal and the
corresponding intermolecular interactions.
Fig.
2
Crystal packing of [26,27-2H6]24-epi-cathasterone in
a
projection along the crystallographic y-axis showing the O–H ؒ ؒ ؒ O
bridging system as broken lines connecting the donor and acceptor O
atoms (symmetry relationships for atoms labelled a, b, w
bЈ x, 1 ϩ y, Ϫ1 ϩ z; w*
x, y, z; aЈ,
1 ϩ x, y, z; b# 1 ϩ x, 1 ϩ y, Ϫ1 ϩ z).16
The head-to-tail packing of a pair of neighbouring molecules
A and B is fixed by two H bonds between the hydroxylic groups
at C(3) of one molecule and C(22) of the other molecule,
respectively (cf. Fig. 1), including two of the four H-donor
functions of the pair of molecules. Such pairs are stacked by
translation along the crystallographic x-axis. To make full use
of their H-bonding potential in the packing would probably
lead to steric problems in the sense described for the case of
† CCDC reference number(s) 183401. See http://www.rsc.org/suppdata/
p1/b2/b203323b/ for crystallographic files in .cif or other electronic
format.
‡ The molecular structure presented in10 and the corresponding data
in the Cambridge Structural Database11 show the compound to be
(22R,24S)-3β-acetoxy-22-hydroxy-5α-ergostan-6-one (3-acetyl-22,24-
di-epi-cathasterone), not 3-acetyl-24-epi-cathasterone as given in Ref.
10.
J. Chem. Soc., Perkin Trans. 1, 2002, 2022–2027
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Table 2 Geometrical H-bond parameters for [26,27-2H6]24-epi-cathasterone. (Labels and the appropriate symmetry operators correspond to
Fig. 1 and 2)
Distance/Å
Atoms
Angle/Њ
D–H ؒ ؒ ؒ A
D–H
H ؒ ؒ ؒ A
D ؒ ؒ ؒ A
D–H ؒ ؒ ؒ A
O(2a)–H(2ao) ؒ ؒ ؒ O(3b)
O(3aЈ)–H(3aoЈ) ؒ ؒ ؒ O(1w)
O(2bЈ)–H(2boЈ) ؒ ؒ ؒ O(3aЈ)
O(3b)–H(3bo) ؒ ؒ ؒ O(2w)
O(1w)–H(1w1) ؒ ؒ ؒ O(2a)
O(1w)–H(2w1) ؒ ؒ ؒ O(2b#)
O(2w)–H(1w2) ؒ ؒ ؒ O(2b)
O(2w*)–H(2w2*) ؒ ؒ ؒ O(2a)
0.820
0.820
0.820
0.820
0.960
0.960
0.960
0.960
1.990
1.965
2.079
2.045
1.943
2.058
1.993
2.160
2.773(8)
2.778(9)
2.854(9)
2.824(9)
2.871(9)
3.015(8)
2.877(10)
3.088(9)
159.4
171.0
157.7
158.6
161.9
174.1
152.2
162.2
Table 3 1H NMR data (δ, multiplicity, coupling constants/Hz) for
protons and 13C chemical shifts for carbons of [26,27-2H6]24-epi-
cathasterone
atom ring and the other one containing the two O(2) ؒ ؒ ؒ O(w)
bridges between two adjacent 6-O atom rings with significantly
longer O ؒ ؒ ؒ O distances, thus forming the 4-O atom ring of
the ribbon.
Position
δ13C
δ1Hα/βa
NMR
1
2
36.6
1.25/1.78
1.85/1.40
Structure elucidation and unambiguous assignment of 1H
and 13C NMR signals of 8 were done by means of one- and
two-dimensional NMR experiments including one-bond
30.7
70.7
30.0
56.7
210.9
46.7
37.9
53.8
40.9
21.5
39.4
42.8
56.6
23.9
27.6
52.7
11.9
13.1
41.4
11.8
71.1
40.3
34.9
32.1
20.0b
17.8b
15.1
3
4
5
6
7
8
9
3.581 dddd(11.3/11.3/4.6/4.6)
1.90/1.48
2.217 dd (12.5/2.6)
1
(GHSQC) and multiple-bond (GHMBC) H, 13C shift correl-
–
ation. The position of the CD3 groups was verified by HMBC
correlation of the residual proton signal of Me-26 and Me-27
(δ 0.872 d (6.8 Hz); δ 0.823 d (6.8 Hz), relative intensity each
ca. 0.45 H). Both 1H doublets show correlations with C-24 and
C-25. The 13C chemical shift of the C-25 HMBC correlation
peak belonging to the residual proton signals of Me-26/Me-27
is downfield shifted by 0.4 ppm in comparison to the C-25
HMBC correlation peak belonging to the proton signals H-
23A/H-23B/Me-28 of the deuteriated compound. This can be
explained by the isotope effect of the six 2H atoms on C-25 in 8.
The 1H and 13C NMR data of 8 are summarized in Table 3.
1.966 dd (13.2/13.2) α 2.324 dd (13.2/4.6) β
1.79
1.24
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
1.62/1.35
1.26/2.029 ddd (12.6/3.0/3.0)
–
1.28
1.53/1.102 dddd (12.0/12.0/12.0/6.2)
1.95/1.33
1.50
0.677 s
0.759 s
1.36
0.917 d (6.8)
3.745 ddd (9.4/3.2/1.5)
1.55 A 1.010 ddd (13.7/9.6/3.3) B
1.48
Infrared and Raman spectra
Although both techniques do not exhibit the potential to
elucidate the structure of the molecules independently of other
procedures, they may give further characterisation, particularly
on molecular interactions. Therefore we have incorporated
these techniques into our study and the results are given below.
The IR spectrum shows some dominant bands as given in the
Experimental section. On cooling additional features become
significant. Thus the OH region shows 3 strong overlapping
bands, namely (after using the fitting procedure) at ν/cmϪ1 3333,
3233, and 3146, whereas the band near 3407 cmϪ1 disappears
in the background and may be assigned as traces of water in
KBr. The band near 3333 cmϪ1 shows a large half width of ca.
200 cmϪ1, double of each of both other bands. Moreover a
broad band appears with decreasing temperature at 850 cmϪ1
(ν1/2 = ca. 200 cmϪ1).
1.52
–; 0.872 d (6.8)b
–; 0.823 d (6.8)b
0.816 d (6.7)
a Values without multiplicity are chemical shifts of the GHSQC.
b Signal of the residual non deuteriated compound
monoalcohols by Brock and Duncan.12 Thus, the stacks are
held together by hydrogen bonds to the crystal water molecules
(cf. Fig. 2) stabilizing the packing as a “gluing factor” as
reported by Krygowski et al.13 Neighbouring stacks in [01–1]
direction as shown in Fig. 2 are connected by additional H
bonds to the water molecules forming hydrogen-bonded sheets
parallel to the crystallographic (011) plane.
The hydrogen-bond system itself may be described as ribbon-
like, consisting of alternating fused 4- and 6-O atom rings
including all of the six O and the eight H atoms of the
hydroxylic groups and the water molecules within the structure.
For an exact determination of the hydrogen-bonding pattern,
the actual H-atom positions should be known. The current
structure analysis allows only a (not uniquely definable)
localization by geometrical considerations, but the topology of
the H-bond system requires, besides their H-donor function,
the O(2) atoms of both molecules to act as double acceptors,
but the O(3) atoms as well as the water-O atoms as acceptors
for one H bond only. Judging from the donor–acceptor
distances (Table 2), two groups of moderate H bonds may be
distinguished, one with the six smaller distances within the 6-O
As it was to be expected, the Raman spectrum is extremely
rich in sharp lines. All in all, the Raman spectrum appears very
well-resolved, whereas the IR spectrum does not exhibit similar
sharp bands probably due to the influence of hydrogen
bonding.
Because of the manifoldness of similar C–C groups their
vibration frequencies are nearly the same. An analogous
situation is likely to happen for the CH groups. Therefore we
could not expect to find sufficiently characteristic features in the
IR spectrum to deduce the given structure of the compound.
Nevertheless, the well-performed deuteriation and the existence
of the C᎐O group are as obvious as the band near 1058 cmϪ1,
᎐
probably representing the C–OH bond. The large difference
between the CO vibration of Raman spectra and IR band of
7 cmϪ1 should be mentioned. But there should exist some
information on hydrogen bonding. The three bands, presented
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J. Chem. Soc., Perkin Trans. 1, 2002, 2022–2027

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in the OH region at liquid nitrogen temperature, which surpass
on cooling all other bands in intensity, may be taken as an
indication of different species of hydrogen bonding. As we
learn from the X-ray data the donor acceptor distances
(oxygen–oxygen) are different in the case of water, and add-
itionally, both water molecules differ in A–D distances up to 0.3
Å. Similar relationships are valid for the C–OH groups, which
also form long distance (3.015 Å) and short distance (ca. 2.8 Å)
hydrogen bonds. The large distances are to be found in the four
membered ring. In the six membered ring the donor–acceptor
distances between 2.77 and 2.87 Å are similar. Taking the
distances as the deciding factor for the similarity of bond
strength, then a remarkable coupling should exist between the
vibrations of the six membered ring but the longer oxygen–
oxygen distance in the four membered ring should lead to a
higher frequency of the OH-vibration. Therefore we assign the
high frequency OH-band to the vibration of the long distance
interactions in the four membered ring (O(2w*)–O(2a) and
O(2b#)–O(1w)). The difference in these bond lengths (3.088 Å
and 3.015 Å) may lead to two bands overlapping each other
and forming the unresolved broad band near 3333 cmϪ1. The
coupling between the other bonds belonging to the COH group
as well as to a water molecule should lead to the rather narrow
bands near ν/cmϪ1 3233 and 3146, exhibiting rather similar half
widths of ca. 100 cmϪ1. The band near 850 cmϪ1 represents
an overlap of the different deformation frequencies of the
The preparative HPLC was carried out on a Knauer
instrument with a YMC-column on ODS, 5 µm, 20 × 150 mm,
MeCN–H2O as eluent and UV detection at 210 nm. YMC-
column on ODS-A, 5 µm, 4,6 × 250 mm, MeCN–H2O as eluent
was used for analytical HPLC.
(22E, 24S)-3ꢀ-Acetoxy-24-ethyl-5ꢁ-cholest-22-en-6-one 2
(22E,24S)-3β,5-Cyclo-24-ethylcholest-22-en-6-one 1 (4.05 g)
was dissolved in glacial acetic acid (90 ml). H2SO4 (22.5 ml, 2.5
M) was added to this solution. The mixture was stirred and
heated under reflux for 2 h. The solution was cooled and diluted
with crushed ice and water. The precipitate was collected,
washed with water and then dissolved in ether. The ether phase
was washed with aq. NaHCO3 and water, dried over Na2SO4
and concentrated in vacuo. The residue (4.45 g) was dissolved in
pyridine (7 ml) and acetic anhydride (7 ml). The mixture was
stirred at room temperature for 16 h and then heated under
reflux for 1 h. The solution was cooled and diluted with crushed
ice and water. After addition of HCl the desired product was
extracted with CHCl3. The chloroform extract was washed
with water, dried over Na2SO4 and concentrated in vacuo. The
residue was recrystallized from acetone to give 3.5 g (75.4%)
compound 2. TLC (silufol 2 × n-hexane–EtOAc 9 : 1 v/v) Rf
0.38 mp 146–148 ЊC (from hexane); EIMS: m/z 470 (Mϩ, 30.7),
427 (Mϩ Ϫ CH3CO, 5.7), 367 (41.4), 358 (Mϩ Ϫ C8H16(fission
C-20/C-22 ϩ 1H), 52.1), 349 (24.3), 329 (Mϩ Ϫ C10H21(fission
C-17/C-20 ϩ 2H), 100), 316 (359 Ϫ CH3CO, 57.1), 303 (50), 299
(35.7), 271 (30.7), 245 (22.8), 149 (58.6), 123 (33.6), 95 (52.1), 83
(63.6%); 1H NMR: δH 0.681 (3H, s, H-18), 0.772 (3H, s, H-19),
0.792 (3H, d, (6.7) H-27*), 0.803 (3H, t, (7.3) H-29), 0.843 (3H,
d, (6.4) H-26*), 1.021 (3H, d, (6.6) H-21), 2.029 (3H, s,
CH3COO–), 4.670 (1H, m, H-3), 5.018 (1H, dd, (15.2/8.5)) and
5.146 (1H, dd, (15.2/8.5)) (H-22 and H-23); * exchangeable;13C
NMR: δC 12.3 (2C), 13.1, 19.0, 21.2 (2C), 21.4, 21.6, 24.1, 25.4,
26.2, 26.9, 28.8, 31.9, 36.4, 38.0, 39.4, 40.4, 41.0, 42.9, 46.7,
51.2, 53.9, 55.9, 56.5, 56.8, 72.8 (C-3), 129.5 and 137.8 (C-22
and C-23), 170.4 (COO–), 210.1 (C-6).
hydrogen bonds. The sharpness and the position of the C᎐O
᎐
band and its existence in the Raman spectrum denies it taking
part in the hydrogen bond mechanism. All in all, the steric
relations determine this rather special structure.
In spite of its excellent quality in the Raman spectrum there
are too many well-resolved lines between 1400 cmϪ1 and 500
cmϪ1, namely 42 (17 of these with similar intensities in the order
of magnitude of that of the CO line) for us to discuss. Only the
region 2050 cmϪ1 to 2230 cmϪ1 gives a well understandable
combination of lines, proving the well-performed deuteriation.
Experimental
(22E,24S)-3ꢀ-Acetoxy-6,6-(ethylenedioxy)-24-ethyl-5ꢁ-cholest-
22-ene 3
Methods and materials
2,2-Dimethyl-1,3-dioxolane (60 ml) and pTsOH (200 mg) were
added to compound 2 (5.879 g). The mixture was stirred and
heated to 110 ЊC under reflux for 16 h. The formed acetone was
removed by distillation. The reaction was stopped by addition
of K2CO3 (355 mg). The excess of 2,2-dimethyl-1,3-dioxolane
was removed in vacuo and the residue was dissolved in ether.
The ether phase was washed with brine and dried with Na2SO4.
The solvent was removed and the residue was purified by
flash chromatography on SiO2. The product was eluated with
n-hexane–EtOAc (92 : 8 v/v). Amorphous 3 (5 g, 77.8%) was
yielded.
TLC (silufol 2 × n-hexane–EtOAc 9 : 1 v/v); Rf 0.43; EIMS:
m/z 514 (Mϩ, 19.3), 375 (Mϩ Ϫ C10H19 (fission C-17/C-20), 1.4),
317 (C21H33O2, 100), 178 (5.7), 99 (7.1), 83 (5.0%);1H NMR: δH
0.687 (3H, s, H-18), 0.794 (3H, d, (6.5) H-27*), 0.801 (3H, t,
(7.3) H-29), 0.844 (3H, d, (6.5) H-26*), 0.956 (3H, s, H-19),
1.009 (3H, d, (6.6) H-21), 2.029 (3H, s, CH3COO–), 3.740 m
and 3.910 m (2 × OCH2), 4.683 (1H, m, H-3), 5.006 (1H, dd,
15.1/8.5) and 5.145 (1H, dd, 15.1/8.3) (H-22 and H-23),
*exchangeable; 13C NMR: δC 12.3 (2C), 14.2, 19.1, 21.1 (2C),
21.3, 21.5, 24.3, 25.3, 25.4, 27.3, 28.9, 31.9, 33.4, 36.9, 38.0,
39.7, 40.5, 41.3, 42.5, 50.5, 51.2, 53.5, 56.0 (2C), 64.2 and 65.4
(2 × CH2O), 73.9 (C-3), 109.4 (C-6), 129.2 and 138.1 (C-22 and
C-23), 170.3 (COO–).
All commercial reagents were used without further purification.
For flash column chromatography Merck silica gel 60
(particle size 0.040–0.063 mm, 230–400 mesh ASTM) was used.
Melting points (uncorrected) were determined on a Boetius
heating table.
High resolution EI-MS was obtained from a MasSpec of
the firm Micromass. EIMS (DIS): 70 eV, AMD 402 (AMD
Intectra.), positive ion ESI mass spectra.
[26,27-2H6](22S,24S)-3β,22-Dihydroxy-5α-ergostan-6-one
was recrystallized from MeOH–water mixture to give compara-
tively small single crystals suitable for X-ray crystal structure
analysis. A crystal with dimensions 0.27 × 0.18 × 0.07 mm was
mounted on a Stoe Stadi4 diffractometer to determine lattice
parameters and intensity data of X-ray reflections at ambient
conditions.14
Because crystallisation only takes place in the presence of
water and crystals are a prerequisite for structure elucidation by
X-ray analysis, IR and Raman spectra were also performed
using these crystals. Infrared spectra are gained using KBr
method (0.37 mg in 500 mg KBr) on the Bruker instrument IFS
25. For hydrogen bond characterisation the sample was cooled
by liquid nitrogen. The Raman spectrum was obtained from
one crystal of about 0.8 mg on the Bruker IFS 66. 100 runs were
added to give the final spectrum.
NMR: 1D: VARIAN GEMINI spectrometer 300, 300.24
MHz and 75.5 MHz, solvents CD3OD and CDCl3, 2D:
(GHMBC, GHSQC and 1H–1H-COSY) NMR VARIAN
UNITY 500 spectrometer, 499.83 MHz, solvent CD3OD and
CDCl3, TMS as internal standard.
(24S)-3ꢀ-Acetoxy-6,6-(ethylenedioxy)-22,23-dihydroxy-24-
ethyl-5ꢁ-cholestane 4
A
mixture consisting of amorphous
3 (2.93 g), N-
methylmorpholine N-oxide (3.34 g), NaHCO3 (241 mg),
J. Chem. Soc., Perkin Trans. 1, 2002, 2022–2027
2025

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MeSO2NH2 (545 mg), THF (50 ml), water (12.5 ml) and OsO4
(5 ml, 2.5% in tBuOH) was stirred for 4 days at 40 ЊC. The
starting compound disappeared as shown by TLC (silufol
n-hexane–EtOAc 7 : 3 v/v). The spot at Rf 0.50 for the starting
compound was absent and two new spots appeared at Rf 0.28
and Rf 0.09. Solid Na2SO3 (6.5 g) and water (45 ml) were added
and the mixture was stirred for 1 h. The solvent was removed
and the residue was extracted with EtOAc. The EtOAc phase
was washed with brine and dried with Na2SO4. The solvent was
removed in vacuo. The residue was purified by flash chroma-
tography (SiO2 n-hexane–EtOAc 8 : 2 to 6 : 4 v/v). Both com-
pounds (87.7%) were isolated in 11 : 1 proportion (Rf 0.28 :
0.09). 2.52 g mp 97–100 ЊC (from n-hexane) and 215 mg mp
128–130 ЊC (from n-hexane).
dered ammonium bromide (8 g), copper powder (0.5 g) and
hydrobromic acid (48% w/w; 48 ml). The mixture was stirred
and warmed up to 30 ЊC for 1.5 h. Then in the infrared
spectrum of the upper layer the OH band (3400 cmϪ1) dis-
appeared. The mixture was cooled, filtered and the residue
was washed with n-hexane. The hexane phase was washed
with 48% hydrobromic acid until the lower layer showed no
violet coloration. The hexane layer was dried (Na2SO4) and
fractionated in vacuo. Yield was 15.2 g (49.7%) 1-bromo-
3-[2H3]methyl[4,4,4-2H3]buta-1,2-diene bp 55–60 ЊC/70 mmHg.
νmax (CCl4/cmϪ1 644 (vs νCBr), 1166 (s δCH), 1959 (s νC᎐C᎐
᎐ ᎐
C).
3-[2H3]Methyl[4,4,4-2H3]but-1-yne
The spectroscopic data given below are for the substance
with mp 97–100 ЊC.
A slurry of lithium aluminium hydride (1.88 g) in diethylcar-
bitol§ (60 ml) purified by distillation from lithium aluminium
hydride was stirred under argon atmosphere and cooled in
an ice–salt bath. To this slurry 15.2 g 1-bromo-3-[2H3]-
methyl[4,4,4-2H3]buta-1,2-diene were added dropwise. The
reaction mixture was stirred for 20 h, during this time the
mixture was allowed to warm up to room temperature. 8 ml
water were added and 3-[2H3]methyl[4,4,4-2H3]but-1-yne
(bp 30–35 ЊC) was distilled from the crude mixture. 4.77 g
EIMS: m/z 548 (Mϩ, 20.7), 434 (Mϩ Ϫ C7H14 (fission C-22/
C-23 ϩ 1H), 54.3), 404 (Mϩ Ϫ C8H16O2 (fission C-22/C-23 ϩ
1H), 22.8), 373 (Mϩ Ϫ C10H23O2 (fission C-17/C-20–2H),
34.3), 351 (C21H35O4, 100), 237 (C14H21O3, 69.3), 178 (20), 99
(C5H7O2, 72.8%);1H NMR: δH 0.710 (3H, s, H-18), 0.955 (3H, s,
H-19), 2.031 (3H, s, CH3COO–), 3.617 (2H, br s, H-22, H-23),
3.753 m and 3.902 m (2 × CH2O), 4.693 (1H, m, H-3);13C
NMR: δC 65.4 and 69.5 (2 × CH2O), 72.2 and 73.9 (C-22 and
C-23), 76.6 (C-3), 109.4 (C-6), 170.6 (COO–).
Ϫ1
᎐
(64.9%). νmax (neat)/cm : 2226 (m νC᎐C), 3313 (vs νCH of
C᎐CH).
᎐
᎐
᎐
(20S)-3ꢀ-Acetoxy-6,6-(ethylenedioxy)-20-formyl-5ꢁ-pregnane 5
Preparation of the organoaluminium reagent 6
Compound 4 (1.59 g) was dissolved in THF (67 ml). To this
stirred solution dry pyridine (333 µl), solid NaHCO3 (243 mg)
and finally periodic acid (660 mg) were added under argon. The
mixture was stirred for 24 h at room temperature under argon.
The precipitated HIO3 was removed by filtration. The filtrate
was concentrated in vacuo. To the residue 5% aq. NaHSO3 (33.3
ml) was added and the mixture was stirred for 30 min. Then
10% aq. Na2CO3 was added and the mixture was stirred for
another 30 min. The desired product was extracted with CH2Cl2
(3 × 35 ml). The CH2Cl2 phase was washed successively with 5%
saturated aq. NaHCO3 (35 ml) and with brine (35 ml) and then
dried with Na2SO4. The solvent was evaporated under reduced
pressure to give 1.055 g (84.2%) amorphous aldehyde 5 which
we did not purify.
4 ml of a solution of trimethylaluminium in n-heptane (2 M)
were added to a stirred suspension of bis(cyclopentadienyl)-
zirconium dichloride (Cp2ZrCl2) (1 g) in dry CH2Cl2 (10 ml)
under argon. The mixture was stirred for 30 min at room
temperature to give a homogeneous yellow solution. To this
stirred mixture 3-[2H3] methyl[4,4,4-2H3]but-1-yne (609 mg =
0.92 ml) was dropped under argon and the stirring was con-
tinued for 105 min at room temperature. Then the excess of
Me3Al and of the solvent were removed under reduced pressure
at 30 ЊC. To the residue dry n-hexane (5 ml) was added to
precipitate Cp2ZrCl2. The supernatant was transferred to
another flask under argon. This n-hexane-extraction of the
organoaluminate was repeated two more times. 2.6 ml of a solu-
tion of n-BuLi in n-hexane (1.6 M) was added dropwise under
argon to the stirred and cooled (Ϫ10 ЊC) n-hexane solution of
the organoaluminate 6. After 30 min the bath temperature was
raised to 2–5 ЊC and the mixture was stirred for 1 h at that
temperature.
3-[2H3]Methyl[4,4,4-2H3]but-1-yn-3-ol
Into stirred liquid ammonia (700 ml) under argon containing
hydrated ferric nitrate (244 mg) as a catalyst sodium (15,73 g) in
small pieces was added during 30 min. Then triphenylmethane
as an indicator for the conversion of sodamide to sodium
acetylide was added to the solution. In the next step acetylene
purified by bubbling through sulfuric acid was added until the
solution lost its red colour. Then to this intensively stirred
solution dry acetone[2H6] (50 ml in 30 min) was added drop-
wise, with continued addition of acetylene. This mixture was
stirred for 14 h and the ammonia was allowed to evaporate. To
the residue diethyl ether (100 ml) and a solution of aqueous
ammonium chloride (68 g in 186 ml water) were added. The
layers were separated and the aqueous phase was extracted
three times with diethyl ether (300 ml). All ether layers were
added to each other and were dried with sodium sulfate. The
product was isolated by fractional distillation over succinic
acid. Yield was 33 g (55%) 3-[2H3]methyl[4,4,4-2H3]but-1-yn-3-
ol bp 98–104 ЊC. The deuteriation rate was found to be 86.6%,
because the acetylene in the steel flask is dissolved in acetone,
sodium acetylide reacted at a low rate with swept away acetone,
although the acetylene was added through sulfuric acid in order
to remove the acetone.
[26,27-2H6]3ꢀ-Acetoxy-6,6-(ethylenedioxy)-22R-hydroxy-24-
methyl-5ꢁ-cholest-23-ene 7
703 mg of the aldehyde 5 was dissolved in dry THF (5 ml) and
then added dropwise to the stirred and cooled (Ϫ10 to Ϫ30 ЊC)
solution of the organoaluminate 6 in n-hexane under argon.
After 5 min the bath temperature was raised to 2–5 ЊC. The
mixture was stirred overnight at room temperature. The next
day the mixture was diluted with THF (5 ml) and quenched
with aq. NH4Cl at ice cooling. Then the mixture was extracted
with ether and EtOAc. The extract was washed with water,
dried with Na2SO4 and then the solvent was removed under
reduced pressure. The residue was purified by flash chroma-
tography with n-hexane–EtOAc (7 : 3 v/v) as a eluent to give 238
mg (28.0%) raw product. This raw product was purified by pre-
parative HPLC (ODS MeCN–H2O 95 : 5).
EIMS: m/z 522 (Mϩ, 0.2), 504 (Mϩ Ϫ H2O, 8.6), 404 (Mϩ Ϫ
C7H6O2H6 (fission C-20/C-22–1H), 100), 373 (Mϩ
Ϫ
C9H13O2H6 (fission C-17/C-20 ϩ 2H), 42.8), 178 (17.8), 119
1-Bromo-3-[2H3]methyl[4,4,4-2H3]buta-1,2-diene
3-[2H3]Methyl[4,4,4-2H3]but-1-yn-3-ol (18 g) was added to a
stirred mixture of powdered cuprous bromide (10 g), pow-
§ The IUPAC name for diethylcarbitol is di(ethylene glycol) diethyl
ether.
2026
J. Chem. Soc., Perkin Trans. 1, 2002, 2022–2027

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(C7H7O2H6, 39.3), 99 (40.7%); 1H NMR: δH 0.683 (3H, s, H-18),
0.729 (1H, ddd (12.1/10.7/4.1) H-9), 0.950 (3H, d (6.7) H-21),
0.957 (3H, s, H-19), 1.611 (3H, d, (1.2) H-28), 2.029 (3H, s,
CH3COO–), 2.185 (1H, s, H-25), 3.751 m and 3.918 m (2 ×
CH2O), 4.461 (1H, dd (7.9/1.6) H-22), 4.696 (1H, m, H-3),
5.318 (1H, dq (7.9/1.2) H-23).
Acknowledgements
The authors are indebted to the Fonds der chemischen
Industrie for financial support, Dr J. Schmidt for the mass
spectra, and Dr B. Schneider for the high resolution mass
spectrum.
[26,27-2H6]24-epi-Cathasterone 8
References
1 For reviews see: (a) G. Adam, J. Schmidt and B. Schneider,
Brassinosteroid in Prog. Chem. Org. Nat. Prod., ed. W. Herz,
H. Falk, G. W. Kitby, R. E. Moore and Ch. Tamm, Springer,
Berlin, 1999, vol. 78, pp. 1–46; (b) V. A. Khripach, V. N. Zhabinskii,
A. E. de Groot, Brassinosteroids: A new class of Plant Hormones,
Academic Press, New York, 1999.
2 S. Fujioka, T. Inoue, S. Takatsuto, T. Yanagisawa, T. Yokota and
A. Sakurai, Biosci. Biotechnol. Biochem., 1995, 59, 1543–1547;
S. Fujioka, T. Inoue, S. Takatsuto, T. Yanagisawa, T. Yokota and
A. Sakurai, Biosci. Biotechnol. Biochem., 1995, 59, 1973–1975.
3 T. Hai, B. Schneider, A. Porzel and G. Adam, Phytochemistry, 1996,
41, 197–201.
4 J. Schmidt, F. Böhme and G. Adam, Z. Naturforsch. C: Biosci.,
1996, 51, 897–9.
5 D. Lichtblau, PhD Thesis, University of Halle, 1999.
6 M. Aburatani, T. Takeuchi and K. Mori, Agric. Biol. Chem., 1987,
51, 1909–1913.
7 K. Mori, M. Sakakibara and K. Okada, Tetrahedron, 1984, 40,
1767–1781.
8 T. C. McMorris, R. G. Chavez and P. A. Patil, J. Chem. Soc., Perkin
Trans 1, 1996, 295–302.
9 R. Sterzycki, Synthesis, 1979, 724–725.
10 B. Voigt, A. Porzel, C. Bruhn, C. Wagner, K. Merzweiler and
G. Adam, Tetrahedron, 1997, 53, 17039–17054.
11 Cambridge Structural Database (CSD), V. 5.22, October 2001, The
Cambridge Crystallographic Data Centre (CCDC), Cambridge,
2001.
12 C. P. Brock and L. L. Duncan, Chem. Mater., 1994, 6, 1307–1312.
13 T. M. Krygowski, S. J. Grabowski and J. Konarski, Tetrahedron,
1998, 54, 11311–11316.
14 STADI4, Diffractometer Control Program for Windows, Stoe & Cie
GmbH, Darmstadt, Germany, 1996; X-RED, Data Reduction
Program, Stoe & Cie GmbH, Darmstadt, Germany, 1996.
15 G. M. Sheldrick, Acta Crystallogr. Sect. A: Found. Crystallogr.,
1990, 46, 467; G. M. Sheldrick, SHELXL-97, Program for the
Refinement of Crystal Structures, University Göttingen, Germany,
1997.
16 XP/PC, Molecular graphics program package for display and
analysis of stereochemical data, V. 4.2 for MS-DOS, Siemens
Analytical X-ray Instruments, Inc., Madison, Wisconsin, USA,
1990.
A small amount of the catalyst (10% Pt/C) was suspended in
benzene (15 ml) and with H2 hydrogenated for 5 h. Then 28 mg
of compound 7 dissolved in benzene (1 ml) were added and the
mixture was hydrogenated for 12 h. The catalyst and the solvent
were removed. Then the residue was dissolved in aq. acetone
(1 ml) to cleave the dioxolane-type acetal by transacetalization
with pyridinium toluene-p-sulfonate as a catalyst. After
addition of pyridinium toluene-p-sulfonate (7.5 mg) the mix-
ture was refluxed for 3 h. The solvent was then removed in vacuo
and the residue dissolved in EtOAc. After the washing with
water and aq. NaHCO3 and drying with Na2SO4 the solvent
was removed under reduced pressure. The residue was dissolved
in dry methanol and 0. 5 M NaOMe (100 µl). After 3 h the
reaction was stopped with acetic acid and the mixture was
purified by flash chromatography (SiO2 EtOAc–n-hexane 1 : 1
to 7 : 3 v/v) and after them by preparative HPLC (ODS MeCN–
H2O 95 : 5). 3.5 mg (14.9%) [26,27-2H6]24-epi-cathasterone were
obtained.
Mp 184–189 ЊC (MeOH–H2O); EIMS: m/z 438 (Mϩ, 0.7),
2
420 (1.0), 347 (2.8), 318 (Mϩ Ϫ C7H8 H6 (fission C-20/
C-22–1H), 100), 300 (5.0), 248 (10.0), 139 (17.8%); m/z
(EI) 438.398514 (MϩؒC28H42D6O3 requires 438.398006),
420.387871 (Mϩ Ϫ H2OؒC28H40D6O2 requires 420.387442).
X-Ray: Crystal data, C28H42D6O3ؒH2O, M = 456.72, triclinic,
space group P1 (no. 1), a = 7.4742(10), b = 14.290(3),
c = 14.776(3) Å, α = 62.376(11), β = 77.377(19), γ = 79.801(12)Њ,
V = 1359.2(4) Å3, Z = 2, T = 293(2) K, µ(Mo-Kga) = 0.71 mmϪ1,
10882 reflections measured, 5359 unique reflections (Rint
=
0.179) used in the calculations,15 final wR(F2) = 0.152 for
all data, final R = 0.072 for 1912 reflections with I > 2σ(I ).
IR: ν/cmϪ1 3407, 2934, 2209, 1713, 1422, 1383, 1252, 1175,
1059, and 963. RAMAN: Only the most dominant lines are
given: ν/cmϪ1 2218, 2207, 2123, 2071, 1720, 1440, 1344, 1267,
1
1175, 1126, 1057, 995, and 838. H NMR and 13C NMR in
Table 3
J. Chem. Soc., Perkin Trans. 1, 2002, 2022–2027
2027