Synthesis of deuterium-labeled (24R)-methyl brassinosteroids

Article abstract of DOI:10.1002/jlcr.1874

Synthesis of labeled brassinosteroids (24-epibrassinolide and its biosynthetic precursors) containing three deuterium atoms in the terminal part of the side chain is reported. Labeling was achieved by three-step reductive transformation of carbethoxy group into methyl using lithium aluminium deuteride. The proposed method ensured high isotopic purity of (24R)-methyl brassinosteroids containing a label in a position which is not subjected to its loss. Copyright 2011 John Wiley & Sons, Ltd. Synthesis of labeled brassinosteroids (24-epibrassinolide and its biosynthetic precursors) containing three deuterium atoms in the terminal part of the side chain is reported. Labeling was achieved by three-step reductive transformation of carbethoxy group into methyl using lithium aluminium deuteride. Copyright

Full text of DOI:10.1002/jlcr.1874

Research Article
Received 1 November 2010,
Revised 28 December 2010,
Accepted 9 January 2011
Published online 17 February 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1874
Synthesis of deuterium-labeled (24R)-methyl
brassinosteroids
Ã
Vladimir A. Khripach, Vladimir N. Zhabinskii, Olga V. Gulyakevich,
Yurii V. Ermolovich, and Olga V. Konstantinova
Synthesis of labeled brassinosteroids (24-epibrassinolide and its biosynthetic precursors) containing three deuterium atoms
in the terminal part of the side chain is reported. Labeling was achieved by three-step reductive transformation of
carbethoxy group into methyl using lithium aluminium deuteride. The proposed method ensured high isotopic purity of
(24R)-methyl brassinosteroids containing a label in a position which is not subjected to its loss.
Keywords: brassinosteroids; epibrassinolide; epicastasterone; episecasterone; episecasterol
Introduction
Experimental
24-Epibrassinolide holds a special place among natural brassino- Melting points were recorded on a Boetius micro-melting point
1
steroids (BS).1 It belongs to the most active representatives apparatus and are uncorrected. H and 13C NMR spectra were
of this group of phytohormones, and at the same time it obtained using a Bruker AVANCE 500 (Bruker Biospin, Rhein-
can be relatively easily prepared from available sterols.2–4 As a stetten, Germany) spectrometer in CDCl3 operating at 500 MHz
result, now it is the most practically useful compound of this for 1H and 125 MHz for 13C. Assignment of 1H and 13C
group; for a number of years preparations with 24-epibrassinolide resonances was performed by the combined use of 1D and
(Figure 1) as an active ingredient have been used in 2D experiments, including COSY, HSQC, HMBC, TOCSY, and
agriculture.5,6 Research in the recent years has revealed its NOESY methods using standard pulse sequences supplied in the
potential usefulness in medicine also.7–9 Further studies, both instrument manufacturer’s software package. Mass spectra were
in agricultural and medicinal directions, require the availability performed on a LCQ Fleet mass spectrometer (Thermo Electron
of labeled 24-epibrassinolide. Recently, we have published Corporation, USA) with an APCI source. Spectra were collected
synthesis of [7-2H2]epibrassinolide.10 This compound proved in the positive ion mode and analyzed by the Xcalibur software.
to be useful for biochemical and physiological investigations, Chemicals were purchased from Aldrich and Fluka and used as
but because of its relatively low isotopic purity (82%) it could received. Reactions were monitored by TLC using aluminum or
not be used in some experiments. Based on the experience plastic sheets, silica gel 60 F254 precoated (Merck Art. 5715).
we have gained in brassinolide biosynthetic studies,11–15 Column chromatography was carried out on Kieselgel 60 (Merck
labeling the terminal part of the steroidal side chain was Art. 7734).
considered as an appropriate solution of the problems
connected with low isotopic purity. The present work is an [26ꢀ2H3](22E,24R)-6b-Methoxy-3a,5-cyclo-24-methyl-5a-
cholest-22-ene (4)
extension of our studies16 toward synthesis of labeled brassi-
nosteroids for biochemical and other biological applications and
To a solution of (22E,24R)-6b-methoxy-3a,5-cyclo-24-methyl-5a-
deals with the preparation of [26-2H3]epibrassinolide and its
cholest-22-en-26-oic acid ethyl ester (3)17 (1.22 g, 2.58 mmol) in
biosynthetic precursors.
THF-diethyl ether (7:1, 80 ml), LiAlD4 (217 mg, 5.16 mmol) was
added portionwise. The reaction was stirred at an ambient
temperature for 1.5 h. Then water (0.22 ml), 15% NaOH (0.22 ml)
and again water (0.65 ml) were consecutively added to the
mixture. The precipitate was filtered off and the filtrate was
evaporated to dryness. Purification of the residue by SiO2
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus,
Kuprevich str., 5/2, 220141 Minsk, Belarus
*Correspondence to: Vladimir N. Zhabinskii, Institute of Bioorganic Chemistry,
Kuprevich str., 5/2, 220141 Minsk, Belarus.
E-mail: vz@iboch.bas-net.by
Figure 1. The Structure of 24-epibrassinolide.
J. Label Compd. Radiopharm 2011, 54 332–336
Copyright r 2011 John Wiley & Sons, Ltd.

V. A. Khripach et al.
chromatography using petroleum ether/EtOAc (10:1 ) 20:1) pyridine (11 mg, 0.09 mmol) was kept at 451C for 4.5 h. Then
provided alcohol 4 (930 mg) as an oil.
water was added and the mixture was extracted with CHCl3.
Triethylamine (0.74 ml, 5.3 mmol) and MsCl (0.25 ml, 3.1 mmol) The organic fraction was dried over Na2SO4 and evaporated.
were added to a stirred solution of alcohol 4 from the previous The residue was purified by SiO2 chromatography using petro-
stage (930 mg, 2.17 mmol) in 25 ml of CH2Cl2 at 01C. The reaction leum ether/EtOAc (15:1 ) 10:1) to afford diacetate 6 (510 mg,
mixture was kept at these conditions for 4 h, and then diluted 93%) as an oil. 1H-NMR: d 0.43 (dd, J = 8.0, 5.1 Hz, 1 H), 0.65
with water. The layers were separated in a separatory funnel and (dd, J = 4.5, 4.5 Hz, 1 H), 0.72 (s, 3 H, 18-Me), 0.83 (d, J = 7.1 Hz, 3
the aqueous portion was extracted with CHCl3. The combined H), 0.93 (d, J = 6.7 Hz, 3 H), 1.00 (d, J = 6.7 Hz, 3 H), 1.02 (s, 3 H,
organic portions were dried over Na2SO4 and evaporated. The 19-Me), 2.03 (s, 3 H, OAc), 2.04 (s, 3 H, OAc), 2.77 (t, J = 2.7 Hz,
residue was purified by SiO2 chromatography using petroleum 1 H, C6-H), 3.32 (s, 3 H, OMe), 5.08 (dd, J = 7.5, 4.6 Hz, 1 H,
ether/EtOAc (40:1 ) 15:1) to give crude mesylate 4a (850 mg) C23-H), 5.26 (d, J = 7.7 Hz, 1 H, C22-H). 13C-NMR: d 10.82,
which was used in the next step without additional purification. 11.90, 13.03, 13.37, 19.26, 20.89, 20.96, 21.46, 22.41, 22.74,
To a vigorously stirred solution of mesylate 4a (850 mg, 24.05, 24.92, 26.57, 28.16, 30.49, 33.33, 34.87, 35.22, 37.72,
1.67 mmol) in THF (18 ml) and diethyl ether (7 ml), LiAlD4 38.63, 40.18, 42.63, 43.30, 47.90, 53.06, 56.36, 56.56, 74.80, 77.60,
(212 mg, 5.05 mmol) was added. The mixture was stirred at 82.31, 170.55.
room temperature for 2 h, then another portion of LiAlD4
(212 mg, 5.05 mmol) was added. After stirring for additional 2 h, [26ꢀ2H3](22R,23R,24S)-24-Methycholest-5-en-3b,22,23-triol
22,23-diacetate (7)
water (0.43 ml), 15% NaOH (0.43 ml) and again water (1.3 ml)
were added. The precipitate was filtered off and the filtrate was
evaporated. Column chromatography of the residue using
petroleum ether/EtOAc (25:1) as eluent gave olefin 5 (600 mg,
A solution of diacetate 6 (443 mg, 0.83 mmol) and TsOH
(94 mg, 0.55 mmol) in dioxane (21 ml) and water (6 ml) was
stirred under argon at 801C for 3 h. Then Et3N (0.91 ml, 6.5 mmol)
was added and solvents were evaporated to dryness. Column
chromatography of the residue using CHCl3/MeOH (20:1) as
1
56% from 3) as an oil. H-NMR: d 0.44 (dd, J = 7.5, 5.3 Hz, 1 H),
0.66 (dd, J = 4.2, 4.2 Hz, 1 H), 0.74 (s, 3 H, 18-Me), 0.82 (d,
J = 6.7 Hz, 3 H), 0.92 (d, J = 6.7 Hz, 3 H), 1.01 (d, J = 6.7 Hz, 3 H),
1.03 (s, 3 H, 19-Me), 2.77 (br. s., 1 H, C6-H), 3.33 (s, 3 H, OMe),
5.12–5.25 (m, 2 H). 13C-NMR: d 12.45, 13.07, 17.62, 19.29, 19.57,
19.88, 20.94, 21.47, 22.76, 24.17, 24.96, 28.64, 30.47, 32.85, 33.34,
35.06, 35.27, 40.19, 42.67, 42.76, 43.39, 48.06, 56.17, 56.56, 56.60,
82.42, 131.69, 135.88.
eluent gave diacetate
7 (429 mg, 99%). Mp 203–2051C
(petroleum ether/EtOAc). 1H-NMR: d 0.69 (s, 3 H, 18-Me), 0.83
(d, J = 6.7 Hz, 3 H), 0.93 (d, J = 6.7 Hz, 3 H), 1.01 (d, J = 6.7 Hz, 3 H),
1.02 (s, 3 H, 19-Me), 2.04 (s, 3 H, OAc), 2.05 (s, 3 H, OAc),
3.51–3.58 (m, 1 H, C3-H), 5.08 (dd, J = 7.4, 4.8 Hz, 1 H, C23-H),
5.22–5.30 (m, 1 H, C22-H), 5.35 (d, J = 5.1 Hz, 1 H, C6-H). 13C-NMR:
d 10.88, 11.57, 13.44, 19.39, 20.88, 20.92, 21.08, 22.39, 24.22,
26.67, 28.11, 29.70, 31.67, 31.80, 31.97, 36.49, 37.27, 37.77, 38.81,
39.75, 42.26, 42.31, 50.08, 53.05, 56.62, 71.77, 74.83, 77.61,
[26ꢀ2H3](22R,23R,24R)-6b-Methoxy-3a,5-cyclo-24-methyl-
5a-cholestan-22,23-diol (6a)
A mixture of olefin 5 (690 mg, 1.66 mmol), K3Fe(CN)6 (1.64 g, 121.63, 140.74, 170.55.
4.99 mmol), K2CO3 (690 mg, 4.99 mmol), methanesulfonamide
(475 mg, 4.99 mmol), K2OsO4.2H2O (12 mg, 0.033 mmol), hydro- [26ꢀ2H3](22R,23R,24R)-24-Methyl-3a,5-cyclo-5a-cholestan-
6-on-22,23-diol diacetate (8)
quinidine 1,4-phthalazinediyl diether (129 mg, 0.166 mmol), tert-
butyl alcohol (20 ml) and water (20 ml) was stirred at room
temperature for 14 days. Then Na2SO3 (1.5 g) was added and
stirring was continued for 24 h. The layers were separated in a
separatory funnel and the aqueous part was extracted with
CHCl3. The combined organic portions were dried over Na2SO4
and evaporated. The residue was purified by column chromato-
graphy on SiO2 using petroleum ether/EtOAc (20:1 ) 5:1)
to give:
A solution of alcohol 7 (484 mg, 0.93 mmol) and TsCl (644 mg,
3.38 mmol) in pyridine (3 ml) was kept at an ambient tempera-
ture overnight. Then the mixture was diluted with water and
extracted with CHCl3. The organic layer was dried over Na2SO4
and evaporated to dryness to give crude tosylate 7a (584 mg).
To a solution of the tosylate 7a (584 mg) from the previous
stage in acetone (62 ml), a solution of AcOK (242 mg) in water
(5 ml) was added. The reaction mixture was heated under reflux
for 21 h. Then it was cooled down to room temperature and
Jones reagent (0.72 ml) was added. The mixture was stirred for
30 min, and then isopropyl alcohol (0.62 ml) was added to
remove the excess of oxidizing agent. The reaction mixture was
diluted with water and extracted with CHCl3. The organic layer
was dried over Na2SO4 and evaporated. The residue was purified
by SiO2 chromatography using petroleum ether/EtOAc
(20:1 ) 2:1) to afford diacetoxyketone 8 (300 mg, 62%). Mp
(a)starting olefin 5 (55 mg);
(b)diol 6a (505mg, 73% based on consumed olefin 5). Mp
127–1301C (petroleum ether/EtOAc). 1H-NMR: d 0.44 (dd, J = 8.0,
5.1Hz, 1 H), 0.65 (m, 1 H), 0.73 (s, 3 H), 0.85 (d, J= 6.7 Hz, 3 H),
0.92 (d, J = 6.7Hz, 3 H), 0.97 (d, J = 6.7Hz, 3 H), 1.03 (s, 3 H 19-
Me), 2.78 (t, J = 2.6Hz, 1 H, C6-H), 3.33 (s, 3 H, OMe), 3.41
(q, J = 5.1 Hz, 1 H, C23-H), 3.71 (t, J =4.5 Hz, 1 H, C22-H). 13C-NMR:
d 10.87, 12.07, 12.45, 13.07, 19.26, 21.45, 22.05, 22.77, 24.10,
24.94, 26.75, 28.00, 30.55, 33.34, 35.06, 35.20, 40.20, 40.25, 41.37,
42.65, 43.35, 47.94, 52.86, 56.35, 56.56, 72.84, 76.39, 82.38.
1
146–1491C (petroleum ether/EtOAc). H-NMR: d 0.73 (s, 3 H, 18-
Me), 0.83 (d, J = 7.1 Hz, 3 H), 0.93 (d, J = 6.7 Hz, 3 H), 1.00 (s, 3 H,
19-Me), 1.01 (d, J = 6.7 Hz, 3 H), 2.03 (s, 3 H, OAc), 2.05 (s, 3 H,
OAc), 5.08 (dd, J = 7.4, 4.8 Hz, 1 H, C23-H), 5.26 (d, J = 6.7 Hz, 1 H,
C22-H). 13C-NMR: d 10.81, 11.61, 11.71, 13.41, 19.65, 20.85, 20.93,
22.38, 22.83, 23.95, 25.87, 26.62, 28.00, 33.45, 34.77, 35.28, 37.70,
38.71, 39.63, 42.58, 44.64, 45.98, 46.26, 46.70, 52.92, 56.79, 74.64,
[26ꢀ2H3](22R,23R,24R)-6b-Methoxy-3a,5-cyclo-24-methyl-
5a-cholestan-22,23-diol diacetate (6)
A mixture of diol 6a (463 mg, 1.03 mmol), pyridine (3.5 ml), 77.48, 170.51, 170.56, 209.47. MS (APCI1) [C32H48D3O5] m/z (MH)
1
acetic anhydride (0.37 ml, 3.92 mmol) and 4-N,N-dimethylamino-
calcd 518.39, found 518.6.
J. Label Compd. Radiopharm 2011, 54 332–336
Copyright r 2011 John Wiley & Sons, Ltd.
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V. A. Khripach et al.
[26ꢀ2H3](22R,23R,24R)-24-Methy-5a-cholest-2-en-6-on-
22,23-diol diacetate (9)
[26ꢀ2H3](22R,23R,24R)-24-Methy-5a-cholestan-6-on-
2a,3a,22,23-tetraol ([26ꢀ2H3]epicastasterone) (12)
A solution of steroid 8 (154 mg, 0.297 mmol) and pyridinium A mixture of olefin 10 (38 mg, 0.088 mmol), N-methylmorpho-
bromide (158 mg, 0.98 mmol) in dimethylacetamide (3.2 ml) line-N-oxide (17 mg), osmium tetroxide (0.3 mg), acetone (2 ml)
was heated under argon at 165–1701C for 5 h. Then it was and water (0.1 ml) was stirred at room temperature for 7 h. Then
cooled and the solvent was evaporated under reduced it was diluted with water (3 ml), the precipitate was filtered off,
pressure. The resulting oil was mixed with water and extracted washed with water and dissolved in acetone. The solvent was
with CHCl3. The organic layer was dried over Na2SO4 and evaporated in vacuo and the residue was purified by SiO2
evaporated. The residue was purified by SiO2 chromatography chromatography using CHCl3/MeOH (15:1) to give tetraol 12
1
using petroleum ether/EtOAc (15:1 ) 12:1) to give enone (21 mg, 51%). Mp 229–2311C (EtOAc). H-NMR: 0.70 (s, 3 H, 18-
9 (120 mg, 78%). Mp 180–1831C (petroleum ether). 1H-NMR: d Me), 0.77 (s, 3 H, 19-Me), 0.87 (d, J = 7.1 Hz, 3 H), 0.93 (d,
0.69 (s, 3 H, 18-Me), 0.71 (s, 3 H, 19-Me), 0.83 (d, J = 7.1 Hz, J = 7.1 Hz, 3 H), 1.00 (d, J = 6.7 Hz, 3 H), 2.70 (d, J = 12.2 Hz, 1 H,
3 H), 0.93 (d, J = 7.1 Hz, 3 H), 1.01 (d, J = 6.7 Hz, 3 H), 2.04 C5-H), 3.39–3.45 (m, 1 H, C23-H), 3.71 (br. s., 1 H, C22-H), 3.76 (d,
(s, 3 H, OAc), 2.05 (s, 3 H, OAc), 5.08 (dd, J = 7.4, 4.8 Hz, 1 H, J = 8.0 Hz, 1 H, C2-H), 4.05 (br. s., 1 H, C3-H). 13C-NMR: d 10.94,
C23-H), 5.25 (d, J = 7.4 Hz, 1 H, C22-H), 5.57 (dd, J = 9.3, 1.6 Hz, 11.90, 12.49, 13.56, 21.30, 22.00, 23.94, 26.45, 26.94, 27.79, 37.80,
1 H, C2-H), 5.69 (dd, J = 9.9, 2.2 Hz, 1 H, C3-H). 13C-NMR: d 10.82, 39.56, 40.31, 40.40, 41.66, 42.57, 42.92, 46.75, 50.81, 52.84, 53.87,
11.62, 13.41, 13.49, 20.86, 20.93, 21.09, 21.69, 22.38, 23.85, 56.66, 68.38, 68.49, 72.75, 76.46, 211.65. MS (APCI1)
26.64, 27.90, 37.68, 38.74, 39.35, 39.44, 39.97, 42.70, 46.86, [C28H46D3O5] m/z (MH)1 calcd 468.38, found 468.2.
52.99, 53.34, 53.81, 56.58, 74.67, 77.51, 124.45, 124.99, 170.54,
211.79. MS (APCI1) [C32H48D3O5] m/z (MH)1 calcd 518.39,
found 518.2.
[26ꢀ2H3](22R,23R,24R)-24-Methy-5a-cholestan-6-on-
2a,3a,22,23-tetraol 22,23-diacetate (13)
The title compound was obtained from enone 9 in 76% yield
according to the procedure described for the preparation of
([26-2H3]epicastasterone) (12). Mp 96–981C (petroleum ether).
1H-NMR: 0.66 (s, 3 H, 18-Me), 0.75 (s, 3 H, 19-Me), 0.82
(d, J = 7.1 Hz, 3 H), 0.92 (d, J = 7.1 Hz, 3 H), 1.00 (d, J = 6.7 Hz, 3
H), 2.03 (s, 3 H, OAc), 2.05 (s, 3 H, OAc), 2.67 (dd, J = 12.7, 2.7 Hz, 1
H, C5-H), 3.73–3.80 (m, 1 H, C2-H), 4.05 (d, J = 2.2 Hz, 1 H, C3-H),
5.07 (dd, J = 7.4, 4.8 Hz, 1 H, C23-H), 5.24 (d, J = 7.7 Hz, 1 H, C22-H).
13C-NMR: d 10.77, 11.66, 13.37, 13.53, 20.88, 20.93, 21.11, 22.36,
23.82, 26.25, 26.58, 27.86, 37.62, 38.64, 39.28, 40.12, 42.51, 42.77,
46.60, 50.65, 52.87, 53.57, 56.42, 68.22, 68.34, 74.60, 77.52,
170.64, 212.01.
[26ꢀ2H3](22R,23R,24R)-24-Methy-5a-cholest-2-en-6-on-
22,23-diol (10)
Diacetate 9 (58 mg, 0.112 mmol) was dissolved in a solution of
KOH in MeOH (5%, 3 ml). The mixture was heated under
reflux for 1 h, then cooled down and acidified by adding a
mixture AcOH–MeOH (1:4) to reach pH 7. Solvents were
evaporated in vacuo and the residue was purified by SiO2
chromatography using CHCl3/MeOH (25:1) to give diol 10
(40 mg, 83%). Mp 148–1501C (petroleum ether/EtOAc).
1H-NMR:
0.86 (d, J = 7.1 Hz,
d
0.69 (s,
3
H, 18-Me), 0.72 (s,
H), 0.92 (d, J = 6.7 Hz,
3
H, 19-Me),
3 H), 0.99
3
(d, J = 6.7 Hz, 3 H), 3.42 (q, J = 5.2 Hz, 1 H, C23-H), 3.68–3.74
(m, 1 H, C22-H), 5.55–5.61 (m, 1 H, C2-H), 5.69 (ddd, J = 9.9, 5.0,
2.1 Hz, 1 H, C3-H). 13C-NMR: d 10.88, 11.77, 12.45, 13.50,
21.14, 21.71, 22.03, 23.88, 26.79, 27.74, 37.78, 39.35, 39.49,
40.04, 40.19, 41.41, 42.69, 46.95, 52.68, 53.34, 53.84, 56.60, 72.71,
76.41, 124.50, 124.95, 211.97. MS (APCI1) [C28H44D3O3] m/z (MH)
[26ꢀ2H3](22R,23R,24S)-24-Methy-5a-cholestan-6-on-
2a,3a,22,23-tetraol tetraacetate (14)
A mixture of the diol 13 (80 mg, 0.145 mmol), Ac2O (0.1 ml,
1.06 mmol), pyridine (1 ml) and 4-N,N-dimethylaminopyridine
(3 mg) was kept at room temperature for 24 h. The solvents were
evaporated under reduced pressure. The residue was purified by
SiO2 chromatography using CHCl3/MeOH (100:1) to give
1
calcd 434.37, found 434.2.
1
[26ꢀ2H3](22R,23R,24R)-2a,3a-Epoxy-24-methy-5a-cholestan-
6-on-22,23-diol ([26ꢀ2H3]secasterol) (11)
tetraacetate 14 (90 mg, 97%) as an oil. H-NMR: d 0.67 (s, 3 H,
18-Me), 0.81 (d, J = 6.7 Hz, 3 H), 0.83 (s, 3 H, 19-Me), 0.92 (d,
J = 6.7 Hz, 3 H), 1.00 (d, J = 6.7 Hz, 3 H), 1.99 (s, 3 H, OAc), 2.04 (s, 3
H, OAc), 2.05 (s, 3 H, OAc), 2.09 (s, 3 H, OAc), 4.91–4.98 (m, 1 H,
C2-H), 5.07 (dd, J = 7.4, 4.8 Hz, 1 H, C23-H), 5.24 (d, J = 7.1 Hz, 1 H,
C22-H), 5.36–5.41 (m, 1 H, C3-H). 13C-NMR: d 10.77, 11.68, 13.40,
13.53, 20.85, 20.92, 21.03, 21.11, 21.19, 22.35, 23.81, 24.79, 26.61,
27.86, 37.51, 37.67, 38.68, 39.24, 42.38, 42.80, 46.40, 51.78, 52.96,
53.61, 56.44, 68.09, 69.08, 74.63, 77.48, 169.98, 170.24, 170.27,
170.57, 210.52.
A solution of olefin 10 (29 mg, 0.067 mmol) and m-chloroper-
benzoic acid (70%, 35 mg, 0.14 mmol) in CHCl3 (1 ml) was
stirred at room temperature for 1.5 h. Then it was washed
successively with 12% NH4OH and brine and dried over
Na2SO4. After solvent removal and purification on a silica gel
column using CHCl3/MeOH (20:1), epoxide 11 (25 mg, 83%)
was obtained as white crystals. Mp 165–1671C (petroleum
ether/EtOAc). 1H-NMR: d 0.67 (s, 3 H, 18-Me), 0.71 (s, 3 H,
19-Me), 0.84 (d, J = 6.7 Hz, 3 H), 0.91 (d, J = 7.1 Hz, 3 H), 0.98
(d, J = 6.7 Hz, 3 H), 3.11–3.15 (m, 1 H, C2- or C3-H), 3.26–3.29
(m, 1 H, C3- or C2-H), 3.38–3.44 (m, 1 H, C23-H), 3.69 (br. s., 1 H,
[26ꢀ2H3](22R,23R,24R)-24-Methy-B-homo-7-oxa-5a-chole-
stan-6-on-2a,3a,22,23-tetraol tetraacetate (15)
C22-H). 13C-NMR: d 10.84, 11.71, 12.43, 15.00, 20.99, 21.04, A solution of trifluoroperbenzoic acid was prepared by adding
22.03, 23.84, 26.75, 27.68, 37.51, 37.81, 38.42, 39.31, 40.18, trifluoroacetic anhydride (3.5 ml) to a stirred emulsion of H2O2 (30%,
41.36, 42.58, 46.84, 49.86, 50.14, 52.37, 52.57, 52.99, 56.36, 72.61, 0.4 ml) in CH2Cl2 (8.4 ml) at 01C. An aliquot of the obtained
76.34, 211.46. MS (APCI1) [C28H44D3O4] m/z (MH)1 calcd 450.37, trifluoroacetic acid solution (5.87 ml) was added to a stirred solution
found 450.5.
of tetraacetate 14 (80 mg, 0.126 mmol) in CH2Cl2 (4 ml) at 01C.
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Copyright r 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 332–336

V. A. Khripach et al.
Stirring was continued for 5 h at these conditions, and then the Results and discussion
mixture was diluted with water and extracted with CHCl3. The
Synthesis of the target compounds was based on the ester 3
organic layer was dried over Na2SO4 and evaporated. The residue
was purified by SiO2 chromatography using petroleum ether/EtOAc
(3:1 ) 2:1) to give tetraacetate 15 (45 mg, 55%). Mp 103–1051C
available in five steps from commercial stigmasterol 2.17
Labeling with deuterium was achieved at the very beginning
of the synthetic sequence. Ester 3 was first submitted to the
reduction with lithium aluminum deuteride to give alcohol 4
containing two deuteriums in the molecule (Scheme 1). Its
mesylation followed by similar reduction with LiAlD4 afforded
the three-deuterio derivative 5 as enantioisotopomeric pair of
compounds. Asymmetric dihydroxylation of D22-double bond in
olefin 5 using Sharpless protocol led, after acetylation, to
(22R,23R)-diacetate 6. A methoxy at C-6 was replaced by oxo
group in four steps to give cycloketone 8 to ensure further
construction of brassinosteroid cyclic part.
1
(petroleum ether/EtOAc). H-NMR: d 0.72 (s, 3 H, 18-Me), 0.81–0.91
(m, 6 H), 0.94 (d, J= 7.1 Hz, 3 H), 0.98 (d, J= 6.7 Hz, 3 H), 1.00 (s, 3 H,
19-Me), 2.01 (s, 3 H, OAc), 2.04 (s, 3 H, OAc), 2.05 (s, 3 H, OAc), 2.12 (s,
3 H, OAc), 3.00 (dd, J= 12.2, 4.5 Hz, 1 H, C5-H), 4.00–4.09 (m, 1 H, C7-
H), 4.09–4.17 (m, 1 H, C7-H), 4.88 (ddd, J= 12.7, 4.6, 2.9 Hz, 1 H, C2-H),
5.07 (dd, J=7.2, 5.0Hz, 1 H, C23-H), 5.23 (d, J= 6.1 Hz, 1 H, C22-H),
5.37 (m, 1 H, C3-H). 13C-NMR: d 10.82, 11.50, 13.37, 15.44, 20.82,
20.90, 21.02, 21.11, 22.28, 22.34, 22.64, 24.73, 26.71, 27.83, 29.34,
31.57, 37.80, 38.41, 38.82, 38.91, 39.20, 39.51, 42.05, 42.53, 51.28,
53.03, 58.42, 67.95, 68.94, 70.41, 74.57, 169.90, 170.18, 170.53, 174.97.
MS (APCI1) [C36H54D3O10] m/z (MH)1 calcd 652.41, found 652.3.
Heating of 3a,5-cyclo-6-ketone 8 in boiling dimethylaceta-
mide in the presence of pyridine hydrobromide led to the
rearranged product 9 containing a D2-double bond (Scheme 2).
[26ꢀ2H3](22R,23R,24R)-24-Methy-B-homo-7-oxa-5a-chole-
stan-6-on-2a,3a,22,23-tetraol (½26 ꢀ2H3ꢁepibrassinolide) (16)
A solution of tetraacetate 15 (40 mg, 0.061 mmol) in 5% KOH in
MeOH (4 ml) was heated under reflux for 1.5 h. Then the reaction
mixture was cooled down to room temperature and 25% HCl
(1 ml) was added. After keeping for 1 h, the mixture was diluted
with water and extracted with CHCl3. The organic layer was
dried over Na2SO4 and concentrated in vacuo. Crystallization
from EtOAc/petroleum ether provided 16 (28 mg, 94%) as white
crystals. Mp 230–2321C. 1H-NMR: d 0. 0.71 (s, 3 H, 18-Me), 0.86 (d,
J = 7.1 Hz, 3 H), 0.92 (d, J = 7.1 Hz, 3 H), 0.93 (s, 3 H, 19-Me), 0.98
(d, J = 6.4 Hz, 3 H), 3.13 (dd, J = 12.3, 4.0 Hz, 1 H), 3.39–3.45 (m, 1
H, C23-H), 3.67 (m, 1 H, C22-H), 3.67–3.78 (m, 1 H, C2-H), 4.01–4.16
(m, 3 H, C2- and C7-H). 13C-NMR: d 10.87, 11.64, 12.38, 15.47,
22.03, 22.24, 24.76, 26.80, 27.70, 31.03, 38.32, 39.23, 39.57, 40.21,
40.89, 41.38, 41.50, 42.49, 50.89, 51.24, 52.58, 58.17, 68.07, 68.15,
70.44, 72.57, 76.41, 176.16. MS (APCI1) [C28H46D3O6] m/z (MH)1
calcd 484.37, found 484.4.
Scheme 2.
Scheme 1.
J. Label Compd. Radiopharm 2011, 54 332–336
Copyright r 2011 John Wiley & Sons, Ltd.
www.jlcr.org

V. A. Khripach et al.
Scheme 3.
Removal of acetyl protecting groups in 9 gave diol 10, which is a
deuterated analogue of 24-episecasterol. The latter is expected
to be a biosynthetic precursor of 24-epibrassinolide in a
pathway similar to that described for secasterol and brassino-
lide. Recently, non-deuterated 24-episecasterol was prepared
and found to be cytotoxic in human breast carcinoma MCF-7
cells.18 The two electrophilic reactions, epoxidation and
dihydroxylation of the olefin 10, gave epoxide 11 and 24-
epicastasterone 12, correspondingly. Compound 11 is a 2a,3a-
epimer of natural brassinosteroid 24-episecasterone found in
Lychnis viscaria seeds.19 Although it has not been described as a
natural product before, such a possibility cannot be excluded.13
Although 24-epibrassinolide 16 could be obtained in one step
by the Baeyer–Villiger oxidation of 24-epicastasterone 12, our
previous experience on brassinosteroid synthesis showed that
more lengthy route with preliminary protection of hydroxy
groups before lactonization gave in general more pure final
product. That is why we performed synthesis of 24-epibrassi-
nolide 16 via endiacetate 9. Its dihydroxylation with OsO4
afforded dioxydiacetate 13, which was further acetylated to
epicastasterone tetraacetate 14 (Scheme 3). It was subjected to
the Baeyer–Villiger oxidation, and after chromatographic purifica-
tion of the lactone 15 and saponification of the latter, deuterated
24-epibrassinolide 16 was obtained. The isotopic purity of
deuterated brassinosteroids was controlled by mass spectra.
Thus, for [26-2H3]epibrassinolide 16 the composition ratio of
2H3:2H2:2H1:2H0 was found to be 99.2:0.3:0.2:0.3.
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Acknowledgement
The work was supported by the Belarusian Republican Founda-
tion for Basic Research (project no. X10P-118).
www.jlcr.org
Copyright r 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 332–336