Synthesis of hexadeuterated 23-dehydroxybrassinosteroids

Article abstract of DOI:10.1016/S0039-128X(02)00071-5

Two hexadeuterated brassinosteroids (BS) ([26,27-²H₆]-23-dehydroxycastasterone and [26,27-²H₆]-cathasterone) containing a hydroxy group at C₂₂ instead of the 22R,23R-diol function characteristic for most compounds of this class were prepared for biochemical studies. The corresponding non-deuterated compounds are considered intermediates in brassinolide biosynthesis. The carbon skeleton of the side chain with proper stereochemistry at C₂₄ was prepared from commercially available (2R)-3-hydroxy-2-methylpropanoate. This low molecular fragment was coupled to the tetracyclic steroidal fragment through the reaction of the appropriate sulfone with C₂₂ aldehyde. Formation of the necessary configuration of the 22-hydroxy group was achieved by hydride reduction of the corresponding ketone. Deuterium atoms at C₂₆ and C₂₇ originated from [²H₃]methyl iodide used for alkylation of the intermediate sulfone.

Full text of DOI:10.1016/S0039-128X(02)00071-5

Steroids 67 (2002) 1101–1108
Synthesis of hexadeuterated 23-dehydroxybrassinosteroids
Vladimir A. Khripacha,∗, Vladimir N. Zhabinskiia, Andrey P. Antonchicka,
Olga V. Konstantinovaa, Bernd Schneiderb
Laboratory of Chemistry of Steroids, Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus,
a
Kuprevich Street 5/2, 220141 Minsk, Belarus
Max-Planck-Institute for Chemical Ecology, Beutenberg Campus, Winzerlaer Street 10, D-07745 Jena, Germany
b
Received 6 March 2002; received in revised form 4 June 2002; accepted 11 June 2002
Abstract
Two hexadeuterated brassinosteroids (BS) ([26,27-2H6]-23-dehydroxycastasterone and [26,27-2H6]-cathasterone) containing a hydroxy
group at C22 instead of the 22R,23R-diol function characteristic for most compounds of this class were prepared for biochemical studies. The
corresponding non-deuterated compounds are considered intermediates in brassinolide biosynthesis. The carbon skeleton of the side chain
with proper stereochemistry at C24 was prepared from commercially available (2R)-3-hydroxy-2-methylpropanoate. This low molecular
fragment was coupled to the tetracyclic steroidal fragment through the reaction of the appropriate sulfone with C22 aldehyde. Formation
of the necessary configuration of the 22-hydroxy group was achieved by hydride reduction of the corresponding ketone. Deuterium atoms
at C26 and C27 originated from [2H3]methyl iodide used for alkylation of the intermediate sulfone.
© 2002 Elsevier Science Inc. All rights reserved.
Keywords: Brassinosteroids; Labeled compounds; Brassinolide; Plant growth hormone
1. Introduction
‘early C6-oxidation pathway’ and the ‘late C6-oxidation
pathway’ operate independently [5,6]. In this respect, the
main objective of the present work was the elaboration
of a synthetic approach to and preparation of deuterated
23-dehydroxybrassinosteroids. Such compounds contain-
ing six deuterium atoms in the side chain are essential for
biosynthetic studies of relevant brassinolide precursors [7].
The presence of many functional groups in the brassi-
nolide molecule implies that its biosynthesis is a multistep
process. Detailed knowledge of the biosynthetic pathway is
very important for a better understanding of the physiologi-
cal processes and subtle mechanisms of the action of brassi-
nosteroids (BS) in plants. Most BS known to date contain a
22R,23R-diol function, which is essential for their biologi-
cal activity [1,2]. Identification of cathasterone lacking one
of the hydroxy groups and application of its labeled analog
in feeding experiments [3] showed that brassinolide biosyn-
thesis proceeds via initial hydroxylation at C22 followed
by introduction of the hydroxy group at C23 (Scheme 1).
This finding has been confirmed by identification of the
CPD gene as a microsomal cytochrome P450-dependent
23␣-hydroxylase [4]. However, in addition to the estab-
lished biosynthetic sequences, hitherto unknown alternative
subpathways or species-specific biosynthetic routes may
occur. It is known, for example, that even within one
biological species, different biosynthetic routes to brassi-
nolide exist. Thus, in Catharanthus roseus, the so-called
2. Experimental
2.1. General
Melting points were recorded on a Boetius micromelt-
ing point apparatus and are uncorrected. IR spectra were
recorded on an UR-20 spectrophotometer. 1H and 13C
NMR spectra were recorded on a Bruker AC-200 (200 MHz
for 1H, 50 MHz for 13C) spectrometer using TMS as an
internal standard in CDCl3. Accurate mass measurements
were carried out on a Micromass MasSpec mass spec-
trometer operating in the 70 eV EI mode. Samples were
introduced by direct probe for accurate mass measurement
by peak matching. Chemicals were purchased from Aldrich,
Fluka, and Steraloids Chemical Companies and were used
as received. [2H3]Methyl iodide (99.5%) was supplied by
Deutero Gmbh. Reactions were monitored by TLC using
∗
Corresponding author. Tel.: +375-172-648-647;
fax: +375-172-648-647.
E-mail address: hripach@iboch.bas-net.by (V.A. Khripach).
0039-128X/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(02)00071-5

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V.A. Khripach et al. / Steroids 67 (2002) 1101–1108
Scheme 1.
aluminum or plastic sheets precoated with silica gel 60 F254
(Merck Art. 5715). Column chromatography was carried
out on Kieselgel 60 (Merck Art. 7734).
and MeOH (300 ml). A solution of the tosylate 5 (19.0 g,
58 mmol) in MeOH (150 ml) was then added, and the mix-
ture was left to stand at ambient temperature for 14 h, diluted
with water, and extracted with petroleum ether. The organic
layer was dried over Na2SO4 and evaporated to give crude
(2R)-2-methyl-1-phenylsulfanyl-3-tetrahydro-2H-2-pyrany-
loxypropane, which was dissolved in CHCl3 (400 ml), and
m-chloroperbenzoic acid (57 g, 0.33 mol) was added. The
mixture was stirred at room temperature for 3 h, washed
with 25% aqueous NH4OH, water, dried over Na2SO4, and
evaporated. The residue was chromatographed on SiO2 with
petroleum ether–EtOAc (20:1 ⇒ 3:1) to give the sulfone
6 (13.2 g, 76%) as an oil. IR (cm−1): 2940, 2880, 1600,
2.2. 2-[(2R)-2-Methyl-3-(4-methylphenylsulfonyloxy)
propyloxy]tetrahydro-2H-pyran (5)
HCl (36%, 2.5 ml) was added to a solution of (2R)-3-hydr-
oxy-2-methylpropanoate (2) (10 ml, 90 mmol) in dihydro-
pyrane (25 ml). After incubation at ambient temperature for
24 h, pyridine (25 ml) was added. The solvent was partially
evaporated in vacuo, and the residue was dissolved in CHCl3
and filtered through a plug of SiO2 with the aid of EtOAc.
The filtrate was concentrated under reduced pressure to give
crude (2R)-2-methyl-3-tetrahydro-2H-2-pyranyloxypropa-
noate, which was dissolved in Et2O (200 ml). LiAlH4
(13.5 g, 0.35 mol) was added portionwise over 1 h, and
the reaction mixture was stirred for an additional 1 h and
then treated with NaOH solution (13.5 ml, 15%) and water
(50.4 ml). The suspension was filtered, and the filtrate was
dried over Na2SO4 and evaporated to give (2S)-2-methyl-
3-tetrahydro-2H-2-pyranyloxypropan-1-ol. The crude oil
was dissolved in pyridine (140 ml), and TsCl (36 g, 0.19 mol)
was added. The mixture was left to stand at room temper-
ature for 2 h, diluted with water (400 ml), and extracted
with CHCl3. The organic layer was dried over Na2SO4 and
evaporated. The residue was chromatographed on SiO2 with
petroleum ether–EtOAc (20:1 ⇒ 5:1) to give the tosylate
5 (19.0 g, 64%) as an oil. IR (cm−1): 2950, 2875, 1610,
1
1460, 1325, 1155, 980. H NMR δ: 1.12 (dd, 3H, J = 8.5,
2.5 Hz, 2-Me), 2.24–2.46 (m, 1H, C2–H), 2.86–3.16 (m,
2H, CH2–S), 3.30–3.84 (m, 4H, CH2–O), 4.42–4.56 (m,
1H, O–CH–O), 7.50–7.98 (m, 5H, Ph). 13C NMR δ: 17.03,
17.25, 19.36, 19.46, 25.35, 29.34, 29.62, 29.65, 30.45,
59.17, 59.47, 62.18, 62.31, 70.43, 71.17, 127.86, 129.25,
133.54, 140.05. HRMS calc. for C15H20O3S: 280.113317.
Found: 280.113152. EI-MS m/z (%): 85 (100), 101 (45), 143
(40), 197 (52), 215 (14), 240 (0.5), 280 (0.3) [M −H2O]•+
.
2.4. 2-[(2R)-2-Methyl-3-[2H3]methyl-3-phenylsulfonyl-
[4-2H3]butyloxy]tetrahydro-2H-pyran (7)
A solution of BuLi (2.5 M in hexane, 40 ml, 0.1 mol)
was added at −30 ◦C to a solution of sulfone 6 (10.0 g,
30.5 mmol) in THF (250 ml). The mixture was stirred for
15 min, after which a solution of CD3I (3.4 ml, 55 mmol) in
THF (10 ml) was added, and the temperature was gradually
increased to 10 ◦C over 30 min. The mixture was recooled
to −30 ◦C, and a solution of BuLi (2.5 M in hexane, 20 ml,
50 mmol) was added. The mixture was maintained at this
temperature for 15 min, and a second portion of CD3I
(3.4 ml, 55 mmol) in THF (10 ml) was introduced. After
15 min, the mixture was allowed to warm to room tempera-
ture, after which it was diluted with water and extracted with
EtOAc. The organic layer was dried over Na2SO4, evapo-
rated, and the residue was chromatographed on SiO2 with
petroleum ether–EtOAc (20:1 ⇒ 3:1) to give the deuterated
derivative 7 (5.5 g, 50%) as an oil. IR (cm−1): 2950, 2875,
2245, 1450, 1390, 1360, 1305, 1155, 1085, 1040, 1030,
1
1465, 1370, 1195, 1370, 1195, 1185, 1045, 980, 820. H
NMR δ: 0.93 (s, 3H, 2-Me), 2.46 (s, 3H, Ph-CH3), 7.36
(d, 2H, J = 8 Hz, Ph), 7.80 (d, 2H, J = 8 Hz, Ph). 13C
NMR δ: 13.58, 13.66, 19.26, 19.40, 19.72, 19.79, 21.60,
25.41, 25.47, 25.70, 30.44, 30.68, 30.94, 33.49, 33.64,
61.92, 62.13, 62.86, 63.33, 67.95, 68.39, 72.21, 94.57,
98.48, 98.87, 99.10, 127.94, 129.80, 133.12, 144.65. HRMS
calc. for C16H24O5S: 328.134445. Found: 328.124615.
EI-MS m/z (%): 85 (100), 91 (82), 101 (72), 155 (62), 173
(72), 227 (12), 245 (5), 310 (2) [M − H2O]•+, 328 (1)
[M]•+
.
2.3. 2-[(2R)-2-Methyl-3-phenylsulfonylpropyloxy]
tetrahydro-2H-pyran (6)
1
Thiophenol (30.6 ml, 0.3 mol) was added to a solution of
sodium methoxide prepared from sodium (18 g, 0.78 mol)
1010, 980. H NMR δ: 1.12 (dd, 3H, J = 7, 5 Hz, 2-Me),
2.14–2.30 (m, 1H, C2–H), 3.14–3.98 (m, 4H, CH2–O),

V.A. Khripach et al. / Steroids 67 (2002) 1101–1108
1103
4.78 (s, 1H, O–CH–O), 7.42–7.86 (m, 5H, Ph). 13C NMR
δ: 13.91, 14.05, 19.40, 25.32, 30.56, 30.59, 36.40, 36.65,
62.12, 62.20, 65.26, 69.62, 69.94, 98.98, 99.07, 128.62,
130.30, 133.36, 136.26. HRMS calc. for C12H11D6O3S:
247.127502. Found: 247.127529. EI-MS m/z (%): 85 (100),
89 (58), 125 (38), 144 (50), 191 (21), 226 (12), 231 (23),
247 (23).
with petroleum ether. The combined organic extacts were
dried over Na2SO4 and evaporated. The residue was chro-
matographed on SiO2 with petroleum ether–EtOAc (20:1 ⇒
5:1) to give the starting tosylate 9 (0.8 g, 15% recovery) and
the sulfide 10 (3.5 g, 68%) as an oil. IR (cm−1): 2940, 1730,
1590, 1485, 1450, 1385, 1300, 1250, 1150, 1150, 1090,
1030. 1H NMR δ: 1.10 (d, 3H, J = 7 Hz, 2-Me), 2.08–2.26
(m, 1H, C2–H), 2.60 (dd, 1H, J = 13, 11 Hz, C1–H), 3.90
(dd, 1H, J = 13, 1.5 Hz, C1–H), 7.14–7.88 (m, 10H, Ph).
13C NMR δ: 14.88, 37.19, 37.45, 65.86, 126.30, 128.74,
128.90, 130.14, 130.24, 133.51, 136.07. HRMS calc. for
C18H16D6O2S2: 340.143784. Found: 340.143641. EI-MS
m/z (%): 77 (9), 89 (16), 123 (100), 180 (5), 199 (17)
2.5. (2R)-2-Methyl-3-[2H3]methyl-3-phenylsulfonyl-
[4-2H3]butan-1-ol (8)
A mixture of 7 (5.5 g, 16.5 mmol) and HCl (36%, 1 ml) in
MeOH (250 ml) was stirred at room temperature for 40 min.
Pyridine (1 ml) was added, and the solvent was evaporated
under reduced pressure. The residue was chromatographed
on SiO2 with petroleum ether–EtOAc (15:1 ⇒ 1:1) to give
the sulfone 8 (4.1 g, 100%) as an oil. IR (cm−1): 2990, 2900,
2250, 1590, 1450, 1290, 1150, 1090, 1055, 1025. 1H NMR
δ: 1.16 (d, 3H, J = 7 Hz, 2-Me), 2.12–2.28 (m, 1H, C2–H),
2.66 (s, 1H, OH), 3.71 (dd, 1H, J = 12, 6 Hz, C1–H), 3.98
(dd, 1H, J = 12, 4 Hz, C1–H), 7.52–7.94 (m, 5H, Ph). 13C
NMR δ: 13.60, 39.50, 64.63, 65.44, 128.83, 130.43, 133.66,
136.17. HRMS calc. for C12H12D6O3S: 248.135327. Found:
248.135130. EI-MS m/z (%): 88 (88), 107 (100), 143 (65),
[M − PhSO2]+, 340 (21) [M]•+
.
2.8. (2S)-2-Methyl-3-[2H3]methyl[4-2H3]butyl phenyl
sulfide (11)
Mg (2 g, 82 mmol) was added to a vigorously stirred
solution of 10 (3.3 g, 9.7 mmol) in MeOH (200 ml). The
mixture was stirred for 2 h, the resultant precipitate was
filtered, and the filtrate was neutralized with 3N HCl. The
acidified aqueous layer was extracted with petroleum ether,
and the combined organic extracts were dried over Na2SO4,
evaporated, and chromatographed on SiO2 with petroleum
ether–EtOAc (20:1 ⇒ 5:1) to give the sulfide 11 (1.7 g,
88%) as an oil. IR (cm−1): 2970, 2940, 2885, 2220, 2080,
1600, 1490, 1450, 1390, 1100, 1065, 1030. 1H NMR δ: 0.96
(d, 3H, J = 7 Hz, 2-Me), 1.52–1.72 (m, 1H, C2–H), 2.72
(dd, 1H, J = 12.5, 8.5 Hz, C1–H), 3.02 (dd, 1H, J = 13,
5 Hz, C1–H), 7.08–7.42 (m, 5H, Ph). 13C NMR δ: 15.12,
18.99, 30.95, 38.31, 38.88, 40.39, 125.49, 125.67, 128.74,
128.97, 137.52. HRMS calc. for C12H12D6S: 200.150583.
Found: 200.149872.
144 (62), 190 (9), 218 (4), 248 (0.2) [M]•+
.
2.6. (2R)-2-Methyl-3-[2H3]methyl-3-phenylsulfonyl
[4-2H3]butyl 4-methyl-1-benzenesulfonate (9)
TsCl (19.0 g, 0.1 mol) was added to a solution of 8 (4.1 g,
16.5 mmol) in pyridine (100 ml). The mixture was left to
stand at ambient temperature for 3 h, diluted with water,
and extracted with CHCl3. The organic layer was dried over
Na2SO4, evaporated, and the residue was chromatographed
on SiO2 with petroleum ether–EtOAc (20:1 ⇒ 5:1) to give
the tosylate 9 (6.1 g, 92%) as an oil. IR (cm−1): 2990,
2250, 1610, 1455, 1370, 1300, 1195, 1185, 1155, 1105,
2.9. (2S)-2-Methyl-3-[2H3]methyl[4-2H3]butyl phenyl
sulfone (12)
1
970. H NMR δ: 1.12 (d, 3H, J = 7 Hz, 2-Me), 2.26–2.46
(m, 1H, C2–H), 2.46 (s, 3H, OTs–CH3), 4.08 (dd, 1H,
J = 10, 7.5 Hz, C1–H), 4.54 (dd, 1H, J = 10, 3.5 Hz,
C1–H), 7.32–7.88 (m, 9H, Ph and OTs). 13C NMR δ: 13.12,
21.54, 36.47, 64.43, 65.20, 72.16, 127.60, 129.80, 129.84,
130.22, 132.59, 133.74, 135.45, 144.88. HRMS calc. for
C13H13D6O3S: 261.143152. Found: 261.143053. EI-MS
m/z (%): 89 (100), 125 (9), 144 (9), 155 (36), 174 (6), 247
MCPBA (5.8 g, 34 mmol) was added to an ice-bath cooled
solution of sulfide 11 (1.7 g, 8.5 mmol) in CHCl3 (100 ml).
The mixture was stirred for 3 h at room temperature and
then, washed with NH4OH (25% solution) and water. The
organic layer was extracted with petroleum ether. The com-
bined organic phases were washed with saturated NaHCO3,
dried over Na2SO4, and evaporated under reduced pressure.
The residue was chromatographed on SiO2 with petroleum
ether–EtOAc (15:1 ⇒ 3:1) to give the sulfone 12 (1.2 g,
(6) [M − Ts]+, 261 (13) [M − PhSO2]+, 402 (0.2) [M]•+
.
2.7. (2R)-2-Methyl-3-[2H3]methyl-1-phenylsulfanyl-
3-phenylsulfonyl[4-2H3]butane (10)
1
61%) as an oil. H NMR δ: 1.02 (d, 3H, J = 7 Hz, 2-Me),
1.90–2.10 (m, 1H, C2–H), 2.88 (dd, 1H, J = 14, 8.5 Hz,
C1–H), 3.10 (dd, 1H, J = 14, 3.5 Hz, C1–H), 7.45–7.96 (m,
5H, Ph). 13C NMR δ: 15.95, 31.95, 33.72, 60.50, 127.88,
129.27, 133.54, 140.15. HRMS calc. for C12H12D6O2S:
232.140412. Found: 232.140400. EI-MS m/z (%): 77 (67),
90 (100), 125 (13), 143 (97), 156 (7), 184 (5), 232 (3)
Thiophenol (17.5 ml, 0.17 mol) was added to a solution of
MeONa prepared from sodium (10 g, 0.44 mol) and MeOH
(150 ml). A solution of tosylate 9 (6.1 g, 15.2 mmol) in
MeOH (100 ml) was added, and the mixture was left to stand
at room temperature for 40 h after which it was extracted
[M]•+
.

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V.A. Khripach et al. / Steroids 67 (2002) 1101–1108
2.10. (24S)-[26,27-2H6]23-Phenylsulfonyl-6,6-
ethylenedioxy-24-methyl-3α,5-cyclo-5α-cholestan- 22-one
(14)
stirred at room temperature for 14 h and filtered through
SiO2. The filtrate was evaporated, and the residue was
chromatographed on SiO2 with petroleum ether–EtOAc
(20:1 ⇒ 5:1) to give 15 (350 mg, 65%) as an oil. IR
(cm−1): 2270, 2265, 2220, 1720, 1470, 1385, 1170, 1110,
1080. 1H NMR δ: 0.73 (s, 3H, 18-Me), 1.02 (s, 3H, 19-Me),
3.68–4.08 (m, 4H, –CH2–O–).
A solution of BuLi in hexane (2.5 M, 5 ml, 12.5 mmol)
was added to a stirred solution of sulfone 12 (0.9 g,
3.9 mmol) in THF (15 ml) under an argon atmosphere at
−40 ◦C). The mixture was stirred for 30 min at −40 ◦C,
cooled to −80 ◦C, and a solution of (20S)-6,6-ethylenedioxy-
20-formyl-3␣,5-cyclo-5␣-pregnane (1) (2.0 g, 5.7 mmol)
in THF (20 ml) was added. After stirring at −70 ◦C for
1 h, the mixture was allowed to warm to room tempera-
ture. NH4Cl (2 g) was added, and the mixture was diluted
with water and extracted with EtOAc. The organic layer
was dried over Na2SO4 and evaporated. The residue was
chromatographed on SiO2 with petroleum ether–EtOAc
2.12. (22S,24R)-[26,27-2H6]22-Hydroxy-24-methyl-3α,
5-cyclo-5α-cholestan-6-one (17)
LiAlH4 (200 mg, 5.3 mmol) was added to a stirred so-
lution of ketone 15 (350 mg, 0.76 mmol) in THF). Stirring
was maintained for 30 min, and then, excess LiAlH4 was
destroyed by careful addition of water (0.8 ml) and NaOH
solution (15%, 0.2 ml). The precipitate was filtered, and
the filtrate was dried over Na2SO4 and evaporated to give
(22S,24R)-[26,27-2H6]22-hydroxy-24-methyl-6,6-ethylene-
dioxy-3␣,5-cyclo-5␣-cholestane 16 (330 mg, 94%) as an
oil, which was used without further purification for the next
step.
(20:1
⇒
3:1) to give 1.4 g (62% based on 12) of
(24S)-[26,27-2H6 ]22-hydroxy-23-phenylsulfonyl-6,6-ethy-
lenedioxy-24-methyl-3␣,5-cyclo-5␣-cholestane (13) as an
oil.
A two-necked flask equipped with a magnetic stirrer
was charged with (COCl)2 (12 ml, 0.14 mol) in CH2Cl2
(40 ml) and cooled to −70 ◦C. DMSO (22 ml, 0.3 mol) was
added, and stirring was continued for 30 min at −70 ◦C.
A solution of hydroxy sulfone 13 (1.0 g, 1.8 mmol) in
CH2Cl2 (20 ml) was added, and the mixture was stirred
at −70 ◦C for 1 h. Et3N (80 ml, 0.58 mol) was added drop-
wise, and the cooling bath was removed. After warming to
room temperature, the mixture was diluted with water and
extracted with CH2Cl2. The organic layer was evapo-
rated, and the residue was chromatographed on SiO2 with
petroleum ether–EtOAc (20:1 ⇒ 5:1) to give the oxo sul-
fone 14 (0.7 g, 70%) as an oil. IR (cm−1): 2950, 2880, 2225,
1720, 1690, 1660, 1455, 1390, 1310, 1150, 1080. 1H NMR
δ: 0.72 (s, 3H, 18-Me), 1.00 (s, 3H, 19-Me), 3.52–4.10 (m,
4H, –CH2–O–), 7.46–7.96 (m, 5H, Ph). 13C NMR δ: 11.41,
11.68, 12.04, 12.19, 13.55, 18.98, 19.68, 22.60, 22.87,
23.06, 23.93, 24.05, 24.91, 25.91, 27.72, 29.82, 33.25,
33.48, 34.21, 34.86, 35.35, 37.62, 39.25, 39.65, 40.17,
41.12, 42.61, 44.70, 45.59, 46.00, 46.33, 46.73, 47.36,
51.86, 56.85, 63.72, 64.66, 64.68, 70.93, 71.06, 109.67,
127.68, 128.09, 128.97, 133.08, 143.90, 209.53. HRMS
calc. for C36H46D6O5S: 602.391208. Found: 602.390976.
EI-MS m/z (%): 87 (79), 165 (74), 343 (100), 358 (17),
407 (16), 462 (35), 465 (37), 527 (10), 547 (38), 587 (9)
Alcohol 16 (270 mg, 0.58 mmol) was dissolved in ace-
tone (137 ml), and a solution of TsOH (16.5 mg) in water
(14 ml) was added. The mixture was stirred at room tem-
perature for 2 h before pyridine (2 ml) was added, and the
solvent was evaporated under reduced pressure. The residue
was chromatographed on SiO2 with petroleum ether–EtOAc
(20:1 ⇒ 6:1) to give the alcohol 17 (180 mg, 73%) as an oil.
1H NMR δ: 0. 73 (s, 3H, 18-Me), 0.84 (d, 3H, J = 6.5 Hz,
21- or 28-Me), 0.92 (d, 3H, J = 6.5 Hz, 28- or 21-Me),
1.02 (s, 3H, 19-Me), 4.72–4.84 (m, 1H, C22–H). 13C NMR
δ: 11.27, 11.68, 12.00, 15.83, 19.70, 22.91, 24.02, 25.92,
27.78, 31.57, 33.49, 34.88, 35.35, 39.41, 39.76, 42.66, 44.79,
46.08, 46.35, 46.77, 52.50, 56.92, 71.62, 209.68. HRMS
calc. for C28H40D6O2: 420.387442. Found: 420.388802.
EI-MS m/z (%): 121 (32), 136 (41), 161 (29), 229 (12), 285
(27), 300 (100), 329 (9), 402 (8) [M − H2O]•+, 420 (50)
[M]•+
.
2.13. (22S,24R)-[26,27-2H6]22-Acetoxy-24-methyl-3α,
5-cyclo-5α-cholestan-6-one (18)
A mixture of alcohol 17 (140 mg, 0.33 mmol), Ac2O
(0.5 ml) and pyridine (1 ml) was stirred at room temperature
for 50 h, after which it was diluted with water, and extracted
with EtOAc. The organic layer was dried over Na2SO4
and evaporated. The residue was chromatographed on SiO2
with petroleum ether–EtOAc (20:1 ⇒ 10:1) to give the
[M − CH3]+, 602 (54) [M]•+
.
2.11. (24R)-[26,27-2H6]6,6-Ethylenedioxy-24-methyl-3α,
5-cyclo-5α-cholestan-22-one (15)
1
acetate 18 (154 mg, 100%) as an oil. H NMR δ: 0.70 (s,
3H, 18-Me), 0.82 (d, 3H, J = 6.5 Hz, 21- or 28-Me), 0.95
(d, 3H, J = 6.5 Hz, 28- or 21-Me), 0.98 (s, 3H, 19-Me),
2.00 (s, 3H, OAc), 4.96–5.08 (m, 1H, C22–H). 13C NMR
δ: 11.59, 11.83, 12.60, 15.49, 19.64, 21.23, 22.87, 23.99,
25.88, 28.07, 31.64, 33.47, 34.81, 34.99, 35.21, 35.73,
38.20, 39.69, 42.62, 44.69, 46.05, 46.26, 46.71, 52.48,
56.86, 74.84, 170.69, 209.44.
Strips of aluminum foil (2.5 g) were treated with NaOH
solution (15%) for 15 min, and the alkali was decanted off.
The foil was washed consecutively with water, ethanol, a so-
lution of HgCl2 (0.5%), and ethanol. The mercury-activated
aluminum was added to a solution of oxo sulfone 14
(700 mg, 1.16 mmol) in EtOH (150 ml). The mixture was

V.A. Khripach et al. / Steroids 67 (2002) 1101–1108
1105
2.14. (22S,24R)-[26,27-2H6]22-Acetoxy-3β-bromo-
24-methyl-5α-cholestan-6-one (19)
(2 ml) at 30–40 ◦C for 30 min. The resulting mixture was
extracted with CHCl3, the extract was dried over Na2SO4,
and the solvent was evaporated. The resultant crude product
21 (46 mg) was treated with a solution of KOH in MeOH
(5%, 2 ml) and heated under reflux for 1 h. After neutral-
ization with AcOH, the solvent was evaporated under re-
duced pressure. The residue was chromatographed on SiO2
with petroleum ether–EtOAc (5:1 ⇒ 0:1) to give the triol
HBr (48%, 0.24 ml) was added to a solution of 18 (75 mg,
0.16 mmol) in AcOH (2 ml). The mixture was stirred for
30 min, diluted with water, and extracted with CHCl3. The
organic layer was washed consecutively with water, sat-
urated NaHCO3, and brine, and then, it was dried over
Na2SO4 and evaporated. The residue was chromatographed
on SiO2 with petroleum ether–EtOAc (20:1 ⇒ 10:1) to give
22 (27 mg, 69%); mp: 243–246 ◦C (CHCl3). H NMR δ:
1
0.72 (s, 3H, 18-Me), 0.78 (s, 3H, 19-Me), 0.84 (d, 3H,
J = 6.5 Hz, 21- or 28-Me), 0.90 (d, 3H, J = 6 Hz, 28- or
21-Me), 2.68 (d, 1H, J = 12 Hz, C5–H), 3.70–3.86 (m, 2H,
C22– and C2–H), 4.08 (m, 1H, C3–H). 13C NMR δ: 11.26,
11.97, 13.55, 15.81, 21.26, 23.91, 26.31, 27.66, 31.58, 35.28,
37.75, 39.43, 40.17, 42.60, 42.89, 46.74, 50.73, 52.48, 53.75,
56.62, 68.43, 71.64, 212.06. HRMS calc. for C28H42D6O4:
454.392921. Found: 454.391823. Calc. for C28H40D6O3:
436.382356. Found: 436.383017. EI-MS m/z (%): 81 (18),
93 (16), 121 (17), 163 (10), 247 (11), 289 (15), 316 (57),
1
the bromide 19 (88 mg, 100%) as an oil. H NMR δ: 0.66
(s, 3H, 18-Me), 0.80 (s, 3H, 19-Me), 2.04 (s, 3H, OAc),
3.94 (m, 1H, C3–H), 5.00–5.12 (m, 1H, C22–H). 13C NMR
δ: 11.80, 12.56, 13.08, 15.50, 21.30, 23.89, 27.95, 31.63,
32.34, 33.40, 35.00, 35.78, 37.80, 38.22, 39.19, 39.38,
40.64, 42.89, 46.50, 50.47, 52.53, 53.84, 56.58, 58.96,
74.82, 170.72, 209.50. HRMS calc. for C28H3981BrD6O:
484.300992. Found: 484.301857. Calc. for C28H3979BrD6O:
482.303038. Found: 482.303368. EI-MS m/z (%): 95 (64),
103 (63), 149 (28), 270 (19), 336 (16), 350 (100), 352 (98),
377 (63), 379 (67), 380 (75), 382 (58), 462 (15), 482 (35),
334 (100), 345 (5), 336 (3) [M − H2O]+, 454 (3) [M]•+
.
484 (42), 542 (1) [M]•+, 544 (1) [M]•+
.
2.17. (22S,24R)-[26,27-2H6]3β,22-Diacetoxy-24-
methyl-5α-cholestan-6-one (23)
2.15. (22S,24R)-[26,27-2H6]22-Acetoxy-24-methyl-
5α-cholest-2-en-6-one (20)
A solution of H2SO4 (3N, 0.075 ml) was added to a solu-
tion of compound 18 (59 mg, 0.13 mmol) in AcOH (1.5 ml).
The mixture was stirred at 115 ◦C for 1 h, cooled, diluted
with water, and extracted with CHCl3. The organic layer
was washed with saturated NaHCO3, dried over Na2SO4,
and evaporated. The residue was chromatographed on SiO2
with petroleum ether–EtOAc (20:1 ⇒ 10:1) to give the di-
acetate 23 (45 mg, 68%) as an oil. 1H NMR δ: 0.70 (s,
3H, 18-Me), 0.78 (s, 3H, 19-Me), 0.84 (d, 3H, J = 6 Hz,
21- or 28-Me), 0.98 (d, 3H, J = 6.5 Hz, 28- or 21-Me),
2.02 (s, 6H, AcO), 4.66 (m, 1H, C3–H), 4.98–5.12 (m, 1H,
C22–H). 13C NMR δ: 11.83, 12.57, 13.04, 15.49, 21.32,
21.47, 23.90, 26.10, 26.83, 27.95, 31.65, 34.98, 35.71, 36.42,
37.93, 38.15, 39.40, 40.91, 42.89, 46.56, 52.50, 53.81, 56.47,
56.53, 72.82, 74.84, 170.62, 170.77, 210.26. HRMS calc.
for C30H42D6O3: 462.398006. Found: 462.397766. EI-MS
m/z (%): 95 (43), 103 (38), 107 (26), 121 (23), 149 (15), 177
(11), 229 (11), 271 (14), 300 (47), 330 (100), 357 (72), 402
A mixture of bromide 19 (88 mg, 0.16 mmol), Li2CO3
(300 mg) and DMF (3 ml) was refluxed for 1 h, allowed
to cool to ambient temperature, diluted with water, and
extracted with CHCl3. The organic layer was dried over
Na2SO4 and evaporated. The residue was chromatographed
on SiO2 with petroleum ether–EtOAc (20:1 ⇒ 12:1) to
give: (a) (22S,24R)-[26,27-2H6]22-acetoxy-24-methyl-5␣-
1
cholest-2-en-6-one (20) (40 mg, 53%) as an oil. H NMR
δ: 0.68 (s, 3H, 18-Me), 0.72 (s, 3H, 19-Me), 0.84 (d, 3H,
J = 6.5 Hz, 21- or 28-Me), 0.96 (d, 3H, J = 6.5 Hz, 28- or
21-Me), 2.05 (s, 3H, OAc), 5.00–5.12 (m, 1H, C22–H), 5.64
(m, 2H, C2– and C3–H). 13C NMR δ: 11.75, 12.58, 13.52,
15.51, 21.14, 21.30, 21.71, 23.90, 27.96, 31.66, 35.00,
35.75, 37.74, 38.19, 39.40, 39.48, 40.02, 42.74, 46.92,
52.53, 53.42, 53.83, 56.65, 63.16, 74.88, 124.48, 125.01,
170.77, 211.90. HRMS calc. for C30H42D6O3: 462.398006.
Found: 462.397206. EI-MS m/z (%): 93 (21), 107 (19), 121
(17), 229 (7), 243 (5), 270 (17), 297 (16), 300 (18), 402
(12), 434 (17), 447 (68) [M − CH3]+, 462 (100) [M]•+; (b)
(22S,24R)-[26,27-2H6 ]22-acetoxy-24-methyl-3␣,5-cyclo-
5␣-cholestan-6-one (18) (20 mg, 27%) as an oil.
(15), 447 (10), 462 (78) [M − AcOH]+, 522 (1.5) [M]•+
.
2.18. (22S,24R)-[26,27-2H6]22-Acetoxy-3β-hydroxy-24-
methyl-5α-cholestan-6-one (24)
Diacetate 23 (43 mg, 0.082 mmol) was treated with a so-
lution of KOH in MeOH (2%, 8 ml) at room temperature for
50 min. AcOH was added to neutralize the mixture, and the
solvent was evaporated under reduced pressure. The residue
was chromatographed on SiO2 with petroleum ether–EtOAc
(10:1 ⇒ 1:1) to give monoacetate 24 (37 mg, 94%) as an oil.
1H NMR δ: 0.66 (s, 3H, 18-Me), 0.76 (s, 3H, 19-Me), 0.84
(d, 3H, J = 6.5 Hz, 21- or 28-Me), 0.96 (d, 3H, J = 6.5 Hz,
2.16. (22S,24R)-[26,27-2H6]2α,3α,22-Trihydroxy-24-
methyl-5α-cholestan-6-one (22)
OsO4 (33 mg, 13 mmol) was added to a solution of enone
20 (40 mg, 0.086 mmol) in pyridine (0.8 ml). The mixture
was stirred at room temperature for 1 h and then, treated with
a solution of Na2SO3 (0.2 g), H2SO4 (0.04 ml), and water

1106
V.A. Khripach et al. / Steroids 67 (2002) 1101–1108
28- or 21-Me), 2.04 (s, 3H, –CH3–CO–), 3.60 (m, 1H,
C3–H), 3.92 (br.s, 1H, –OH), 5.00–5.12 (m, 1H, C22–H).
13C NMR δ: 11.82, 12.54, 13.13, 15.49, 20.68, 21.30, 21.53,
23.93, 27.97, 29.91, 30.58, 31.65, 34.89, 35.74, 36.68, 37.93,
38.19, 39.46, 40.94, 42.89, 46.62, 52.53, 53.90, 56.62, 56.77,
70.64, 74.92, 170.69, 210.98. HRMS calc. for C28H40D6O2:
420.387442. Found: 420.388942. EI-MS m/z (%): 95 (53),
247 (12), 261 (16), 288 (100), 315 (80), 330 (7), 360 (5),
405 (10), 420 (68) [M − AcOH]+, 462 (4) [M − H2O]+,
22␣-hydroxy group and carbon side chain characteristic of
brassinolide has not been reported before.
In this paper, we describe preparation of [26,27-2H6]23-
dehydroxybrassinosteroids as depicted in the retrosynthetic
Scheme 2. Synthesis of related [26,27-2H6] sterols has been
reported [16,17], but none of these methods provided a
2
facile entry into H-labeled BS. The standard approach to
22␣-alcohols is reaction of organometallic reagents with
22C-aldehydes (1) [18,19]. However, it suffers from the for-
mation of a considerable amount (up to 40%) of isomeric
22␤-alcohol. In this respect, better results were anticipated
by the hydride reduction of 22-ketones [18]. Formation of
the desired stereochemistry at C24 required the use of a
chiral synthetic building block. Our approach made use of
(2R)-3-hydroxy-2-methylpropanoate (2) [20].
Synthesis of the C23–C28 fragment of the side chain was
performed as depicted in Scheme 3. The hydroxy group in 2
was protected as a tetrahydropyranyl ether, and via a num-
ber of steps, the carbomethoxy group was transformed into a
methylene phenylsulfone moiety. Introduction of deuterium
was achieved by twice repeated treatment of the sulfone 6
with butyl lithium and [2H3]methyl iodide. At this stage, the
necessary carbon skeleton was prepared, and efforts were
directed toward formation of functionality, which was re-
quired for the coupling of the low-molecular fragment to the
steroidal aldehyde.
Sulfones are known to be of great synthetic utility in
carbon–carbon bond forming reactions in the construction
of steroidal side chains [19], including those characteristic
of BS [20–22]. The same procedure was used for introduc-
tion of another phenylsulfone group. However, prior to the
oxidation of the sulfide 11, the phenylsulfone group at C25
had to be removed. This was achieved by treatment of 10
with magnesium in methanol.
480 (2) [M]•+
.
2.19. (22S,24R)-[26,27-2H6]3β,22-Dihydroxy-24-
methyl-5α-cholestan-6-one (25)
Monoacetate 24 (8 mg, 16.6 ␮mol) was treated with a
solution of KOH in MeOH (5%, 5 ml) and heated at 45 ◦C
for 5 h. The mixture was neutralized by the addition of
AcOH, and the solvent was evaporated. The residue was
chromatographed on SiO2 with petroleum ether–EtOAc
1
(5:1 ⇒ 1:1) to give the diol 25 (6 mg, 82%) as an oil. H
NMR δ: 0.72 (s, 3H, 18-Me), 0.78 (s, 3H, 19-Me), 0.84 (d,
3H, J = 6 Hz, 21- or 28-Me), 0.92 (d, 3H, J = 6.5 Hz, 28-
or 21-Me), 3.62 (m, 1H, C3–H), 3.72–3.86 (m, 1H, C22–H).
13C NMR δ: 11.28, 12.02, 13.19, 15.91, 21.63, 23.98,
27.70, 29.72, 30.13, 30.81, 31.66, 35.34, 36.79, 38.04,
39.49, 39.64, 40.97, 42.99, 46.73, 52.67, 54.04, 56.78,
56.88, 70.73, 71.76, 210.63. HRMS calc. for C28H40D6O2:
420.387442. Found: 420.386767. EI-MS m/z (%): 95 (33),
139 (29), 248 (21), 285 (21), 300 (24), 318 (100), 347 (6),
420 (10) [M − H2O]+, 438 (3) [M]•+
.
3. Results and discussion
Construction of the necessary side chain was performed as
depicted in Scheme 4. Addition of the lithium salt of sulfone
12 to the aldehyde 1, prepared in five steps from stigmas-
terol [23,24], gave a mixture of hydroxy sulfones 13. It has
been shown [25] that a similar reaction produced a mixture
of all possible isomers at C22 and C23. Taking into account
that the total amount of 22␣-alcohols was rather substantial,
removal of phenylsulfonyl group at this stage seemed to be
reasonable. However, all attempts of desulfurization of 13
failed, and the only isolated product was the ꢀ22-derivative,
corresponding to a Julia olefination. Conversely, a similar
Preparation of BS analogs containing a hydroxy group
only at C22 in the side chain instead of the 22R,23R-diol
function has been described earlier. These experiments
were aimed at the synthesis of more simple derivatives than
natural BS for practical purposes [8], for structure–activity
relationship [9–11] and biosynthetic [12,13] studies, and
to develop synthetic methodologies for steroidal side chain
synthesis [14]. However, control over the substituent at
C24 was exercised [15] only in one study. It is known that
most natural BS contain a 24␣-methyl group that is essen-
tial for biological activity. Synthesis of compounds with a
Scheme 2.

V.A. Khripach et al. / Steroids 67 (2002) 1101–1108
1107
Scheme 3.
Scheme 5.
lem was solved using Swern oxidation. Desulfurization of
14 with aluminum amalgam gave the desired 22-ketone 15
without epimerization of the adjacent chiral center. Hydride
reduction of the ketone 15 led to the 22␣-alcohol 16. This
gave the required side chain, and for further transformations
in the cyclic skeleton, the hydroxy group was protected as
the acetate.
Scheme 4.
Construction of the required cyclic moiety was accom-
plished according to previously reported procedures [28] for
the synthesis of [26-2H3]BS (Scheme 5). Hydrobromic acid
assisted cyclopropane ring opening in 18 followed by de-
hydrobromination of 19 gave the ꢀ2-olefin 20, which was
reaction is well known for keto sulfones [26,27]. The initial
attempt to oxidize 13 with CrO3 in pyridine into the corre-
sponding ketone gave poor results; the reaction proceeded
slowly (2–3 days), and the yield of 14 was low. The prob-
Scheme 6.

1108
V.A. Khripach et al. / Steroids 67 (2002) 1101–1108
further subjected to hydroxylation to give the 2␣,3␣-diol 21.
Upon treatment with base, compound 21 gave the deuterated
23-dehydroxy analog of castasterone 22.
Treatment of 18 with acetic acid in the presence of sulfu-
ric acid gave the diacetate 23 (Scheme 6). Stepwise saponi-
fication gave the monoacetate 24 and then the 23-dehydroxy
analog of teasterone 25.
In conclusion, a new method for preparation of cathas-
terone side chain have been developed. Two new brassinolide
biosynthetic intermediates (as hexadeuterated derivatives
22 and 25) have been synthesized. The obtained com-
pounds are considered as effective tools for elucidation of
alternative subpathways in brassinolide biosynthesis.
[9] Voigt B, Bruhn C, Wagner C, Merzweiler K, Adam G. Synthesis
of 24-epicathasterone and related new brassinosteroids. Proc Plant
Growth Regul Soc Am 1997;24:138–9.
[10] Voigt B, Porzel A, Bruhn C, Wagner C, Merzweiler K, Adam G.
Synthesis of 24-epicathasterone and related brassinosteroids with
modified side chain. Tetrahedron 1997;53:17039–54.
[11] Jin F, Xu Y, Huang W. 2,2-Difluoro enol silyl ethers: convenient
preparation and application to the synthesis of novel fluorinated
brassinosteroids. J Chem Soc, Perkin Trans I 1993:795–9.
[12] Takatsuto S, Kuriyama H, Noguchi T, Suganuma H, Fujioka S,
Sakurai A. Synthesis of cathasterone and its related putative inter-
mediates in brassinolide biosynthesis. J Chem Res Synopsis 1997:
418–9.
[13] Takatsuto S, Watanabe T, Gotoh C, Kuriyama H, Noguchi T, Fujioka
S. A convenient synthesis of (22S)-22-hydroxycampesterol and some
related steroids. J Chem Res Synopsis 1998:176–7.
[14] Werner F, Parmentier G, Luu B, Dinan L. A convenient stereo-
selective synthesis of castasterone and its analogs using arsenic
ylides. Tetrahedron 1996;52:5525–32.
Acknowledgments
[15] Nagano H, Matsuda M, Yajima T. Stereoselective synthesis of 24-
alkyl-22-hydroxy sterols based on chelation-controlled radical reac-
tions. J Chem Soc, Perkin Trans I 2001:174–82.
[16] Kirk DN, Varley MJ, Makin HLJ, Trafford DJH. Synthesis of
(26,27-2H6)cholesterol and derivatives substituted in the side chain.
J Chem Soc, Perkin Trans I 1983:2563–7.
[17] Holm T, Crossland I. A simple preparation of hexadeuteriocholes-
terol. J Labelled Compd Radiopharm 1996;38:803–8.
[18] Piatak DM, Wicha J. Various approaches to the construction of
aliphatic side chains of steroids and related compounds. Chem Rev
1978;78:199–241.
We thank Dr. N.B. Khripach and E.V. Skorodumov
(Minsk) for recording the NMR spectra, Dr. N.J. Oldham
(Jena) for the high resolution mass measurements, and
Dr. D.J. Fowler (Jena) for linguistic support in the prepa-
ration of this manuscript. The authors are grateful to
the Belorussian Foundation for Fundamental Research
and the Deutsche Forschungsgemeinschaft for financial
support.
[19] Zhabinskii VN, Olkhovick VK, Khripach VA. Methods of stereo-
selective construction of steroidal side chains. Zhurn Org Khim
1996;32:327–63.
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