Synthesis of fatty acyl derivatives of 24-epibrassinolide

Article abstract of DOI:10.1016/j.jsbmb.2013.01.016

A number of fatty acid (palmitic, myristic and lauric) esters (both 3α- and 3β-isomers) of epibrassinolide has been prepared as reference compounds for metabolic studies. Selective protection of the three of four hydroxyl groups of epibrassinolide was successively performed first as cyclic 22,23-methylboronates and then as 2α-benzyl ethers. α,β- Inversion of C-3 hydroxyl group was achieved through a consecutive oxidation-reduction reactions or by a nucleophilic substitution of the 3α-mesylates. Treatment of the 3α- and 3β-alcohols with palmitic, myristinic or lauric acid chlorides gave the corresponding esters. The hydrolysis of 22,23-methylboronates was performed after their transformation into 2-hydroxy-1,3,2-dioxaborolanes using a cation exchange column with DOWEX 50WX8 in NH₄⁺ form. Hydrogenolysis of the benzyl ethers catalyzed by palladium yielded the target compounds. This article is part of a Special Issue entitled Synthesis and biological testing of steroid derivatives as inhibitors .

Full text of DOI:10.1016/j.jsbmb.2013.01.016

Journal of Steroid Biochemistry & Molecular Biology 137 (2013) 345–354
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry and Molecular Biology
journal homepage: www.elsevier.com/locate/jsbmb
Synthesis of fatty acyl derivatives of 24-epibrassinolide
Vladimir A. Khripach∗, Vladimir N. Zhabinskii, Dmitrii V. Tsavlovskii
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevich str., 5/2, 220141 Minsk, Belarus
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of fatty acid (palmitic, myristic and lauric) esters (both 3␣- and 3␤-isomers) of epibrassinolide
has been prepared as reference compounds for metabolic studies. Selective protection of the three of four
hydroxyl groups of epibrassinolide was successively performed first as cyclic 22,23-methylboronates
and then as 2␣-benzyl ethers. ␣,␤-Inversion of C-3 hydroxyl group was achieved through a consecutive
oxidation-reduction reactions or by a nucleophilic substitution of the 3␣-mesylates. Treatment of the
3␣- and 3␤-alcohols with palmitic, myristinic or lauric acid chlorides gave the corresponding esters. The
hydrolysis of 22,23-methylboronates was performed after their transformation into 2-hydroxy-1,3,2-
Received 30 October 2012
Received in revised form 15 January 2013
Accepted 31 January 2013
Keywords:
Brassinosteroids
Epibrassinolide metabolites
Fatty acid esters
Cyclic methylboronates
+
dioxaborolanes using a cation exchange column with DOWEX 50WX8 in NH4 form. Hydrogenolysis of
the benzyl ethers catalyzed by palladium yielded the target compounds.
This article is part of a Special Issue entitled ‘Synthesis and biological testing of steroid derivatives as
inhibitors’.
© 2013 Elsevier Ltd. All rights reserved.
1. Introduction
and, namely, in its selective modifications with fatty acids mim-
icking metabolic pathways. Therefore, the task of the present work
Since the isolation of the first steroidal plant hormone brassino-
lide in 1979 [1], a lot of knowledge has been accumulated on the
biosynthesis of this class of phytohormones called brassinosteroids
[2–5]. Relatively little, however, is known about their metabolic
fate. At least in part it is because of poor availability of brassi-
nosteroid metabolites (real or supposed), which could be used as
reference compounds in biochemical studies.
was the preparation of epibrassinolide metabolites (including sup-
posed ones), having a fatty acid ester moiety at C-3, for subsequent
metabolic studies.
2. Experimental
2.1. General
Esterification is one of the metabolic processes occurring with
brassinosteroids [6] that contribute to their homeostasis. A num-
ber of fatty acid conjugates 1a–c and 2a–c (Fig. 1) were identified
on exogenous application of epicastasterone and epibrassinolide
to cell suspension culture of Ornithopus sativus [7]. The acyl com-
ponents for both brassinosteroid conjugate types were found to
be palmitic, myristic and lauric acids. Teasterone myristate 3a [8]
and teasterone laurate 3b [9] were identified in studies of lily cell
cultures. These acyl conjugates were supposed to be the reversible
teasterone storage forms during the biosynthesis of brassinolide
[10]. BAHD acyltransferase-like protein may be involved in transfer
of the acyl groups to brassinosteroids in Arabidopsis [11].
Melting points were recorded on a Boetius micro-melting
point apparatus and are uncorrected. 1H and 13C NMR spec-
tra were obtained using a Bruker AVANCE 500 (Bruker Biospin,
Rheinstetten, Germany) spectrometer in CDCl3 (if not stated oth-
erwise) operating at 500 MHz for 1H and 125 MHz for 13C. 1H
chemical shifts were determined relative to the residual solvent
peaks (CHCl3, ı 7.26; C5D5N, ı 7.58), and coupling constants
are reported in Hz. Carbon spectra were referenced internally
to solvent signals, using values ı 77.00 for CDCl3 and ı 135.91
for C5D5N. Mass spectra were performed on a LCQ Fleet mass
spectrometer (Thermo Electron Corporation, USA) with an APCI
or ESI source. Spectra were collected in a positive ion mode
and analyzed by Xcalibur software. Chemicals were purchased
from Aldrich and Fluka and used as received unless otherwise
noted. All solvents were purified according to standard meth-
ods [13]. Reactions were monitored by TLC using aluminium or
plastic sheets, silica gel 60 F254 precoated (Merck Art. 5715). Col-
umn chromatography was carried out on Kieselgel 60 (Merck Art.
7734).
Since bringing epibrassinolide into agricultural practice as crop-
yield-increasing and plant-protecting agent [2,12], we became
interested in studying all aspects of this steroidal phytohormone
∗
Corresponding author. Tel.: +375 172 678 647; fax: +375 172 678 647.
E-mail address: khripach@iboch.bas-net.by (V.A. Khripach).
0960-0760/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jsbmb.2013.01.016

346
V.A. Khripach et al. / Journal of Steroid Biochemistry & Molecular Biology 137 (2013) 345–354
Fig. 1. Structures of natural fatty acid derivatives of brassinosteroids: 3␤-palmitate- (1a), 3␤-myristate- (1b) and 3␤-laurate- (1c) of 3,24-bisepicastasterone; 3␤-palmitate-
(2a), 3␤-myristate- (2b) and 3␤-laurate- (2c) of 3,24-bisepibrassinolide; 3␤-myristate- (3a) and 3␤-laurate- (3b) of teasterone.
2.2. Synthesis of compounds
24.93, 27.07, 27.73, 29.10, 29.23, 29.33, 29.45, 29.66, 30.82, 31.90,
34.43, 37.74, 38.41, 39.17, 39.25, 40.97, 41.06, 42.40, 44.35, 51.08,
52.23, 58.11, 66.46, 70.40, 71.36, 81.94, 82.36, 172.83, 175.83. MS
(APCI+) m/z (%): 743.4 ([M+H]+, 100).
2.2.1. (22R,23R,24R)-2˛-(tert-Butyldimethylsilyl)-3˛-palmitoyl-
22,23-dihydroxy-24-methyl-B-homo-7-oxa-5˛-cholestan-6-one
22,23-methylboronate (7)
(22R,23R,24R)-2˛-Palmitoyl-3˛,22,23-trihydroxy-24-methyl-B-
homo-7-oxa-5˛-cholestan-6-one 22,23-methylboronate 9 (10 mg,
A
mixture of the silyl ether 6 (150 mg, 0.24 mmol, pre-
pared according to [14]), palmitoyl chloride (158 ␮L, 142 mg,
0.52 mmol), N,N-dimethylaminopyridine (32 mg, 0.26 mmol), and
pyridine (0.9 mL) was stirred at room temperature overnight. Then
solvents were evaporated in vacuo and the residue was purified by
column chromatography on silica gel (toluene–EtOAc = 20:1 ⇒ 7:1)
to afford unreacted starting alcohol 6 (62 mg) and ester 7 (84 mg,
41%, 69% based on reacted starting material) as an oil. 1H NMR ı:
21%) as an oil. 1H NMR ı: 0.28 (s, 3H,
B CH3), 0.70 (d, J = 6.6 Hz,
3H, CHCH3), 0.70 (s, 3H, C18-H), 0.84 (d, J = 6.8 Hz, 3H, CHCH3),
0.85–0.90 (m, 6H, CH3), 0.92 (d, J = 6.9 Hz, 3H, CHCH3), 0.94
(s, 3H, C19-H), 2.39 (t, J = 7.3 Hz, 2H, OCOCH2C14H29), 2.96 (dd,
J = 12.3, 4.3 Hz, 1H, C5-H), 3.78 (dd, J = 8.8, 5.2 Hz, 1H, C23-H), 3.83
(ddd, J = 12.0, 4.0, 2.5 Hz, 1H, C3-H), 4.01–4.16 (m, 3H, C7- and
C22-H), 5.23 (br.s, 1H, C2-H). 13C NMR ı: 9.13, 11.45, 11.58, 14.11,
15.51, 16.44, 21.03, 22.19, 22.68, 24.78, 25.07, 27.10, 27.75, 29.15,
29.35, 29.53, 29.68, 31.91, 34.50, 38.25, 39.14, 39.23, 41.10, 42.05,
42.26, 42.44, 44.37, 51.10, 52.24, 58.41, 67.36, 70.58, 71.28, 81.96,
82.36, 174.57, 175.45. MS (APCI+) m/z (%): 743.2 ([M+H]+, 100).
0.03 (s, 3H, CH3 Si ), 0.05 (s, 3H, CH3 Si ), 0.28 (s, 3H,
B CH3),
0.69 (d, J = 6.6 Hz, 3H, CHCH3), 0.70 (s, 3H, C18-H), 0.80–0.94
(m, 21H), 1.21–1.29 (m, 26H, OCOCH2 (CH2)13 CH3), 2.34 (t,
J = 7.4 Hz, 2H, OCOCH2C14H29), 2.92 (dd, J = 12.2, 4.4 Hz, 1H, C5-H),
3.71 (ddd, J = 11.6, 4.0, 2.7 Hz, 1H, C2-H), 3.77 (dd, J = 8.7, 5.2 Hz, 1H,
C23-H), 4.03 (dd, J = 12.3, 9.3 Hz, 1H, 1H, C7-H␣), 4.07–4.15 (m, 2H,
C7- and C22-H), 5.21 (br.s, 1H, C3-H). 13C NMR ı: −4.95, −4.86, 9.12,
11.43, 11.57, 14.10, 15.66, 16.43, 18.14, 21.02, 22.20, 22.67, 24.78,
25.17, 25.76, 27.09, 27.72, 29.17, 29.34, 29.40, 29.52, 29.67, 31.90,
34.58, 38.22, 39.13, 39.26, 41.06, 41.95, 42.42, 43.30, 44.36, 51.06,
52.26, 58.33, 67.41, 70.11, 70.52, 81.95, 82.35, 172.88, 175.67. MS
(APCI+) m/z (%): 857.4 ([M+H]+, 100).
2.2.3. Benzylation of (22R,23R,24R)-2˛,3˛,22,23-tetrahydroxy-
24-methyl-B-homo-7-oxa-5˛-cholestan-6-one
22,23-methylboronate (5) (synthesis of compounds (10–12))
Variant A.
A mixture of diol 5 (146 mg, 0.29 mmol, pre-
pared according to [14]), benzyl bromide (44.2 ␮L, 0.063 mg,
0.37 mmol) and NaH (11 mg, 0.46 mmol) in dry THF (1.5 mL)
was stirred at 0 ◦C for 2 h. Then it was diluted with satu-
rated NH4Cl (2 mL) and extracted with EtOAc (3× 5 mL). The
combined organic layers were dried (Na2SO4), and concen-
trated. The residue was purified by column chromatography
on silica gel (toluene–EtOAc = 10:1 ⇒ 1:2) to give dibenzyl com-
pound 12 (14 mg, 7%), monobenzyl compound 11 (10 mg, 6%),
monobenzyl compound 10 (22 mg, 13%) and starting diol 5
(94 mg, 64%).
2.2.2. Desilylation of
(22R,23R,24R)-2˛-(tert-butyldimethylsilyl)-3˛-palmitoyl-22,23-
dihydroxy-24-methyl-B-homo-7-oxa-5˛-cholestan-6-one
22,23-methylboronate (7) (synthesis of compounds (8) and (9))
Silyl ether 7 (54 mg, 0.063 mmol) was dissolved in 1 M solu-
tion of TBAF in THF (540 ␮L, 0.54 mmol), the reaction mixture was
stirred at room temperature overnight, and then worked up with
saturated solution of NH4Cl (1 mL). Aqueous layer was extracted
with EtOAc (3× 1 mL). The organic layer was dried (Na2SO4) and
evaporated. The residue was chromatographed on silica gel (petrol
ether-EtOAc = 10:1 ⇒ 7:3) to give compounds 8 and 9.
(22R,23R,24R)-2˛,3˛-Dibenzyloxy-22,23-dihydroxy-24-methyl-
B-homo-7-oxa-5˛-cholestan-6-one
22,23-methylboronate
12
(14 mg, 7%) as an oil. 1H NMR ı: 0.28 (s, 3H,
B
CH3), 0.69
(s, 3H, C18-H), 0.70 (d, J = 6.9 Hz, 3H, CHCH3), 0.84 (d, J = 6.8 Hz,
3H, CHCH3), 0.88 (s, 3H, C19-H), 0.89 (d, J = 6.8 Hz, 3H, CHCH3),
0.92 (d, J = 6.9 Hz, 3H, CHCH3), 3.07 (dd, J = 12.0, 4.5 Hz, 1H, C5-H),
3.43 (ddd, J = 12.2, 4.2, 2.2 Hz, 1H, C2-H), 3.78 (dd, J = 8.8, 5.2 Hz,
1H, C23-H), 3.94 (br.s, 1H, C3-H), 3.99–4.09 (m, 2H, C7-H), 4.13
(d, J = 4.9 Hz, 1H, C22-H), 4.47 (d, J = 11.9 Hz, 1H, CH2Ph), 4.55 (d,
J = 11.9 Hz, 1H, CH2Ph), 4.65 (d, J = 12.4 Hz, 1H, CH2Ph), 4.72 (d,
J = 12.4 Hz, 1H, CH2Ph), 7.26–7.41 (m, 10H, Ph). 13C NMR ı: 9.14,
11.44, 11.49, 11.59, 15.73, 16.47, 21.04, 22.21, 24.78, 27.11, 27.78,
29.53, 38.18, 39.17, 39.25, 40.09, 41.14, 41.99, 42.44, 44.37, 51.03,
52.03, 52.21, 58.10, 70.38, 70.50, 71.44, 71.84, 76.13, 81.96, 82.36,
(22R,23R,24R)-3˛-Palmitoyl-2˛,22,23-trihydroxy-24-methyl-B-
homo-7-oxa-5˛-cholestan-6-one 22,23-methylboronate 8 (24 mg,
51%) as an oil. 1H NMR ı: 0.27 (s, 3H,
B CH3), 0.69 (s, 3H, C18-H),
0.69 (d, J = 6.3 Hz, 3H, CHCH3), 0.83 (d, J = 6.8 Hz, 3H, CHCH3),
0.85–0.89 (m, 6H, CH3), 0.91 (d, J = 6.9 Hz, 3H, CHCH3), 0.96 (s,
3H, C19-H), 1.21–1.29 (m, 26H, OCOCH2 (CH2)13 CH3), 2.31
(t, J = 7.4 Hz, 2H, OCOCH2C14H29), 3.16 (dd, J = 12.2, 4.3 Hz, 1H,
C5-H), 3.77 (dd, J = 8.7, 5.2 Hz, 1H, C23-H), 4.03–4.15 (m, 4H, C2-,
C22- and C7-H), 4.84 (ddd, J = 12.2, 4.3, 2.2 Hz, 1H, C3-H). 13C NMR
ı: 9.11, 11.42, 11.57, 14.10, 15.30, 16.42, 21.02, 22.21, 22.66, 24.75,

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347
127.47, 127.52, 127.57, 128.38, 139.04, 176.38. MS (APCI+) m/z (%):
685.5 ([M+H]+, 100).
CHCH3), 2.15 (d, J = 8.3 Hz, 2H), 2.25 (dq, J = 10.8, 6.9 Hz, 1H), 2.32
(dt, J = 14.8, 4.1 Hz, 1H), 2.53 (t, J = 13.5 Hz, 1H), 3.62 (dd, J = 12.0,
4.2 Hz, 1H, C5-H), 3.96 (dd, J = 9.0, 4.7 Hz, 1H), 4.16–4.01 (m, 3H),
4.30 (d, J = 4.4 Hz, 1H), 4.44 (s, 1H). 13C NMR (C5D5N) ı: 9.62, 12.03,
12.27, 16.27, 16.88, 21.51, 22.86, 25.30, 27.88, 28.48, 33.52, 38.94,
40.08, 42.11, 42.28, 42.95, 43.17, 45.24, 51.65, 53.26, 58.66, 68.82,
69.14, 70.63, 81.42, 82.07, 177.02.
(22R,23R,24R)-3˛-Benzyloxy-2˛,22,23-trihydroxy-24-methyl-B-
homo-7-oxa-5˛-cholestan-6-one 22,23-methylboronate 11 (10 mg,
6%) as an oil. 1H NMR ı 0.28 (s, 3H,
B CH3), 0.69 (s, 3H, C18-H),
0.70 (d, J = 6.8 Hz, 3H, CHCH3), 0.84 (d, J = 6.8 Hz, 3H, CHCH3),
0.88 (d, J = 6.6 Hz, 3H, CHCH3), 0.91 (d, J = 6.8 Hz, 3H, CHCH3),
0.92 (s, 3H, C19-H), 2.95 (dd, J = 12.0, 4.5 Hz, 1H, C5-H), 3.62 (ddd,
J = 12.3, 4.5, 3.2 Hz, 1H, C2-H), 3.78 (dd, J = 8.8, 5.0 Hz, 1H, C23-H),
4.00 (dd, J = 12.3, 9.3 Hz, 1H, C3-H), 4.05–4.12 (m, 2H, C7-H), 4.13
(d, J = 5.0 Hz, 1H, C22-H), 4.49 (d, 1H d, J = 11.9 Hz, 1H, CH2Ph),
4.66 (d, J = 11.9 Hz, 1H, CH2Ph), 7.29–7.42 (m, 5H, Ph). 13C NMR
ı: 9.13, 10.74, 11.44, 11.56, 15.36, 16.45, 21.03, 22.15, 24.78, 27.11,
27.75, 28.31, 38.37, 39.16, 39.27, 41.10, 41.55, 42.43, 43.26, 44.37,
51.06, 52.24, 58.16, 67.83, 70.56, 71.13, 75.72, 81.96, 82.37, 127.60,
127.91, 128.57, 176.14. MS (APCI+) m/z (%): 595.7 ([M+H]+, 100).
(22R,23R,24R)-2˛-Benzyloxy-3˛,22,23-trihydroxy-24-methyl-B-
homo-7-oxa-5˛-cholestan-6-one 22,23-methylboronate 10 (22 mg,
13%). Mp 124–126 ◦C (ether-hexane). 1H NMR ı: 0.27 (s, 3H,
2.2.5. (22R,23R,24R)-2˛-Benzyloxy-3˛-palmitoyloxy-22,23-
dihydroxy-24-methyl-B-homo-7-oxa-5˛-cholestan-6-one
22,23-methylboronate (14a)
Variant A. A mixture of the monobenzyl ether 10 (60 mg,
0.10 mmol), palmytoyl chloride (58 ␮L, 53 mg, 0.19 mmol), N,N-
dimethylaminopyridine (3 mg, 0.025 mmol), and pyridine (2 mL)
was stirred at room temperature for 40 h. Then solvents were
evaporated in vacuo and the residue was purified by column chro-
matography on silica gel (toluene–EtOAc = 20:1 ⇒ 12:1) to afford
ester 14a (73 mg, 87%) as an oil. 1H NMR ı: 0.27 (s, 3H,
B CH3), 0.70
(s, 3H, C18-H), 0.70 (d, J = 6.2 Hz, 3H, CHCH3), 0.84 (d, J = 6.8 Hz,
3H, CHCH3), 0.88 (t, J = 6.8 Hz, 3H, OCO (CH2)14 CH3), 0.88 (d,
J = 6.6 Hz, 3H, CHCH3), 0.91 (s, 3H, C19-H), 0.92 (d, J = 6.7 Hz, 3H,
CHCH3), 1.21–1.29 (m, 26H, OCOCH2 (CH2)13 CH3), 2.37 (t,
J = 7.4 Hz, 2H, OCOCH2C14H29), 2.98 (dd, J = 12.2, 4.4 Hz, 1H, C5-
H), 3.46 (ddd, J = 12.2, 4.2, 2.7 Hz, 1H, C2-H), 3.78 (dd, J = 8.7, 5.2 Hz,
1H, C23-H), 4.05 (dd, J = 12.4, 9.3 Hz, 1H, C7-H␣), 4.10 (br.s, 1H, C7-
H␤), 4.13 (d, J = 5.1 Hz, 1H, C22-H), 4.41 (d, J = 11.3 Hz, 1H, CH2Ph),
4.71 (d, J = 11.3 Hz, 1H, CH2Ph), 5.56 (br.s, 1H, C3-H), 7.27–7.36 (m,
5H, Ph). 13C NMR ı: 9.14, 11.45, 11.59, 14.10, 15.68, 16.47, 21.03,
22.23, 22.68, 24.78, 25.15, 27.12, 27.76, 29.14, 29.35, 29.39, 29.52,
29.69, 31.91, 34.55, 38.19, 39.21, 40.77, 41.12, 42.24, 42.45, 44.38,
51.10, 52.23, 58.50, 66.02, 70.43, 70.55, 73.55, 81.96, 82.34, 127.68,
127.86, 128.39, 138.01, 173.03, 175.57. MS (APCI+) m/z (%): 833.5
([M+H]+, 100).
Variant B. A mixture of palmitic acid (181 mg, 0.71 mmol) and
DCC (227 mg, 1.1 mmol) in dry CH2Cl2 (2 mL) was stirred at room
temperature for 20 min. Then it was filtered through a cotton
plug to remove a small amount of suspended matter and poured
into a solution of the monobenzyl ether 10 (168 mg, 0.28 mmol)
in dry CH2Cl2 (3 mL). The mixture was stirred at room tem-
perature for 48 h, and then evaporated till the final volume of
2 mL. The residue was diluted with EtOAc (5 mL) and the result-
ing mixture was filtered through a silica gel pad, concentrated
in vacuo, and purified by flash silica gel column chromatography
(toluene–EtOAc = 20:1 ⇒ 12:1). Fractions containing the ester 14a
were combined and evaporated, the residue was dissolved in EtOAc
(2 mL) and left in refrigerator for 30 min. The mixture was filtered
through a Schott filter to remove precipitated contaminants and
filtrate was evaporated to give the ester 14a (226 mg, 89%) as an
oil.
B
CH3), 0.69 (s, 3H, C18-H), 0.70 (d, J = 6.2 Hz, 3H, CHCH3), 0.84
(d, J = 6.8 Hz, 3H, CHCH3), 0.88 (s, 3H, C19-H), 0.92 (d, J = 6.9 Hz, 3H,
CHCH3), 3.15 (dd, J = 11.9, 4.3 Hz, 1H, C5-H), 3.45 (d, J = 11.0 Hz,
1H, C2-H), 3.78 (dd, J = 8.3, 5.0 Hz, 1H, C23-H), 4.08 (m, 2H, C7-H),
4.13 (d, J = 5.0 Hz, 1H, C22-H), 4.17 (s, 1H, C3-H), 4.55 (d, J = 11.6 Hz,
1H, CH2Ph), 4.60 (d, J = 11.6 Hz, 1H, CH2Ph), 7.39–7.28 (m, 5H,
Ph). 13C NMR ı: 9.14, 11.44, 11.60, 15.54, 16.46, 21.03, 22.23, 24.77,
27.11, 27.75, 30.32, 38.13, 38.91, 39.25, 39.28, 41.04, 41.12, 42.44,
44.37, 51.11, 52.22, 58.21, 65.18, 70.36, 70.47, 75.44, 81.96, 82.35,
127.64, 127.93, 128.57, 138.01, 176.20. MS (APCI+) m/z (%): 595.2
([M+H]+, 100).
Variant B. A mixture of diol 5 (100 mg, 0.20 mmol), freshly
prepared silver oxide (69 mg, 0.30 mmol), and benzyl bromide
(47 ␮L, 0.067 mg, 0.39 mmol) in dry CH2Cl2 (2 mL) was stirred at
room temperature for 4 h. Then it was filtered through a short
pad of silica gel followed by washing with CH2Cl2 (5 mL) and
EtOAc (5 mL). After concentration under vacuum, the crude product
was separated into four components by column chromatogra-
phy on silica gel (toluene–EtOAc = 10:1 ⇒ 1:2) to give dibenzyl
compound 12 (10 mg, 7%), monobenzyl compound 11 (23 mg,
19%), monobenzyl compound 10 (67 mg, 57%) and starting diol 5
(12 mg, 12%).
Variant C. A solution of diol 5 (210 mg, 0.42 mmol) and dibutyl tin
oxide (130 mg, 0.52 mmol) in dry toluene (30 mL) was refluxed in
a round bottomed flask equipped with a Dean-Stark apparatus for
20 h. Then toluene (20 mL), tetrabutylammonium bromide (80 mg,
0.25 mmol), and benzyl bromide (74 ␮L, 0.11 mg, 0.62 mmol) were
added and reflux was continued for 2 h. After cooling to room
temperature, solvents were removed by rotary evaporation. The
residue was separated into two components by column chro-
matography on silica gel (toluene–EtOAc = 10:1 ⇒ 1:2) to give
monobenzyl compound 11 (28 mg, 11%) and monobenzyl com-
pound 10 (193 mg, 78%).
2.2.6. (22R,23R,24R)-2˛-Benzyloxy-3˛-myristoyloxy-22,23-
dihydroxy-24-methyl-B-homo-7-oxa-5˛-cholestan-6-one
22,23-methylboronate (14b)
2.2.4. (22R,23R,24R)-2˛,3˛,22,23-Tetrahydroxy-24-methyl-B-
homo-7-oxa-5˛-cholestan-6-one 22,23-boronate
(13)
The title compound (73 mg) was prepared in 88% yield as an oil
from the monobenzyl ether 10 and myristoyl chloride as described
above for the preparation of the ester 14a (variant A). 1H NMR
A
solution of methylboronate
5
(98 mg, 0.19 mmol) and
ı: 0.27 (s, 3H,
B CH3), 0.70 (s, 3H, C18-H), 0.70 (d, J = 6.4 Hz,
trimethylamine N-oxide dihydrate (65 mg, 0.59 mmol) in diglyme
(8 mL) was stirred under reflux for 45 min. The mixture was cooled
to ambient temperature and the solvents were evaporated in vacuo.
The residue was purified by column chromatography on silica gel
(CHCl3–MeOH = 40:1 ⇒ 10:1) to give epibrassinolide 4 (2 mg, 2%)
and boronate 13 (95 mg, 96%). Mp 300 ◦C (ether, decomp). 1H NMR
(C5D5N) ı: 0.64 (s, 3H, C18-H), 0.74 (d, J = 6.9 Hz, 3H, CHCH3), 0.91
(d, J = 6.9 Hz, 6H, CHCH3), 1.06 (s, 3H, C19-H), 1.10 (d, J = 6.5 Hz, 3H,
3H, CHCH3), 0.84 (d, J = 6.8 Hz, 3H, CHCH3), 0.88 (t, J = 6.8 Hz,
3H, OCO (CH2)12 CH3), 0.88 (d, J = 6.6 Hz, 3H, CHCH3), 0.91 (s,
3H, C19-H), 0.92 (d, J = 6.8 Hz, 3H, CHCH3), 1.23–1.27 (m, 22H,
OCOCH2 (CH2)11 CH3), 2.37 (t, J = 7.4 Hz, 2H, OCOCH2C12H25),
2.99 (dd, J = 12.2, 4.4 Hz, 1H, C5-H), 3.46 (ddd, J = 12.1, 3.9, 2.6 Hz, 1H,
C2-H), 3.78 (dd, J = 8.7, 5.2 Hz, 1H, C23-H), 4.05 (dd, J = 12.3, 9.5 Hz,
1H, C7-H␣), 4.10 (br.s, 1H, C7-H␤), 4.13 (d, J = 5.1 Hz, 1H, C22-H),
4.41 (d, J = 11.3 Hz, 1H, CH2Ph), 4.71 (d, J = 11.3 Hz, 1H, CH2Ph),

348
V.A. Khripach et al. / Journal of Steroid Biochemistry & Molecular Biology 137 (2013) 345–354
5.56 (br.s, 1H, C3-H), 7.27–7.35 (m, 5H, Ph). 13C NMR ı: 9.14, 11.45,
11.59, 14.10, 15.68, 16.46, 21.03, 22.22, 22.67, 24.77, 25.15, 27.11,
27.75, 29.14, 29.38, 29.51, 29.67, 31.90, 34.55, 38.19, 39.20, 40.76,
41.12, 42.23, 42.44, 44.37, 51.09, 52.22, 58.49, 66.01, 70.43, 70.55,
73.54, 81.95, 82.33, 127.68, 127.86, 128.39, 138.01, 173.03, 175.57.
MS (APCI+) m/z (%): 805.4 ([M+H]+, 100).
2.2.9. (22R,23R,24R)-2˛-Benzyloxy-3˛-myristoyloxy-22,23-
dihydroxy-24-methyl-B-homo-7-oxa-5˛-cholestan-6-one
(16b)
The title compound (41 mg) was prepared in 67% yield as an oil
from the methylboronate 14b as described above for the prepa-
ration of the diol 16a. 1H NMR ı: 0.70 (s, 3H, C18-H), 0.81–0.94
(m, 18H), 1.22–1.29 (m, 22H, OCOCH2 (CH2)11 CH3), 2.37 (m,
2H, OCOCH2C12H25), 2.99 (dd, J = 12.2, 4.3 Hz, 1H, C5-H), 3.41
(t, J = 5.1 Hz, 1H, C23-H), 3.46 (ddd, J = 12.4, 4.2, 2.6 Hz, 1H, C2-H),
3.68 (d, J = 3.5 Hz, 1H, C22-H), 4.01–4.08 (m, 1H, C7-H␣), 4.14 (dd,
J = 15.4, 5.2 Hz, 1H, C7-H␤), 4.40 (d, J = 11.3 Hz, 1H, CH2Ph), 4.71 (d,
J = 11.3 Hz, 1H, CH2Ph), 5.57 (s, 1H, C3-H), 7.27–7.35 (m, 5H, Ph).
13C NMR ı: 10.82, 11.63, 12.37, 14.12, 15.68, 17.27, 22.11, 22.24,
22.68, 24.74, 25.13, 27.00, 27.68, 29.13, 29.38, 29.51, 29.68, 31.91,
34.53, 38.17, 39.19, 39.45, 40.23, 40.74, 41.40, 42.21, 42.46, 47.04,
51.17, 52.53, 58.47, 65.99, 70.41, 70.56, 72.51, 73.53, 76.34, 127.67,
127.85, 128.38, 173.04, 175.61. MS (APCI+) m/z (%): 781.8 ([M+H]+,
100).
2.2.7. (22R,23R,24R)-2˛-Benzyloxy-3˛-lauroyloxy-22,23-
dihydroxy-24-methyl-B-homo-7-oxa-5˛-cholestan-6-one
22,23-methylboronate (14c)
The title compound (68 mg) was prepared in 87% yield as an oil
from the monobenzyl ether 10 and lauroyl chloride as described
above for the preparation of the ester 14a (variant A). 1H NMR
ı: 0.27 (s, 3H,
B CH3), 0.69 (s, 3H, C18-H), 0.70 (d, J = 6.7 Hz,
3H, CHCH3), 0.84 (d, J = 6.8 Hz, 3H, CHCH3), 0.88 (t, J = 6.7 Hz,
3H, OCO (CH2)10 CH3), 0.88 (d, J = 6.5 Hz, 3H, CHCH3), 0.91 (d,
J = 6.6 Hz, 3H, CHCH3), 0.91 (s, 3H, C19-H), 1.23–1.27 (m, 18H,
OCOCH2 (CH2)9 CH3), 2.37 (t, J = 7.4 Hz, 2H, OCOCH2C10H21),
2.98 (dd, J = 12.2, 4.4 Hz, 1H, C5-H), 3.46 (ddd, J = 12.2, 4.1, 2.7 Hz, 1H,
C2-H), 3.78 (dd, J = 8.7, 5.2 Hz, 1H, C23-H), 4.05 (dd, J = 12.3, 9.3 Hz,
1H, C7-H␣), 4.10 (br.s, 1H, C7-H␤), 4.13 (d, J = 5.1 Hz, 1H, C22-H),
4.40 (d, J = 11.3 Hz, 1H, CH2Ph), 4.71 (d, J = 11.3 Hz, 1H, CH2Ph),
5.57 (br.s, 1H, C3-H), 7.27–7.35 (m, 5H, Ph). 13C NMR ı: 9.12, 11.44,
11.58, 14.10, 15.67, 16.44, 21.02, 22.21, 22.66, 24.68, 24.77, 25.14,
27.09, 27.74, 29.04, 29.12, 29.22, 29.32, 29.37, 29.50, 29.57, 29.62,
31.89, 33.81, 34.54, 38.18, 39.18, 40.74, 41.11, 42.22, 42.43, 44.35,
51.07, 52.20, 58.46, 65.99, 70.42, 70.55, 73.53, 81.94, 82.33, 127.67,
127.86, 128.38, 137.97, 173.05, 175.61. MS (APCI+) m/z (%): 777.4
([M+H]+, 100).
2.2.10. (22R,23R,24R)-2˛-Benzyloxy-3˛-lauroyloxy-22,23-
dihydroxy-24-methyl-B-homo-7-oxa-5˛-cholestan-6-one
(16c)
The title compound (41 mg) was prepared in 65% yield as an
oil from the methylboronate 14c as described above for the prepa-
ration of the diol 16a. 1H NMR ı: 0.70 (s, 3H, C18-H), 0.83–0.93
(m, 12H), 0.91 (s, 3H, C19-H), 0.96 (d, J = 6.6 Hz, 3H, CHCH3),
1.22–1.29 (m, 18H, OCOCH2 (CH2)9 CH3), 2.37 (t, J = 7.5 Hz,
2H, OCOCH2C9H21), 2.98 (dd, J = 12.2, 4.4 Hz, 1H, C5-H), 3.40 (t,
J = 5.3 Hz, 1H, C23-H), 3.46 (ddd, J = 12.6, 4.2, 2.6 Hz, 1H, C2-H), 3.68
(d, J = 3.6 Hz, 1H, C22-H), 4.05 (dd, J = 12.3, 9.4 Hz, 1H, C7-H␣), 4.12
(d, J = 11.7 Hz, 1H, C7-H␤), 4.40 (d, J = 11.3 Hz, 1H, CH2Ph), 4.70 (d,
J = 11.3 Hz, 1H, CH2Ph), 5.56 (s, 1H, C3-H), 7.27–7.35 (m, 5H, Ph).
13C NMR ı: 10.81, 11.62, 12.36, 14.11, 15.67, 17.26, 22.11, 22.23,
22.67, 24.73, 25.13, 27.00, 27.67, 29.12, 29.36, 29.49, 29.62, 31.89,
34.53, 38.17, 39.18, 39.44, 40.23, 40.73, 41.40, 42.22, 42.45, 51.17,
52.52, 58.47, 66.00, 70.40, 70.55, 72.50, 73.52, 76.32, 127.66, 127.84,
128.38, 137.99, 173.04, 175.62. MS (APCI+) m/z (%): 753.6 ([M+H]+,
100).
2.2.8. (22R,23R,24R)-2˛-Benzyloxy-3˛-palmitoyloxy-22,23-
dihydroxy-24-methyl-B-homo-7-oxa-5˛-cholestan-6-one
(16a)
A mixture of 14a (65 mg, 0.078 mmol) and trimethylamine
N-oxide dihydrate (27 mg, 0.24 mmol) in diglyme (4 mL) was
stirred under reflux for 45 min. The mixture was cooled to
80 ◦C and evaporated. The residue was purified by column chro-
matography on silica gel (toluene–EtOAc = 10:1) to give an oily
product (56 mg) consisting of a mixture of 15a and 16a. It
was dissolved in EtOH (4 mL) and passed through a column of
2.2.11. (22R,23R,24R)-3˛-Palmitoyloxy-2˛,22,23-trihydroxy-24-
methyl-B-homo-7-oxa-5˛-cholestan-6-one
(17a)
+
Dowex-50W×8, 200–400 mesh, NH4 form resin. The column
A stirred solution of 16a (43 mg, 0.053 mmol) in EtOH (5 mL)
was hydrogenated over Pd/C (10%, 5 mg) at ambient temperature
for 20 h. Then the mixture was filtered through a pad of silica gel
and solvent was evaporated. The residue was chromatographed
on silica gel (toluene–EtOAc = 3:1 ⇒ 2:1) to give triol 17a (28 mg,
73%) as an oil. 1H NMR ı: 0.71 (s, 3H, C18-H), 0.85 (d, J = 7.0 Hz,
3H, CHCH3), 0.87 (d, J = 7.0 Hz, 3H, CHCH3), 0.87 (t, J = 6.9 Hz,
3H, OCO (CH2)14 CH3), 0.92 (d, J = 6.9 Hz, 3H, CHCH3), 0.94 (s,
3H, C19-H), 0.97 (d, J = 6.7 Hz, 3H, CHCH3), 1.22–1.29 (m, 26H,
OCOCH2 (CH2)13 CH3), 2.38 (t, J = 7.3 Hz, 2H, OCOCH2C14H29),
2.95 (dd, J = 12.2, 4.4 Hz, 1H, C5-H), 3.41 (t, J = 5.2 Hz, 1H, C23-H),
3.68 (d, J = 3.4 Hz, 1H, C22-H), 3.82 (ddd, J = 12.5, 4.1, 2.8 Hz, 1H,
C2-H), 4.00–4.09 (m, 1H, C7-H␣), 4.10 (d, J = 12.3 Hz, 1H, C7-H␤),
5.23 (br.s, 1H, C3-H). 13C NMR ı: 10.83, 11.62, 12.37, 14.11, 15.51,
17.28, 22.11, 22.21, 22.68, 24.76, 25.06, 27.02, 27.68, 29.15, 29.35,
29.38, 29.52, 29.65, 29.69, 31.91, 34.49, 38.25, 39.15, 39.49, 40.22,
41.41, 42.05, 42.26, 42.47, 51.20, 52.57, 58.41, 67.35, 70.59, 71.27,
72.51, 76.36, 174.53, 175.46. MS (APCI+) m/z (%): 719.3 ([M+H]+,
100), 701.4 ([M-H2O+H]+, 21), 683.5 ([M-2H2O+H]+, 17), 463.3 ([M-
C15H31CO2H+H]+, 25), 445.4 ([M-C15H31CO2H-H2O+H]+, 32), 427.5
([M-C15H31CO2H-2H2O+H]+, 39).
was eluted with a mixture of EtOH-NH4OH (7:3). The frac-
tions containing the expected product 16a were combined and
concentrated to dryness under reduced pressure. The residue
was chromatographed on silica gel (toluene-EtOAc = 5:1 ⇒ 2:1) to
give diol 16a (50 mg, 79%) as an oil. 1H NMR ı: 0.70 (s, 3H,
C18-H), 0.85 (d, J = 6.2 Hz, 3H, CHCH3), 0.87 (d, J = 7.5 Hz, 3H,
CHCH3), 0.88 (t, J = 6.8 Hz, 3H, OCO (CH2)14 CH3), 0.91 (s, 3H,
C19-H), 0.92 (d, J = 7.2 Hz, 3H, CHCH3), 0.96 (d, J = 6.6 Hz, 3H,
CHCH3), 1.22–1.29 (m, 26H, OCOCH2 (CH2)13 CH3), 2.37 (t,
J = 7.4 Hz, 2H, OCOCH2C14H29), 2.98 (dd, J = 12.2, 4.4 Hz, 1H, C5-
H), 3.41 (t, J = 5.3 Hz, 1H, C23-H), 3.46 (ddd, J = 12.5, 4.1, 2.5 Hz,
1H, C2-H), 3.68 (d, J = 3.7 Hz, 1H, C22-H), 4.05 (dd, J = 12.4, 9.4 Hz,
1H, C7-H␣), 4.09–4.16 (m, 1H, C7-H␤), 4.40 (d, J = 11.3 Hz, 1H,
CH2Ph), 4.71 (d, J = 11.3 Hz, 1H, CH2Ph), 5.57 (s, 1H, C3-H),
7.27–7.35 (m, 5H, Ph). 13C NMR ı: 10.86, 11.62, 11.66, 12.40,
14.09, 15.68, 17.31, 22.10, 22.29, 22.68, 24.78, 25.17, 27.08,
27.71, 29.16, 29.35, 29.39, 29.47, 29.52, 29.70, 31.92, 34.57,
38.22, 39.27, 39.53, 40.28, 40.80, 41.52, 42.28, 42.52, 51.25, 52.64,
58.51, 58.57, 66.12, 70.44, 70.58, 72.57, 73.57, 127.67, 127.83,
128.39, 138.09, 173.00, 175.53. MS (APCI+) m/z (%): 809.3 ([M+H]+,
100).

V.A. Khripach et al. / Journal of Steroid Biochemistry & Molecular Biology 137 (2013) 345–354
349
(m, 5H, Ph). 13C NMR ı: 9.12, 11.45, 11.58, 15.74, 16.42, 21.02, 22.20,
24.75, 27.07, 27.74, 30.72, 38.16, 38.93, 39.15, 39.69, 41.11, 41.89,
42.41, 44.35, 50.98, 52.18, 57.83, 70.44, 70.97, 73.46, 77.95, 81.95,
82.33, 127.85, 128.07, 128.58, 137.32, 175.09. MS (APCI+) m/z (%):
673.2 ([M+H]+, 100), 577.3 ([M-MsOH+H]+, 19).
2.2.12. (22R,23R,24R)-3˛-Myristoyloxy-2˛,22,23-trihydroxy-24-
methyl-B-homo-7-oxa-5˛-cholestan-6-one
(17b)
The title compound (34 mg) was prepared in 97% yield as an
oil from the benzyl ether 16b as described above for the prepa-
ration of the triol 17a. 1H NMR ı: 0.71 (s, 3H, C18-H), 0.84 (d,
J = 7.0 Hz, 3H, CHCH3), 0.87 (d, J = 7.5 Hz, 3H, CHCH3), 0.87 (t,
J = 7.2 Hz, 3H, OCO (CH2)12 CH3), 0.92 (d, J = 6.9 Hz, 3H, CHCH3),
0.93 (s, 3H, C19-H), 0.96 (d, J = 6.7 Hz, 3H, CHCH3), 1.22–1.29
(m, 22H, OCOCH2 (CH2)11 CH3), 2.38 (td, J = 7.4, 1.1 Hz, 2H,
OCOCH2C12H29), 2.95 (dd, J = 12.2, 4.3 Hz, 1H, C5-H), 3.40 (dd,
J = 9.7, 5.1 Hz, 1H, C23-H), 3.68 (d, J = 4.9 Hz, 1H, C22-H), 3.81
(dd, J = 17, 9.6 Hz, 1H, C2-H), 4.04 (dd, J = 12.4, 9.4 Hz, 1H, C7-
H␣), 4.12 (d, J = 12.4 Hz, 1H, C7-H␤), 5.22 (s, 1H, C3-H). 13C
NMR ı: 10.81, 11.61, 12.36, 14.11, 15.50, 17.26, 22.11, 22.20,
22.67, 24.74, 25.04, 26.99, 27.67, 29.13, 29.34, 29.51, 29.64, 31.90,
34.48, 38.23, 39.13, 39.47, 40.22, 41.39, 42.03, 42.22, 42.45, 51.17,
52.54, 58.38, 67.32, 70.58, 71.24, 72.48, 76.33, 174.54, 175.50.
MS (APCI+) m/z (%): 691.5 ([M+H]+, 100), 673.6 ([M-H2O+H]+,
20), 655.6 ([M-2H2O+H]+, 13), 463.4 ([M-C13H27CO2H+H]+, 7),
445.9 ([M-C13H27CO2H-H2O+H]+, 32), 427.6 ([M-C13H27CO2H-
2H2O+H]+, 20).
2.2.15. (22R,23R,24R)-2˛-Benzyloxy-22,23-dihydroxy-24-
methyl-B-homo-7-oxa-5˛-cholestan-3,6-one
(19)
A mixture of alcohol 10 (51 mg, 0.086 mmol), pyridinium dichro-
mate (80 mg, 0.213 mmol) and CH2Cl2 (4 mL) was stirred at room
temperature overnight. The mixture was transferred directly to sil-
ica gel column for purification. Eluting with toluene–EtOAc (10:1)
afforded ketone 19 (50 mg, 98%) as an oil. 1H NMR ı: 0.27 (s, 3H,
B
CH3), 0.69 (d, J = 6.9 Hz, 3H, CHCH3), 0.71 (s, 3H, C18-H), 0.84
(d, J = 6.8 Hz, 3H, CHCH3), 0.88 (d, J = 6.6 Hz, 3H, CHCH3), 0.91
(d, J = 6.9 Hz, 3H, CHCH3), 1.12 (s, 3H, C19-H), 3.27 (dd, J = 12.4,
5.1 Hz, 1H, C5-H), 3.77 (dd, J = 8.8, 5.2 Hz, 1H, C23-H), 4.01 (dd,
J = 12.7, 9.5 Hz, 1H, C7-H), 4.12 (m, 3H, C2-, C7- and C22-H), 4.46 (d,
J = 11.5 Hz, 1H, CH2Ph), 4.88 (d, J = 11.5 Hz, 1H, CH2Ph), 7.24–7.35
(m, 5H, Ph). 13C NMR ı: 9.12, 11.43, 11.61, 15.81, 16.43, 21.02, 23.18,
24.72, 27.08, 27.70, 38.62, 39.11, 39.21, 39.89, 41.09, 42.40, 44.34,
48.02, 49.49, 51.01, 52.13, 57.39, 70.57, 72.22, 81.91, 82.28, 127.93,
128.49, 137.56, 173.59, 208.25.
2.2.13. (22R,23R,24R)-3˛-Lauroyloxy-2˛,22,23-trihydroxy-24-
methyl-B-homo-7-oxa-5˛-cholestan-6-one
(17c)
2.2.16. (22R,23R,24R)-2˛-Benzyloxy-3ˇ,22,23-trihydroxy-24-
methyl-B-homo-7-oxa-5˛-cholestan-6-one
22,23-methylboronate (20)
The title compound (38 mg) was prepared in 91% yield as an oil
from the benzyl ether 16c as described above for the preparation
of the triol 17a. 1H NMR ı: 0.70 (s, 3H, C18-H), 0.84 (d, J = 7.0 Hz,
3H, CHCH3), 0.86 (d, J = 7.3 Hz, 3H, CHCH3), 0.87 (t, J = 7.3 Hz,
3H, OCO (CH2)12 CH3), 0.92 (d, J = 6.9 Hz, 3H, CHCH3), 0.93 (s,
3H, C19-H), 0.96 (d, J = 6.6 Hz, 3H, CHCH3), 1.22–1.29 (m, 18H,
OCOCH2 (CH2)9 CH3), 2.38 (t, J = 7.4 Hz, 2H, OCOCH2C12H29),
2.95 (dd, J = 12.2, 4.2 Hz, 1H, C5-H), 3.40 (t, J = 5.2 Hz, 1H, C23-
H), 3.68 (d, J = 3.7 Hz, 1H, C22-H), 3.82 (d, J = 11.7 Hz, 1H, C2-H),
4.01–4.08 (m, 1H, C7-H␣), 4.12 (d, J = 12.3 Hz, 1H, C7-H␤), 5.23 (s,
Variant A. A mixture of mesylate 18 (160 mg, 0.24 mmol)
and NaNO2 (95 mg, 1.38 mmol) in DMF (4 mL) was stirred at
100 ◦C for 7 h. Then it was cooled to room temperature and
poured into 1% NaCl (25 mL). The resulting mixture was extracted
with EtOAc (3× 15 mL). The organic layer was dried (Na2SO4)
and evaporated. The residue was chromatographed on silica gel
(toluene–EtOAc = 10:1 ⇒ 1:5) to give alcohol 20 (52 mg, 36%) as an
oil. 1H NMR ı: 0.27 (s, 3H,
B CH3), 0.69 (s, 3H, C18-H), 0.69 (d,
1H, C3-H). 13C NMR ı: 10.81, 11.61, 12.36, 14.11, 15.50, 17.26, 22.11,
22.20, 22.67, 24.74, 25.04, 26.99, 27.67, 29.13, 29.33, 29.51, 29.63,
31.89, 34.48, 38.23, 39.13, 39.47, 40.22, 41.39, 42.03, 42.22, 42.45,
51.17, 52.55, 58.39, 67.32, 70.58, 71.24, 72.49, 76.33, 174.54, 175.49.
MS (APCI+) m/z (%): 663.5 ([M+H]+, 100), 645.7 ([M-H2O+H]+, 12),
627.7 ([M-2H2O+H]+, 10), 463.9 ([M-C11H23CO2H+H]+, 12), 445.8
([M-C11H23CO2H-H2O+H]+, 8), 427.5 ([M-C11H23CO2H-2H2O+H]+,
10).
J = 6.5 Hz, 3H, CHCH3), 0.84 (d, J = 6.8 Hz, 3H, CHCH3), 0.88 (d,
J = 6.6 Hz, 3H, CHCH3), 0.91 (d, J = 7.0 Hz, 3H, CHCH3), 0.93 (s, 3H,
C19-H), 2.89 (dd, J = 12.2, 4.5 Hz, 1H, C5-H), 3.29 (ddd, J = 12.5, 9.1,
4.1 Hz, 1H, C2- or C3-H), 3.50 (ddd, J = 12.5, 8.8, 4.5 Hz, 1H, C3- or
C2-H), 3.77 (dd, J = 8.6, 5.2 Hz, 1H, C23-H), 4.01 (dd, J = 12.5, 9.3 Hz,
1H, C7-H␣), 4.10 (d, J = 12.0 Hz, 1H, C7-H␤), 4.13 (d, J = 4.9 Hz, 1H,
C22-H), 4.53 (d, J = 11.4 Hz, 1H, CH2Ph), 4.64 (d, J = 11.4 Hz, 1H,
CH2Ph), 7.28–7.39 (m, 5H, Ph). 13C NMR ı: 9.11, 11.42, 11.57,
16.00, 16.41, 21.02, 22.80, 24.73, 27.06, 27.72, 32.02, 38.77, 39.15,
39.25, 41.07, 42.38, 43.55, 44.34, 46.09, 51.06, 52.19, 58.30, 70.41,
71.71, 72.52, 79.51, 81.93, 82.33, 127.84, 127.91, 128.54, 138.17,
174.82.
2.2.14. (22R,23R,24R)-2˛-Benzyloxy-3˛-methanesulfonyloxy-
22,23-dihydroxy-24-methyl-B-homo-7-oxa-5˛-cholestan-6-one
22,23-methylboronate (18)
Variant B. To a cooled to −15 ◦C solution of ketone 19 (49 mg,
0.083 mmol) in EtOAc–MeOH (1:1), NaBH4 (4.5 mg, 0.12 mmol)
was added under stirring. The cooling bath was removed, and the
reaction mixture was allowed to warm to ambient temperature.
Then it was filtered through a short pad of silica gel and rinsed
with EtOAc (12 mL). The filtrate was evaporated, and the residue
was chromatographed on silica gel (toluene–EtOAc = 10:1 ⇒ 3:1) to
give:
A mixture of the alcohol 10 (200 mg, 0.34 mmol) and MsCl
(0.1 mL, 148 mg, 1.2 mmol) in pyridine (4 mL) was stirred at ambi-
ent temperature for 1 h. Then it was diluted with water (12 mL)
and the formed suspension was stirred for 30 min. The precipitate
was filtered, washed with water (8 mL) and dried under reduced
pressure. The resulting material was dissolved in a mixture of
EtOAc-hexane (1:10, 8 mL) and placed in the refrigerator for 2 h.
The precipitated crystals were filtered, washed on the filter with
hexane (4 mL), and dried at room temperature to afford mesylate
18 (176 mg, 78%). Mp 193–195 ◦C (EtOAc-hexane). 1H NMR ı: 0.28
a) alcohol 20 (18 mg, 37%).
b) alcohol 10 (30 mg, 61%).
(s, 3H,
B CH3), 0.70 (s, 3H, C18-H), 0.70 (d, J = 6.7 Hz, 3H, CHCH3),
0.84 (d, J = 6.8 Hz, 3H, CHCH3), 0.88 (d, J = 6.7 Hz, 3H, CHCH3), 0.90
(s, 3H, C19-H), 0.92 (d, J = 6.9 Hz, 3H, CHCH3), 3.04 (s, 3H, Ms), 3.11
(dd, J = 11.9, 4.8 Hz, 1H, C5-H), 3.48 (ddd, J = 12.3, 4.1, 2.2 Hz, 1H,
C2-H), 3.77 (dd, J = 8.8, 5.2 Hz, 1H, C23-H), 4.06–4.12 (m, 2H, C7-H),
4.14 (d, J = 4.9 Hz, 1H, C22-H), 4.14 (s, J = 4.9 Hz, 1H, C3-H), 4.54 (d,
J = 11.3 Hz, 1H, CH2Ph), 4.68 (d, J = 11.3 Hz, 1H, CH2Ph), 7.39–7.28
2.2.17. (22R,23R,24R)-2˛-Benzyloxy-3ˇ-palmitoyloxy-22,23-
dihydroxy-24-methyl-B-homo-7-oxa-5˛-cholestan-6-one
22,23-methylboronate (21a)
A mixture of the alcohol 20 (52 mg, 0.087 mmol), palmytoyl
chloride (53 ␮L, 48 mg, 0.17 mmol), and pyridine (1 mL) was stirred

350
V.A. Khripach et al. / Journal of Steroid Biochemistry & Molecular Biology 137 (2013) 345–354
at room temperature for 5 h. Then solvents were evaporated
in vacuo and the residue was purified by column chromatogra-
phy on silica gel (toluene–EtOAc = 10:1 ⇒ 8:1) to afford ester 21a
2.2.20. (22R,23R,24R)-2˛-Benzyloxy-3ˇ-palmitoyloxy-22,23-
dihydroxy-24-methyl-B-homo-7-oxa-5˛-cholestan-6-one
(22a)
(49 mg, 67%) as an oil. 1H NMR ı: 0.27 (s, 3H,
B
CH3), 0.69
The title compound (25 mg) was prepared in 59% yield as an oil
from the methylboronate 21a as described above for the prepara-
tion of the diol 16a. 1H NMR ı: 0.69 (s, 3H, C18-H), 0.84 (d, J = 7.1 Hz,
3H, CHCH3), 0.86 (d, J = 6.9 Hz, 3H, CHCH3), 0.87 (t, J = 6.8 Hz,
3H, OCO (CH2)14 CH3), 0.91 (s, 3H, C19-H), 0.91 (d, J = 7.2 Hz,
3H, CHCH3), 0.96 (d, J = 6.6 Hz, 3H, CHCH3), 1.22–1.29 (m, 26H,
OCOCH2 (CH2)13 CH3), 2.96 (dd, J = 11.0, 5.7 Hz, 1H, C5-H), 3.40
(t, J = 5.2 Hz, 1H, C23-H), 3.49 (ddd, J = 11.8, 9.7, 4.2 Hz, 1H, C2-H),
3.67 (d, J = 3.4 Hz, 1H, C22-H), 4.02 (dd, J = 12.4, 9.5 Hz, 1H, C7-H␣),
4.07–4.15 (m, 2H, C7- and C22-H), 4.60 (d, J = 11.8 Hz, 1H, CH2Ph),
4.64 (d, J = 11.8 Hz, 1H, CH2Ph), 4.76 (dt, J = 16.1, 7.9 Hz, 1H, C3-H),
7.27–7.35 (m, 5H, Ph). 13C NMR ı: 10.80, 11.62, 12.35, 14.12, 15.77,
17.24, 22.11, 22.67, 24.69, 24.93, 26.96, 27.64, 29.14, 29.27, 29.34,
29.42, 29.64, 29.67, 30.09, 31.90, 34.62, 38.40, 39.13, 39.42, 40.20,
41.34, 42.40, 44.95, 45.90, 51.10, 52.44, 58.16, 70.52, 72.45, 74.92,
75.71, 76.29, 127.43, 127.61, 128.34, 138.44, 173.34, 174.74. MS
(APCI+) m/z (%): 809.7 ([M+H]+, 100), 553.2 ([M-C15H31CO2H+H]+,
7).
(s, 3H, C18-H), 0.69 (d, J = 6.7 Hz, 3H, CHCH3), 0.84 (d, J = 6.8 Hz,
3H, CHCH3), 0.88 (t, J = 6.8 Hz, 3H, OCO (CH2)14 CH3), 0.89
(d, J = 6.6 Hz, 3H, CHCH3), 0.91 (d, J = 6.6 Hz, 3H, CHCH3), 0.92
(s, 3H, C19-H), 1.21–1.29 (m, 26H, OCOCH2 (CH2)13 CH3), 2.96
(dd, J = 11.1, 5.8 Hz, 1H, C5-H), 3.49 (ddd, J = 11.7, 9.8, 4.3 Hz,
1H, C2-H), 3.77 (dd, J = 8.8, 5.2 Hz, 1H, C23-H), 4.01 (dd, J = 12.4,
9.4 Hz, 1H, C7-H␣), 4.07–4.15 (m, 2H, C7- and C22-H), 4.60 (d,
J = 11.8 Hz, 1H, CH2Ph), 4.65 (d, J = 11.8 Hz, 1H, CH2Ph), 4.76
(td, J = 10.1, 5.9 Hz, 1H, C3-H), 7.27–7.36 (m, 5H, Ph). 13C NMR
ı: 9.11, 11.43, 11.59, 14.12, 15.78, 16.41, 21.03, 22.68, 24.72,
24.94, 27.06, 27.71, 29.05, 29.15, 29.28, 29.35, 29.43, 29.64, 29.67,
30.11, 31.91, 33.70, 34.63, 38.42, 39.15, 41.10, 42.39, 44.34, 44.99,
45.92, 51.01, 52.14, 58.18, 70.52, 72.47, 74.92, 75.74, 81.92, 82.32,
127.44, 127.61, 128.34, 138.47, 173.32, 174.71. MS (APCI+) m/z
(%): 833.4 ([M+H]+, 100), 725.4 ([M-BnOH+H]+, 24), 577.4 ([M-
C
15H31CO2H+H]+, 13).
2.2.18. (22R,23R,24R)-2˛-Benzyloxy-3ˇ-myristoyloxy-22,23-
dihydroxy-24-methyl-B-homo-7-oxa-5˛-cholestan-6-one
22,23-methylboronate (21b)
2.2.21. (22R,23R,24R)-2˛-Benzyloxy-3ˇ-myristoyloxy-22,23-
dihydroxy-24-methyl-B-homo-7-oxa-5˛-cholestan-6-one
(22b)
The title compound (24 mg) was prepared in 74% yield as an oil
from the alcohol 20 and myristoyl chloride as described above for
The title compound (11 mg) was prepared in 62% yield as an oil
from the methylboronate 21b as described above for the prepara-
tion of the diol 16a. 1H NMR ı: 0.70 (s, 3H, C18-H), 0.84 (d, J = 7.0 Hz,
3H, CHCH3), 0.87 (d, J = 6.8 Hz, 3H, CHCH3), 0.87 (t, J = 6.8 Hz,
3H, OCO (CH2)12 CH3), 0.91 (s, 3H, C19-H), 0.92 (d, J = 6.5 Hz,
3H, CHCH3), 0.96 (d, J = 6.6 Hz, 3H, CHCH3), 1.22–1.29 (m, 22H,
OCOCH2 (CH2)11 CH3), 2.34 (t, J = 7.5 Hz, 2H, OCOCH2C12H25),
2.96 (dd, J = 11.1, 5.6 Hz, 1H, C5-H), 3.38–3.42 (m, 1H, C23-H),
3.44–3.53 (m, 1H, C2-H), 3.68 (d, J = 3.4 Hz, 1H, C22-H), 3.97–4.06
(m, 1H, C7-H␣), 4.11 (d, J = 12.4 Hz, 1H, C7-H␤), 4.60 (d, J = 11.8 Hz,
1H, CH2Ph), 4.64 (d, J = 11.8 Hz, 1H, CH2Ph), 4.71–4.80 (m, 1H,
C3-H), 7.27–7.35 (m, 5H, Ph). 13C NMR ı: 10.85, 11.66, 12.39, 14.09,
15.78, 17.29, 22.10, 22.67, 22.80, 24.74, 24.96, 27.05, 27.67, 29.17,
29.28, 29.33, 29.43, 29.64, 30.17, 31.91, 34.65, 38.45, 39.23, 39.52,
40.27, 41.50, 42.48, 45.03, 46.00, 51.19, 52.58, 58.27, 70.55, 72.45,
72.55, 74.95, 75.81, 76.35, 127.45, 127.61, 128.35, 138.54, 173.31,
174.66. MS (APCI+) m/z (%): 781.5 ([M+H]+, 100).
the preparation of the ester 21a. 1H NMR ı: 0.27 (s, 3H,
B CH3),
0.68 (s, 3H, C18-H), 0.69 (d, J = 6.6 Hz, 3H, CHCH3), 0.83 (d,
J = 6.8 Hz, 3H, CHCH3), 0.87 (t, J = 6.9 Hz, 3H, OCO (CH2)14 CH3),
0.88 (d, J = 6.6 Hz, 3H, CHCH3), 0.91 (d, J = 6.6 Hz, 3H, CHCH3), 0.91
(s, 3H, C19-H), 1.21–1.29 (m, 22H, OCOCH2 (CH2)11 CH3), 2.96
(dd, J = 10.7, 6.2 Hz, 1H, C5-H), 3.49 (ddd, J = 11.9, 9.7, 4.3 Hz, 1H, C2-
H), 3.77 (dd, J = 8.7, 5.2 Hz, 1H, C23-H), 4.01 (dd, J = 12.2, 9.7 Hz, 1H,
C7-H␣), 4.06–4.17 (m, 2H, C7- and C22-H), 4.60 (d, J = 11.8 Hz, 1H,
CH2Ph), 4.65 (d, J = 11.8 Hz, 1H, CH2Ph), 4.76 (td, J = 10.0, 5.9 Hz,
1H, C3-H), 7.27–7.36 (m, 5H, Ph). 13C NMR ı: 9.10, 11.42, 11.58,
14.10, 15.77, 16.40, 21.01, 22.66, 22.72, 24.72, 24.92, 27.05, 27.70,
29.14, 29.26, 29.32, 29.41, 29.62, 30.10, 31.89, 34.62, 38.41, 39.14,
41.08, 42.38, 44.33, 44.97, 45.91, 51.00, 52.13, 58.16, 70.50, 72.46,
74.91, 75.74, 81.91, 82.31, 121.09, 127.43, 127.60, 128.33, 129.61,
138.46, 150.78, 173.30, 174.69. MS (APCI+) m/z (%): 805.3 ([M+H]+,
100).
2.2.22. (22R,23R,24R)-2˛-Benzyloxy-3ˇ-lauroyloxy-22,23-
dihydroxy-24-methyl-B-homo-7-oxa-5˛-cholestan-6-one
(22c)
2.2.19. (22R,23R,24R)-2˛-Benzyloxy-3ˇ-lauroyloxy-22,23-
dihydroxy-24-methyl-B-homo-7-oxa-5˛-cholestan-6-one
22,23-methylboronate (21c)
The title compound (18 mg) was prepared in 62% yield as an
oil from the methylboronate 21c as described above for the prepa-
ration of the diol 16a. 1H NMR ı: 0.69 (s, 3H, C18-H), 0.79–0.99
(m, 18H), 1.22–1.29 (m, 18H, OCOCH2 (CH2)9 CH3), 2.96 (dd,
J = 10.9, 5.6 Hz, 1H, C5-H), 3.40 (t, J = 5.1 Hz, 1H, C23-H), 3.44–3.53
(m, 1H, C2-H), 3.67 (d, J = 3.4 Hz, 1H, C22-H), 3.97–4.15 (m, 2H,
C7-H), 4.60 (d, J = 11.8 Hz, 1H, CH2Ph), 4.64 (d, J = 11.8 Hz, 1H,
CH2Ph), 4.76 (td, J = 9.8, 6.1 Hz, 1H, C3-H), 7.27–7.35 (m, 5H, Ph).
13C NMR ı: 10.81, 11.62, 12.36, 14.11, 15.77, 17.24, 22.11, 22.66,
22.75, 24.70, 24.93, 26.97, 27.64, 29.14, 29.27, 29.31, 29.42, 29.59,
30.10, 31.89, 34.62, 38.41, 39.15, 39.44, 40.22, 41.37, 42.42, 44.97,
45.92, 51.12, 52.47, 58.18, 70.52, 72.45, 72.48, 74.92, 75.73, 76.30,
127.43, 127.61, 128.34, 138.46, 173.33, 174.72. MS (APCI+) m/z (%):
753.4 ([M+H]+, 100).
The title compound (40 mg) was prepared in 61% yield as an oil
from the alcohol 20 and lauroyl chloride as described above for the
preparation of the ester 21a. 1H NMR ı: 0.27 (s, 3H,
B CH3), 0.69
(s, 3H, C18-H), 0.69 (d, J = 6.6 Hz, 3H, CHCH3), 0.83 (d, J = 6.8 Hz,
3H, CHCH3), 0.87 (t, J = 6.9 Hz, 3H, OCO (CH2)14 CH3), 0.88 (d,
J = 6.5 Hz, 3H, CHCH3), 0.91 (d, J = 6.0 Hz, 3H, CHCH3), 0.92 (s,
3H, C19-H), 1.21–1.29 (m, 18H, OCOCH2 (CH2)9 CH3), 2.96 (dd,
J = 11.0, 5.8 Hz, 1H, C5-H), 3.49 (td, J = 11.6, 4.3 Hz, 1H, C2-H), 3.77
(dd, J = 8.8, 5.2 Hz, 1H, C23-H), 4.01 (dd, J = 12.4, 9.4 Hz, 1H, C7-H␣),
4.07–4.15 (m, 2H, C7- and C22-H), 4.60 (d, J = 11.8 Hz, 1H, CH2Ph),
4.65 (d, J = 11.8 Hz, 1H, CH2Ph), 4.76 (td, J = 10.0, 6.0 Hz, 1H, C3-
H), 7.27–7.36 (m, 5H, Ph). 13C NMR ı: 9.11, 11.43, 11.59, 14.12,
15.79, 16.41, 21.03, 22.67, 22.73, 24.73, 24.94, 27.06, 27.72, 29.05,
29.15, 29.28, 29.32, 29.42, 29.59, 30.12, 31.89, 33.67, 34.63, 38.43,
39.15, 41.10, 42.39, 44.34, 44.99, 45.92, 51.02, 52.14, 58.18, 70.52,
72.48, 74.93, 75.74, 81.93, 82.32, 127.44, 127.62, 128.35, 138.46,
173.33, 174.71. MS (APCI+) m/z (%): 777.4 ([M+H]+, 100), 577.4
([M-C11H23CO2H+H]+, 10).
2.2.23. (22R,23R,24R)-3ˇ-Palmitoyloxy-2˛,22,23-trihydroxy-24-
methyl-B-homo-7-oxa-5˛-cholestan-6-one
(2a)
The title compound (14 mg) was prepared in 63% yield from the
benzyl ether 22a as described above for the preparation of the triol

V.A. Khripach et al. / Journal of Steroid Biochemistry & Molecular Biology 137 (2013) 345–354
351
17a. Mp 196–198 ◦C (EtOH). Its 1H NMR spectrum matched that
described in the literature [7]. 13C NMR ı: 10.85, 11.65, 12.40, 14.10,
15.87, 17.29, 22.11, 22.68, 22.77, 24.74, 24.96, 27.05, 27.68, 29.12,
29.24, 29.35, 29.44, 29.60, 29.68, 31.92, 34.52, 38.47, 39.18, 39.51,
40.26, 41.46, 42.48, 46.29, 46.91, 51.19, 52.58, 58.30, 68.90, 70.62,
72.55, 76.37, 174.25, 174.57. MS (APCI+) m/z (%): 719.3 ([M+H]+,
91), 701.4 ([M-H2O+H]+, 23), 683.5 ([M-2H2O+H]+, 15), 463.3
([M-C15H31CO2H+H]+, 100), 445.6 ([M-C15H31CO2H-H2O+H]+, 43),
427.2 ([M-C15H31CO2H-2H2O+H]+, 15).
29.23, 29.31, 29.42, 29.58, 29.67, 31.89, 34.50, 38.45, 39.14, 39.47,
40.13, 41.32, 42.45, 46.24, 46.87, 51.15, 52.50, 58.25, 68.86, 70.60,
72.58, 174.26, 174.60.
3. Results and discussion
The preparation of the desired compounds (2a–c, 17a–c)
required the protection of three of the four hydroxyl groups in epi-
brassinolide molecule 4. A solution to this problem was proposed
by the authors previously [14]. The side chain diol function was
protected as 22,23-methylboronate 5 followed by protection of the
equatorial 2␣-hydroxyl group as TBS-ether to give 3␣-alcohol 6
(Scheme 1). However, attempts to use this compound for the prepa-
ration of 3-acyl derivatives proved to be somewhat disappointing.
Acylation of 6 wih palmitoyl chloride in the presence of DMAP pro-
ceeded smoothly to give ester 7, but subsequent desilylation was
accompanied by 1,2-acyl migration with formation of a mixture of
2␣- and 3␣-esters 8 and 9 in 51% and 21% yield, respectively. The
location of an acyl group at C-3 in 8 and at C-2 in 9 was deduced from
the values of coupling constants of the corresponding signals. In 9
it appeared as broadened singlet at ı 5.23. Consequently, this pro-
ton was in equatorial orientation, indicating the location of the acyl
group at axial C-3 position. The large coupling constant (J 12.2 Hz)
of signal at ı 4.84 in 1H NMR spectrum of 8 supported the axial ori-
entation of this proton, thus confirming a C-2 equatorial position
of the acyl group.
2.2.24. (22R,23R,24R)-3ˇ-Myristoyloxy-2˛,22,23-trihydroxy-24-
methyl-B-homo-7-oxa-5˛-cholestan-6-one
(2b)
The title compound (5 mg) was prepared in 63% yield from the
benzyl ether 22b as described above for the preparation of the
triol 17a. Mp 203–205 ◦C (EtOH). Its 1H NMR spectrum matched
that described in the literature [7]. 13C NMR ı: 10.83, 11.64,
12.38, 14.11, 15.86, 17.27, 22.11, 22.68, 22.75, 24.73, 24.94, 27.02,
27.67, 29.11, 29.24, 29.34, 29.43, 29.63, 31.91, 34.50, 38.46, 39.15,
39.48, 40.24, 41.41, 42.45, 46.25, 46.86, 51.16, 52.53, 58.26, 68.87,
70.61, 72.52, 174.27, 174.61. MS (ESI+) m/z (%): 691.0 ([M+H]+,
100), 673.3 ([M-H2O+H]+, 18), 655.3 ([M-2H2O+H]+, 7), 463.1 ([M-
C13H27CO2H+H]+, 56), 445.3 ([M-C13H27CO2H-H2O+H]+, 15), 427.4
([M-C13H27CO2H-2H2O+H]+, 10).
2.2.25. (22R,23R,24R)-3ˇ-Lauroyloxy-2˛,22,23-trihydroxy-24-
methyl-B-homo-7-oxa-5˛-cholestan-6-one
(2c)
The title compound (7 mg) was prepared in 57% yield from the
benzyl ether 22c as described above for the preparation of the triol
17a. Mp 213–215 ◦C (EtOH). Its 1H NMR spectrum matched that
described in the literature [7]. 13C NMR ı: 10.85, 11.64, 12.38, 14.10,
15.86, 17.31, 22.14, 22.67, 22.75, 24.73, 24.94, 26.99, 27.66, 29.11,
Difficulties in removing the TBS group encouraged the search
for an alternative protecting group that could be removed under
conditions avoiding acyl migration. Protection of 2␣-OH in 5 as the
ether 10 (Scheme 2) seemed to be a good choice due to easiness
of the benzyl group removal by hydrogenolysis. Although the axial
hydroxyl at C-3 is more sterically encumbered than the equatorial
one at C-2, the first attempts to prepare the monobenzyl ether
Scheme 1.

352
V.A. Khripach et al. / Journal of Steroid Biochemistry & Molecular Biology 137 (2013) 345–354
Scheme 2.
10 were discouraging. Benzylation of 5 with benzyl bromide and
sodium hydride [15] gave only 13% of the desired product 10 along
with 7% of dibenzyl ether 12, 6% of monobenzyl ether 11 and 64% of
the starting material. Better results (57% of 10) were obtained using
benzyl bromide and silver oxide [16]. Ultimately the best result (78
and 11% yields of the monobenzyl ethers 10 and 11, respectively)
was obtained by the use of dibutyl tin oxide [17].
Another problem, which had to be solved, was connected with
the removal of methylboronate protective group. This reaction
requires basic conditions [14] that are not compatible with the
acyl group at C-3. Attempts to remove 22,23-methylboronate using
the exchange reaction with pinacol [18] (based on the assumption
that pinacolate ester would be more stable) failed. The solution of
the problem was found to be initial oxidation of methylboronate.
The methodology was first tested on the ester 5. Its treatment with
trimethylamine N-oxide gave the corresponding 2-hydroxy-1,3,2-
dioxaborolane 13 (Scheme 3) which proved to be less resistant to
hydrolysis. Because of this reaction reversibility, the hydrolysis was
carried out using a cation exchange column with DOWEX 50W×8 in
NH4+ form. The oxidation of 5 was accompanied by the formation of
a small amount of epibrassinolide 4 that is why the two-step depro-
tection was carried out with other compounds without isolation of
intermediate 2-hydroxy-1,3,2-dioxaborolanes.
With these findings in hand, the preparation of 3␣-acyl deriva-
tives of epibrassinolide 17a–c was straightforward enough as
outlined in Scheme 4. Treatment of the alcohol 10 with palmitic,
myristinic or lauric acid chlorides gave the corresponding esters
14a–c. In addition, for comparison purposes, the ester 14a was pre-
pared by the reaction of palmitic acid and DCC. The yield was a little
bit higher than with acid chlorides, but troublesome formation and
removal of urea by-products have made this method less attractive.
Acylation resulted in a downfield signal shift of the methine proton
(ı 5.56–5.57) adjacent to the acyl group in the 1H NMR spectra of
the esters 14a–c in comparison with that of the starting material
10. This signal appeared as a broad singlet suggesting an equatorial
orientation of the methine proton, and hence a C-3 axial position of
the acyl group. It is also an experimental proof of the benzyl group
location at C-2 in 10 and 14a–c.
The ester groups at C-3 left unreacted in the methylboronate
deprotection sequence involving oxidation of 14a–c followed by
Scheme 3.
Scheme 4.

V.A. Khripach et al. / Journal of Steroid Biochemistry & Molecular Biology 137 (2013) 345–354
353
Scheme 5.
hydrolysis of 2-hydroxy-1,3,2-dioxaborolanes 15a–c. Hydrogenol-
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ysis of the benzyl ethers 16a–c catalyzed by palladium on carbon
yielded compounds 17a–c. Investigation of their 1H NMR spectra
showed no signs of acyl migration in this reaction.
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Acknowledgments
The author wishes to thank Dr. Alaksiej Hurski for his help in
conducting the experiment. We are grateful also to the Belorussian
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