New Synthesis of Castasterone

Article abstract of DOI:10.1007/s10600-018-2272-8

An improved synthesis that could produce gram quantities of castasterone was proposed. The starting material was stigmasterol, the cyclic part of which was transformed in the first synthetic step into the 3α,5-cyclo-6-ketone. The side-chain carbon skeleton in the target compound was constructed with the required stereochemistry of the C-24 methyl via addition of methylacetylene, hydrogenation of the propargyl alcohol over Lindlar catalyst, and Claisen rearrangement. Diols were introduced using Sharpless asymmetric dihydroxylation of the intermediate ?^2,22-dienone in the presence of (DHQD)₂AQN. A unique feature of the synthesis was the avoidance of chromatographic separations of propargyl alcohols with similar chromatographic mobilities because the C-22 diastereomers were enriched in subsequent redox reactions.

Full text of DOI:10.1007/s10600-018-2272-8

DOI 10.1007/s10600-018-2272-8
Chemistry of Natural Compounds, Vol. 54, No. 1, January, 2018
NEW SYNTHESIS OF CASTASTERONE
1,2*
1
1
V. A. Khripach,
V. N. Zhabinskii, A. L. Gurskii,
1
1
1
A. V. Kolosova, O. V. Gulyakevich, O. V. Konstantinova,
1
1
A. V. Antonchik, and A. A. Pap
An improved synthesis that could produce gram quantities of castasterone was proposed. The starting
material was stigmasterol, the cyclic part of which was transformed in the first synthetic step into the
3ꢀ,5-cyclo-6-ketone. The side-chain carbon skeleton in the target compound was constructed with the
required stereochemistry of the C-24 methyl via addition of methylacetylene, hydrogenation of the propargyl
alcohol over Lindlar catalyst, and Claisen rearrangement. Diols were introduced using Sharpless asymmetric
2,22
dihydroxylation of the intermediate ꢁ -dienone in the presence of (DHQD) AQN. A unique feature of the
2
synthesis was the avoidance of chromatographic separations of propargyl alcohols with similar
chromatographic mobilities because the C-22 diastereomers were enriched in subsequent redox reactions.
Keywords: brassinosteroids, castasterone, Claisen rearrangement, diastereomeric enrichment.
The discovery in 1979 of brassinolide (2) in rape pollen became an event [1] that had a great conceptual influence on
hormonal regulation of plant growth and development. About 70 related brassinolides were identified in subsequent years and
were called brassinosteroids (BS). Nanomolar concentrations of them were capable of affecting all of the most important
aspects of plant vital functions [2–5]. The two most active BS, 2 and its biosynthetic precursor castasterone (1), stimulated the
most interest among researchers studying phytohormone activity on the molecular level. Natural raw material cannot be
considered a source for ensuring their availability because they occur at exceptionally low concentrations in plants. Chemical
synthesis remains the only solution to the problem. An unresolved issue is the production of 1 because the transition from 1 to
2 is thoroughly understood [3].
OH
OH
HO
HO
HO
HO
HO
HO
O
H
H
O
O
1
2
The C –C side chain must also be constructed to synthesize 1, in contrast with related BS that can be prepared
23 28
comparatively readily from sterols with the appropriate carbon skeleton (epicastasterone from ergosterol and homocastasterone
from stigmasterol), because a suitable starting material with the castasterone carbon skeleton is not available. Although many
synthetic methods for 1 have been reported [6–19], it is still poorly accessible even for scientific research because of their
deficiencies (multiple steps, low yields, costly reagents, required separation of pure intermediates).
Therefore, the goal of the present work was to develop a synthetic approach that could produce gram quantities of 1.
It would be based on known methods (including those attempted by us previously), the efficiency of which would be improved
by using selective reagents, setting the optimum reaction conditions, and using special techniques to separate isomeric mixtures
and to isolate the target products.
1) Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, 5/2 Kuprevicha St., Minsk, 220141,
Belarus, fax: +375-172 67 86 47, e-mail: khripach@iboch.bas-net.by; 2) National Research Tomsk State University, 36 Prosp.
Lenina, Tomsk, 634050. Translated from Khimiya Prirodnykh Soedinenii, No. 1, January–February, 2018, pp. 101–106. Original
article submitted April 29, 2017.
©
0009-3130/18/5401-0117 2018 Springer Science+Business Media, LLC
117

Stigmasterol (3), which is manufactured on industrial scales from soy oil, was chosen as the starting material. Use of
22
3 to synthesize BS necessitated oxidative cleavage of the ꢁ -bond to produce the corresponding 22-aldehyde and subsequent
replacement of the carbon side chain and construction of an AB-cyclic portion that was stable to the reaction conditions.
Aldehyde 8, which was accessible in five steps and was used earlier to synthesize 27C- [20, 21], 28C- [11], and 29C-BS [22],
was a convenient intermediate for preparing 1.
Jones
a
b
TsO
HO
OH
4
5
3
CHO
c
d
82%
O
O
O
O
O
8
6
7, 70% from
a. TsCl/Py; b. Me CO–H O, KOAc; c. (CH OH) , CH(OEt) , TsOH; d. O
3
3
2
2
2
2
3
The procedure for preparing 8 was slightly updated by us. The usual conditions for the i-steroid rearrangement are
refluxing a solution of tosylate 4 in an Me CO–H O mixture in the presence of KOAc. A drawback of this method is the need
2
2
to use dilute solutions (0.3–0.5%) of the reagents because of the poor solubility of 4. We found that it did not necessarily have
to be completely dissolved. The yield of alcohol 5 was essentially unaffected if the volume of the solvents was decreased by
6–8 times. In this instance, a suspension of 4 was stirred and refluxed until it was completely dissolved.
Another improvement to the procedure for producing 8 was the use of triethyl orthoformate to add the dioxalane
protection. A solution of 6 in CH Cl was held at room temperature for 20 min in the presence of ethylene glycol and triethyl
2
2
orthoformate to produce 7. However, a previous method [11] suggested refluxing for several hours a solution of 6 in C H
6
6
with a Dean–Stark trap for azeotropic removal of H O.
2
Ozonolysis of 7 used a MeOH–CHCl mixture in the presence of small amounts of Sudan III dye, NaHCO , and Py.
3
3
The dye was used to determine the ozonolysis end point. The other reagents were added to avoid acidification of the reaction
mixture, which is especially critical when ozone is generated from atmospheric oxygen.
Construction of the side-chain carbon skeleton was the most difficult part of the castasterone synthesis. Claisen
rearrangement of allyl alcohol 12 [23–26] was especially attractive among the variety of proposed strategies for solving this
problem [6–19]. This reaction typically has high stereoselectivity and yields, does not require inert conditions, and is easily
scaled up. Syntheses of BS based on it usually include addition of methylacetylene to 22-aldehyde 9 to give a mixture of
propargyl alcohols 10 and 11. They had to be separated by chromatography for the present approach because the C-24
stereochemistry of Claisen rearrangement product 14 was determined by the configuration of the C-22 hydroxyl of 10 [27].
A choke point in the method as a whole was the need for preliminary separation of C-22 isomers 10 and 11, which have very
similar chromatographic mobilities. This prevented it from being scaled up. Based on previous research [11], we supposed
that the isomers could also be separated in the later step for 16 and 17 with fully constructed side chains.
OH
OH
OH
HO
16
12
14
CHO
10
OH
OH
9
OH
HO
15
17
11
13
118

In our instance, such an approach would allow the mixture of propargyl alcohols 18 and 19 to be used to synthesize
1 without separating them into the pure components. Also, increasing the fraction of isomer 18 in the mixture would be
desirable because the sequence of conversions for 19 gave a product with the side chain of 24-epicastasterone. Thus, the
mixture of 18 and 19 was oxidized to ketone 20, reduction of which gave mixtures of these same compounds but in different ratios.
OH
OH
O
CHO
[H]
a
b
+
18 + 19
8
18
20
2 : 1
19
c
(18:19/reagent): 1.1:1/LiAlH ; 1:1/Red-Al;
4
OH
2.2:1/NaBH ; 3.7:1/LiBH ;
4
4
1:1/Zn(BH ) ; 9:1/Ca(BH ) ;
16:1/R-Alpine Borane
4 2
4 2
d
e
EtO C
2
76%
23
22, 54% from 8
a. Li-CꢂC-CH ; b. PDC; c. H /Pd, quinoline; d. MeC(OEt) , EtCO H; e. 1. LiAlH , 2. TsCl, 3. LiAlH
4
21
3
2
3
2
4
The best results (18:19 ratio of 16:1) were obtained using R-Alpine-Borane as the reductant, like for 25C- and
5
27C-type ꢁ -22-ketones [28]. The main drawback of this reagent is the relatively high cost. Therefore, derivatives of
borohydrides and aluminum hydrides were also studied for the reduction of 20. Acceptable results (18:19 ratio of 9:1) were
obtained for Ca(BH ) , which was generated in situ from NaBH and CaCl . The mixture of diastereomerically enriched
4 2
4
2
acetylenic alcohols 18 and 19 was converted into olefin 23 by sequential hydrogenation over Lindlar catalyst, Claisen
rearrangement of allylic alcohol 21, and transformation of the ester of 22 into a methyl.
b
c
a
H
O
O
O
O
23
24
25
OH
OCH
3
OH
R
N
1: R =
27: R =
28: R =
HO
HO
HO
HO
O
O
O
N
N
63% from 25
H
OH
OH
O
26: R =
1, 26 - 28
O
N
HO
HO
OCH
3
29
+
–
a. PPTS; b. PyH Br ; c. K OsO
2
4
The dioxolane protection was removed to regenerate the C-6 carbonyl by treating 23 with pyridinium p-toluenesulfonate
(PPTS). Dienone 25 was obtained in one step by refluxing cycloketone 24 in dimethylacetamide (DMA) in the presence of
pyridinium bromide as before [29]. Compound 25 contained two double bonds, cis-hydroxylation of which by OsO was the
4
2
most obvious method for introducing the diol. The reaction of the ꢁ -derivatives with OsO occurred with preferential
4
formation of the required 2ꢀ,3ꢀ-diols [30]. Therefore, the problem with cis-dihydroxylation of 25 consisted of providing the
required stereochemistry for the diol in the side chain. This reaction without a catalyst gave mainly the non-natural
22S,23S-diols [31]. The problem was solved by using quinidine-type catalysts [32, 33]. However, the cis-dihydroxylation
22
yields are often <50% for 24S-alkyl-ꢁ -steroids because the oxidation goes too far. We used (DHQD) AQN
2
119

[29, hydroquinidine(anthraquinon-1,4-diyl)diether] as the catalyst in order to minimize the side formation of the 22,23-diketones
[34]. The cis-dihydroxylation of 25 gave a mixture of 1 and its isomers 26–28. The target product was readily isolated from
the reaction mixture despite its complexity. Analytically pure 1 was obtained via double crystallization from MeOH in 63% yield.
Thus, the developed synthetic scheme produced 1 in 16 chemical steps in 11% overall yield from 3. Its advantage
was the facile scale up because chromatographic separation of intermediates with similar chromatographic mobilities could be
avoided. This allowed gram quantities of the target product to be produced.
EXPERIMENTAL
NMR spectra were recorded on a Bruker Avance DRX-500 instrument (Germany). Chemical shifts were measured
1
13
13
relative to residual solvent resonances (CHCl , ꢃ 7.26 ppm for H and ꢃ 77.00 ppm for C; C H N, ꢃ 135.91 for C). High-
3
5 5
resolution mass spectra were taken on a 6550 iFunnel Q-TOF LC/MS (APCI and ESI, Agilent Technologies). All reactions
involving organometallic reagents were performed under Ar as customary with dried reagents, solvents, and glassware. TLC
used Kieselgel 60 F
chromatographic plates; column chromatography, Kieselgel 60 (VWR, Art. 7734). Melting points
254
were measured on a Kofler block.
(22E,24S)-6-(1,3-Dioxolan-2-yl)-3ꢀ,5-cyclo-24-ethyl-5ꢀ-cholest-22-ene (7). A solution of 3 (100 g, 0.24 mol) in
Py (560 mL) in a 6-L flask was stirred, cooled in an ice bath, treated with TsCl (92.6 g, 0.49 mol), left for 1 d at room
temperature, treated slowly with H O (50 mL), stirred for 30 min to destroy the excess of TsCl, and treated slowly with H O
2
2
(5.6 L) to precipitate the product. The supernatant liquid was separated by back filtration. The resulting crystals were rinsed
with H O (3 ꢄ 5 L). The obtained tosylate 4 was used in the next step without further purification.
2
Crude tosylate 4 in a flask was treated with Me CO (2.5 L), KOAc (138 g, 1.41 mol), and H O (630 mL); refluxed for
2
2
2 d; and evaporated in vacuo. The solid was extracted with EtOAc (2 ꢄ 500 mL). The organic layer was dried over Na SO
2
4
and evaporated at reduced pressure.
The solid was dissolved in Me CO (1.5 L), cooled to 0°C, treated slowly with Jones reagent (92 mL), stirred at 0°C
2
for 20 min, treated with i-PrOH (40 mL) over 40 min, and treated with a mixture of CHCl (1.5 L) and H O (1.5 L).
3
2
The aqueous phase was separated and extracted with CHCl (500 mL). The combined organic phases were washed with H O
3
2
and NaHCO solution until gas evolution stopped, dried over Na SO , and evaporated. The solid was dissolved in EtOAc
3
2
4
(300 mL), filtered through a layer of Kieselgel, and evaporated. Yield of 6, 93.5 g.
Compound 6 was dissolved in CH Cl (1.3 L); stirred; treated with ethylene glycol (36 mL, 0.64 mol), triethyl
2
2
orthoformate (72 mL, 0.43 mol), and p-TsOH (1.9 g, 11 mmol); stirred at room temperature for 20 min; and washed with
saturated NaHCO solution until gas evolution stopped. The organic layer was dried over Na SO and evaporated. The solid
3
2
4
1
was placed onto a column of SiO and eluted by petroleum ether–EtOAc (9:1) to afford 7 (77 g, 70%) as an oil. Í NMR
2
spectrum (500 MHz, CDCl , ꢃ, ppm, J/Hz): 5.14 (1H, dd, J = 15.1, 8.7), 5.01 (1H, dd, J = 15.1, 8.7), 4.02 (1H, dt, J = 7.8, 6.3),
3
3.93–3.87 (1H, m), 3.84 (1H, dd, J = 12.4, 6.6), 3.74 (1H, q, J = 6.5), 2.08–1.99 (1H, m), 1.96 (1H, dt, J = 12.5, 3.2), 1.01 (6H,
d, J = 6.6), 1.00 (4H, s), 0.84 (4H, d, J = 6.4), 0.80 (6H, t, J = 7.1), 0.73 (3H, s), 0.61 (1H, dd, J = 8.1, 4.6), 0.32 (1H, t, J = 4.2).
13
C NMR spectrum (125 MHz, CDCl , ꢃ, ppm): 138.3, 129.2, 109.9, 64.9, 64.6, 56.4, 56.0, 51.2, 47.4, 45.6, 42.7, 40.5, 40.2,
3
40.0, 39.2, 34.1, 33.2, 31.9, 28.9, 25.4, 24.9, 24.2, 23.0, 22.6, 21.2, 21.1, 19.0, 12.3, 12.2, 7.3. ESI-HR-MS m/z 455.3880
+
[M + H] . Calcd for Ñ H O , 455.3884.
31 51
2
(20S)-6-(1,3-Dioxolan-2-yl)-20-formyl-3ꢀ,5-cyclo-5ꢀ-pregnane (8). A solution of 7 (34.0 g, 75 mmol) in CHCl
3
(300 mL) and MeOH (600 mL) was stirred; treated with NaHCO (6.2 g, 74 mmol), Sudan III (20 mg), and Py (3 mL); cooled
3
to –60°C, purged with ozone until the red color started to fade, purged with O , treated with Me S (18 mL, 0.25 mol), left
2
2
overnight to warm to room temperature, and filtered. The filtrate was evaporated. The solid was separated over a column of
1
SiO with elution by petroleum ether–EtOAc (95:5) to afford aldehyde 8 (22.9 g, 82%) as an oil. Í NMR spectrum
2
(500 MHz, CDCl , ꢃ, ppm, J/Hz): 9.56 (1H, d, J = 3.3), 4.04–3.98 (1H, m), 3.92–3.86 (1H, m), 3.86–3.80 (1H, m), 3.77–3.71
3
(1H, m), 2.36 (1H, ddd, J = 10.2, 6.8, 3.3), 1.11 (3H, d, J = 6.8), 1.00 (3H, s), 0.76 (3H, s), 0.61 (1H, dd, J = 8.2, 4.4), 0.32 (1H,
13
t, J = 4.4). C NMR spectrum (125 MHz, CDCl , ꢃ, ppm): 205.3, 109.9, 65.0, 64.8, 55.7, 51.3, 49.6, 47.6, 45.7, 43.6, 40.3,
3
39.9, 39.4, 34.2, 33.4, 27.2, 25.0, 24.7, 23.1, 22.7, 19.1, 13.6, 12.7, 7.4.
(22R)-6-(1,3-Dioxolan-2-yl)-3ꢀ,5-cyclo-26,27-bis-nor-5ꢀ-cholest-23-yn-22-ol (18) and (22S)-6-(1,3-Dioxolan-2-
yl)-3ꢀ,5-cyclo-26,27-bis-nor-5ꢀ-cholest-23-yn-22-ol (19). A solution of methylacetylene (40 mL, 0.53 mol) in THF
(150 mL) was cooled to –60°C, treated slowly with a solution of BuLi (2.1 M) in hexanes (88.3 mL, 0.186 mol), stirred
120

at –60°C for 15 min, treated with a solution of 8 (22.9 g, 61 mmol) in THF (50 mL), left overnight to warm to room temperature,
treated with NH Cl (33 g, 0.62 mol), diluted with H O (0.5 L), and extracted with CHCl (3 ꢄ 200 mL). The combined organic
4
2
3
layers were dried over Na SO and evaporated. The solid was chromatographed over a column of SiO (petroleum
2
4
2
ether–EtOAc, 85:15ꢅ75:25) to afford a mixture of 18 and 19 (23.4 g, 92%) as an oil (18:19 ratio 2:1 based on integrated
intensities of PMR resonances [35]).
Synthesis of 6-(1,3-Dioxolan-2-yl)-3ꢀ,5-cyclo-26,27-bis-nor-5ꢀ-cholest-23-yn-22-one (20) and its Reduction by
Ca(BH ) . A mixture of 18 and 19 (9.4 g, 23 mmol), PDC (17.2, 49 mmol), and CH Cl (250 mL) was stirred at room
4 2
2
2
temperature for 24 h. The precipitate was filtered off. The filtrate was evaporated. The resulting ketone 20 was dissolved in
EtOH (200 mL), treated with CaCl (9.8 g, 88 mmol), cooled to –78°C, treated in portions with NaBH (5.7 g, 151 mmol),
2
4
stirred at –78°C for 10 min, allowed to warm slowly to room temperature, treated with H O (200 mL), and extracted with
2
EtOAc (3 ꢄ 150 mL). The organic layers were dried over Na SO and evaporated. The solid was placed onto a column of
2
4
SiO and eluted by petroleum ether–EtOAc (85:15ꢅ80:20) to afford a mixture of propargyl alcohols 18 and 19 as an oil
2
(18:19 ratio 9:1 based on integrated intensities of PMR resonances [35]).
(22E)-6-(1,3-Dioxolan-2-yl)-3ꢀ,5-cyclo-24-methyl-5ꢀ-cholest-22-en-26-oic Acid Ethyl Ester (22). A solution of
a mixture of 18 and 19 (9:1, 4.32 g, 10.5 mmol) in EtOH (50 mL) and THF (7 mL) was treated with Pd on BaSO (0.65 g, 5%)
4
and quinoline (0.71 mL, 6 mmol), and hydrogenated until H absorption stopped. The catalyst was separated. The filtrate was
2
evaporated. The resulting allylic alcohol 21 was used in the next step without further purification.
A mixture of 21 from the previous step, triethyl orthopropionate (40.3 mL, 0.24 mol), and propionic acid (1.03 mL,
13.8 mmol) in C H (130 mL) was refluxed under Ar for 2 h, cooled to room temperature, diluted with H O, and extracted
6
6
2
with EtOAc. The organic phase was dried over Na SO and evaporated. The solid was placed onto a column of SiO and
2
4
2
1
eluted by petroleum ether–EtOAc (90:10ꢅ85:15) to afford 22 (4.91 g, 94% for the two steps) as an oil. Í NMR spectrum
(500 MHz, CDCl , ꢃ, ppm, J/Hz): 5.25 (1H, dd, J = 15.2, 8.6), 5.09 (1H, dd, J = 15.2, 8.5), 4.16–4.06 (2H, m), 4.04–3.98 (1H,
3
m), 3.92–3.86 (1H, m), 3.86–3.80 (1H, m), 3.76–3.70 (1H, m), 2.39–2.26 (1H, m), 2.25–2.17 (1H, m), 2.05–1.91 (2H, m)
1.78–0.67 (18H, m), 1.25 (3H, t, J = 7.2), 1.06 (3H, d, J = 7.0), 1.00 (3H, s), 1.02–0.93 (6H, m), 0.71 (3H, s), 0.60 (1H, dd,
13
J = 8.2, 4.4), 0.32 (1H, t, J = 4.4). C NMR spectrum (125 MHz, CDCl , ꢃ, ppm): 176.5, 137.9, 130.1, 110.0, 65.0, 64.8,
3
60.2, 56.5, 56.0, 47.6, 45.8, 45.7, 42.9, 40.4, 40.3, 40.3, 40.2, 39.4, 34.3, 33.4, 28.9, 25.1, 24.4, 23.2, 22.7, 20.9, 19.5,
+
19.1, 15.2, 14.4, 12.5, 7.4. ESI-HR-MS m/z 499.3780 [M + H] . Calcd for Ñ H O , 499.3782.
32 51
4
(22E)-6-(1,3-Dioxolan-2-yl)-3ꢀ,5-cyclo-24ꢀ-methyl-5ꢀ-cholest-22-ene (23). A solution of 22 (12.4 g, 25 mmol)
in THF (300 mL) was treated in portions with LiAlH (2.8 g, 75 mmol) and stirred at room temperature for 20 min. The excess
4
of LiAlH was decomposed by successive addition of H O (2.8 mL), NaOH solution (15%, 2.8 mL), and H O (8.4 mL).
4
2
2
The precipitate was filtered off. The filtrate was evaporated. The solid was dissolved in Py (150 mL), treated with TsCl (14.2 g,
75 mmol), held at room temperature for 20 h, diluted with saturated NaCl solution, and extracted with CHCl (3 ꢄ 150 mL).
3
The extract was dried over Na SO and evaporated. The solid was dissolved in Et O (300 mL), stirred, treated in portions with
2
4
2
LiAlH (2.8 g, 75 mmol), stirred at room temperature for 45 min, and treated with H O (2.8 mL), NaOH solution (15%,
4
2
2.8 mL), and H O (8.4 mL) to decompose the excess of LiAlH . The resulting precipitate was filtered off. The filtrate was
2
4
evaporated. The solid was chromatographed over a column of SiO with elution by cyclohexane–EtOAc (30:1ꢅ5:1) to
2
1
isolate 23 (8.3 g, 76%) as an oil. Í NMR spectrum (500 MHz, CDCl , ꢃ, ppm, J/Hz): 5.16 (2Í, m, Í-22, 23), 3.73–4.05 (4Í,
3
13
m, dioxolane), 1.01 (3Í, s, 19-Ìå), 0.73 (3Í, s, 18-Ìå). C NMR spectrum (125 MHz, CDCl , ꢃ, ppm): 135.9, 131.7, 109.8,
3
64.7, 64.5, 55.9, 56.3, 42.9, 45.5, 47.3, 39.9, 42.6, 40.2, 40.1, 39.1, 28.7, 33.1, 34.0, 24.8, 22.9, 20.9, 18.9, 22.5, 17.9, 24.1, 19.5,
20.0, 12.2, 7.1.
(22E)-3ꢀ,5-Cyclo-24ꢀ-methyl-5ꢀ-cholest-22-en-6-one (24). A solution of 23 (7.76 g, 17.6 mmol) in Me CO
2
(370 mL) was treated with H O (8 mL) and PPTS (0.93 g, 3.70 mmol), held at room temperature for 8 h, and evaporated at
2
reduced pressure. The solid was chromatographed over a column of SiO with elution by petroleum ether–toluene to afford 24
2
1
(6.65 g, 95%) as an oil. Í NMR spectrum (500 MHz, CDCl , ꢃ, ppm, J/Hz): 5.21–5.10 (2H, m), 2.47–2.37 (1H, m),
3
2.06–0.68 (23H, m), 1.02–0.99 (6H, m), 0.91 (3H, d, J = 6.9), 0.83 (3H, d, J = 6.8), 0.81 (3H, d, J = 6.8), 0.72 (3H, s).
13
C NMR spectrum (125 MHz, CDCl , ꢃ, ppm): 209.8, 135.9, 132.2, 57.2, 56.0, 46.9, 46.5, 46.3, 44.9, 43.2, 42.7, 40.4,
3
+
39.8, 35.4, 34.9, 33.6, 33.3, 28.9, 26.0, 24.2, 23.0, 21.1, 20.3, 19.8, 19.8, 18.2, 12.4, 11.8. ESI-HR-MS m/z 397.3467 [M + H] .
Calcd for Ñ H O, 397.3465.
28 45
(22E)-24ꢀ-Methyl-5ꢀ-cholesta-2,22-dien-6-one (25). A mixture of 24 (5.13 g, 12.9 mmol), pyridinium bromide
(4.15 g, 26 mmol), and DMA (50 mL) was stirred, heated at 160°C under Ar for 1 h, cooled, and evaporated at reduced
pressure. The solid was partitioned between petroleum ether (40 mL) and H O (20 mL). The aqueous layer was extracted with
2
121

petroleum ether (3 ꢄ 20 mL). The combined organic phases were dried over Na SO and evaporated at reduced pressure.
2
4
The solid was chromatographed over a column of SiO with elution by petroleum ether–EtOAc (90:10) to afford 25 (4.03 g,
2
1
79%), mp 108–109°C (petroleum ether). Í NMR spectrum (500 MHz, CDCl , ꢃ, ppm, J/Hz): 5.70–5.65 (1H, m), 5.58–5.53
3
(1H, m), 5.21–5.10 (2H, m), 2.37–0.60 (22H, m), 1.00 (3H, d, J = 6.6), 0.90 (3H, d, J = 6.9), 0.83 (3H, d, J = 6.8), 0.81 (3H,
13
d, J = 6.8), 0.70 (3H, s), 0.68 (3H, s). C NMR spectrum (125 MHz, CDCl , ꢃ, ppm): 212.1, 135.9, 132.2, 125.1, 124.7, 57.0,
3
56.1, 54.0, 53.6, 47.2, 43.2, 42.9, 40.3, 40.2, 39.5, 39.5, 37.9, 33.3, 28.8, 24.1, 21.9, 21.3, 21.1, 20.3, 19.8, 18.2, 13.6, 12.3.
(22R,23R,24S)-2ꢀ,3ꢀ,22,23-Tetrahydroxy-24-methyl-5ꢀ-cholest-6-one (1) (castasterone, 1). A solution of 25
(4.79 g, 12.1 mmol) in t-BuOH (275 mL) was stirred, treated with H O (275 mL), K [Fe(CN) ] (23.7 g, 72 mmol), K CO
2
3
6
2
3
(9.95 g, 72 mmol), K OsO 42H O (178 mg, 0.48 mmol), (DHQD) AQN (1.04 g, 1.2 mmol), and CH SO NH (3.42 g,
2
4
2
2
3
2
2
36 mmol); stirred at room temperature for 2 d; treated with Na SO (18.3 g, 0.145 mol); stirred for 1 h; and diluted with H O
2
3
2
(1.5 L) and CHCl (500 mL). The aqueous phase was extracted with CHCl –MeOH (4:1, 3 ꢄ 150 mL). The combined organic
3
3
phases were washed with H SO (0.1 N, 3 ꢄ 250 mL) and dried over Na SO . The solid was filtered off. The solvent was
2
4
2
4
evaporated at reduced pressure. Double recrystallization from MeOH (300 mL, 70 mL) afforded white crystals of 1 (3.56 g,
63%), mp 250–256°C (MeOH). The spectral data of 1 agreed with those reported for castasterone [36]. ESI-HR-MS
+
m/z 469.3284 [M – H O + Na] . Calcd for C H O Na, 469.3288.
2
28 46 4
ACKNOWLEDGMENT
The work was sponsored by Russian Science Foundation (RSF) Grant No. 16-16-04057.
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