Synthetic pathway to 22,23-dioxocholestanic chain derivatives and their usefulness for obtaining brassinosteroid analogues

Article abstract of DOI:10.1016/j.steroids.2013.04.014

Recognizing the functionality of the pentacyclic steroidal derivative 7a as important synthon to obtain new brassinosteroid analogs, we have accomplished the derivatization of hecogenin, a sapogenin from the 25R serie containing a carbonyl group at C-12, to a 22,23-dioxocholestanic chain derivative. Starting from hecogenin acetate (5a) or hecogenin tosylate (5b), we obtained two pentacyclic derivatives (7a and 7b) which were subjected to an oxidation reaction on the double bond at C-12(23) to obtain a 22,23- dioxocholestanic chain, with the regeneration of the carbonyl group at C-12. Reduction of the carbonyl groups lead to the 20-epi-12,23-dihydroxy-22-oxo system 11a-b. The absolute configuration of compound 11a was established by X-ray diffraction analysis.

Full text of DOI:10.1016/j.steroids.2013.04.014

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Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthetic pathway to 22,23-dioxocholestanic chain derivatives
and their usefulness for obtaining brassinosteroid analogues
2
3
a,
Víctor Gómez-Calvario a, Ailed Arenas-González a, Socorro Meza-Reyes a, , Sara Montiel-Smith
⇑
⇑
,
4 Q1
José Luis Vega-Báez a, Jesús Sandoval-Ramírez a, María Guadalupe Hernández-Linares b
5
a Benemérita Universidad Autónoma de Puebla, Facultad de Ciencias Químicas, Ciudad Universitaria, Col. San Manuel, C.P. 72570 Puebla, Pue., Mexico
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b Escuela de Ingeniería en Petróleos e Ingeniería Química, Universidad del Istmo, Ciudad Universitaria S/N, C.P. 70760 Sto Domingo Tehuantepec, Oax., Mexico
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a r t i c l e i n f o
a b s t r a c t
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2 4
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Article history:
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Recognizing the functionality of the pentacyclic steroidal derivative 7a as important synthon to obtain
new brassinosteroid analogs, we have accomplished the derivatization of hecogenin, a sapogenin from
the 25R serie containing a carbonyl group at C-12, to a 22,23-dioxocholestanic chain derivative. Starting
from hecogenin acetate (5a) or hecogenin tosylate (5b), we obtained two pentacyclic derivatives (7a and
7b) which were subjected to an oxidation reaction on the double bond at C-12(23) to obtain a 22,23-
dioxocholestanic chain, with the regeneration of the carbonyl group at C-12. Reduction of the carbonyl
groups lead to the 20-epi-12,23-dihydroxy-22-oxo system 11a–b. The absolute configuration of com-
pound 11a was established by X-ray diffraction analysis.
Received 10 October 2012
Received in revised form 21 March 2013
Accepted 21 April 2013
Available online xxxx
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Keywords:
Hecogenin
Pentacyclic steroidal compounds
Oxidative cleavage
Ó 2013 Published by Elsevier Inc.
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22,23-Dioxocholestanic derivatives
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1. Introduction
drugs [11–13]; special attention deserve 12-oxosapogenins such as
botogenin (4, Fig. 2) and hecogenin (5) that have been used, for
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Chiral vicinal diol units are often observed as key fragments of
natural products as for example in carbohydrates [1a], some anti-
biotics [1b], vitamins [1c], steroids [1d], etc. In particular, in the
steroidal group, the brassinosteroids (BSs) have a 2a,3a-diol and
22R,23R-diol on the side chain.
The isolation of the brassinolide (1, Fig. 1) in 1979 [1d] was the
start point of the study of a new class of steroid hormones that plays
an important role in the growth, development and adaptation of
plants in the environment. Since then, many compounds related
to brassinolide, such as castasterone (2) and homobrassinolide (3)
have been identified in many natural sources [2].
Due to the importance of these compounds, a variety of meth-
odologies to obtain brassinosteroids and/or their analogs have
been developed. Initial studies showed that compounds containing
example, in the synthesis of cortisone [14] and betamethasone
[15], respectively. These syntheses require the cleavage of the spi-
roketal rings; this process has been widely investigated by many
research groups [5,16–18].
We have recently published [19] the opening of E and F rings of
12-oxosapogenins 4a and 5a, by acetolysis reaction with Ac2O and
BF3ꢀOEt2 affording the corresponding penta- and hexacyclic com-
pounds (6–9, Scheme 1).
In continuation of our studies we describe herein the use of the
pentacyclic compounds (7a–b) as strategic intermediate for the
preparation of the brassinosteroid analogs 20-epi-12,23-dihy-
droxy-22-oxocholestanic (11a–d).
the functions 2a,3a-diol, lactone or 6-ketone on ring B and
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2. Experimental methods
22R,23R-diol on the side chain (Fig. 1) possess biological activity
[3]. Nevertheless some active analogues have been synthesized
by the modification of the cholestanic chain [4–6] and the A, B
[7–8], and C [9–10] rings.
For many years, sapogenins have been utilized as a cheap raw
material in the pharmaceutical industry for the synthesis of several
2.1. General methods
NMR spectra 1D and 2D 1H and 13C (DEPT, COSY, NOESY, HSQC,
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HMBC) were recorded on
a VARIAN Mercury spectrometer
(400 MHz for 1H, 100 MHz for 13C). Chemical shifts are stated in
ppm (d), and are referred to the residual 1H signal (d = 7.27) or to
the central 13C triplet signal (d = 77.0) for CDCl3. Coupling con-
stants (J) are quoted in Hz. IR spectra were acquired on a Nicolet
⇑
Corresponding authors. Tel.: +52 222 2295500x2838/7382; fax: +52 222
2443106.
FT-IR 380 spectrophotometer (
high resolution mass (HRMS) spectra were recorded on a Jeol-
m
max; cmꢁ1Þ). FAB-MS, EI-MS and
E-mail addresses: maria.meza@correo.buap.mx (S. Meza-Reyes), maria.mon-
tiel@correo.buap.mx (S. Montiel-Smith).
0039-128X/$ - see front matter Ó 2013 Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.steroids.2013.04.014
Please cite this article in press as: Gómez-Calvario V et al. Synthetic pathway to 22,23-dioxocholestanic chain derivatives and their usefulness for obtaining
brassinosteroid analogues. Steroids (2013), http://dx.doi.org/10.1016/j.steroids.2013.04.014

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2.3. (25R)-12-Oxo-5a-spirostan-3b-yl tosylate (5b)
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430.6 mg (1.0 mmol) of hecogenin (5) was dissolved in 3.6 mL
of CH2Cl2 and 0.8 mL of pyridine. Then 1.9 g (10.0 mmol) of tosyl
chloride was added, and the mixture was stirred for 24 h at 4 °C.
The reaction mixture was washed with 5% HCl solution
(2 ꢂ 6 mL), saturated solution of NaHCO3 (2 ꢂ 12 mL), and brine
(2 ꢂ 12 mL), and water (20 mL). The resulting organic phase was
dried over Na2SO4 and concentrated to dryness under vacuum.
The crude reaction was separated by column chromatography
(4:1 hexane–EtOAc) to afford 5b (94%).
Colorless solid, mp 179–181 °C, [
a
]
D
ꢁ17° (c 0.1, CHCl3) (Lit.
Fig. 1. Structures of some brassinosteroids.
ꢁ15.7° [21]). IR
m
max: 1706, 1356, 1446, 1180. 1H NMR d: 7.78
(2H, d, J = 8.4 Hz, Hortho), 7.33 (2H, d, J = 8.0 Hz, Hmeta), 4.36 (2H,
m, H-3 y H-16), 3.48 (1H, ddd, Jgem = 10.4 Hz, J26eq–25ax = 6.0 Hz,
J26eq–24eq = 2.0 Hz, H-26eq), 3.34 (1H, dd, Jgem = J26ax–25ax = 10.4 Hz,
H-26ax), 2.45 (3H, s, CH3-Ts), 1.05 (3H, d, J = 6.8 Hz, CH3-21), 1.03
(3H, s, CH3-18), 0.87 (3H, s, CH3-19), 0.78 (3H, d, J = 6.0 Hz, CH3-
27). 13C NMR d: 213.2 (C-12), 144.4 (Cipso), 134.4 (Cpara), 129.7
(Cmeta), 127.5 (Cortho), 109.2 (C-22), 81.6 (C-3), 79.0 (C-16), 66.8
(C-26), 55.5 (C-14), 55.0 (C-9 and C-13), 53.4 (C-17), 44.4 (C-5),
42.1 (C-20), 37.6 (C-11), 36.1 (C-1), 35.7 (C-10), 34.6 (C-4), 34.1
(C-8), 31.3 (C-7), 31.2 (C-23), 31.0 (C-24), 30.1 (C-25), 28.7 (C-
15), 28.0 (C-2), 27.9 (C-6), 21.6 (CH3-Ts), 17.1 (C-27), 15.9 (C-18),
13.2 (C-21), 11.7 (C-19). HRMS-EI (m/z): [M]+ calcd for
C34H48O6S: 584.3172, found 584.3162.
Fig. 2. Steroidal sapogenins containing a carbonyl group at C-12.
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JMS-700 MS Station spectrometer. Optical rotations were deter-
mined on a Perkin Elmer 241 polarimeter at room temperature
using chloroform solutions. Melting points were measured from
a Mel-Temp apparatus and were not corrected. Analytical TLC
was performed on silica gel ALUGRAMÒ SIL G/UV254 plates and
chromatographic columns were carried out on silica gel Davisil™
grade 633 (200–425 mesh).
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2.4. General procedure for the formation of polycyclic steroidal
derivatives 7a and 7b
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1 mmol of sapogenin derivative (5a or 5b) was dissolved in
10.7 mL of CH2Cl2, then 10.0 mmol of acetic anhydride and
4.5 mmol of BF3ꢀOEt2 were added. The mixture was stirred at room
temperature, and monitored by TLC until complete disappearance
of starting material (20 min). The reaction mixture was poured into
a saturated solution of NaHCO3 in methanol/H2O (1:1) and vigor-
ously shaken (10 min). The organic phase was extracted with
50.0 mL of CH2Cl2, and the reaction mixture was washed with
brine (3 ꢂ 10 mL), followed by saturated solution of NaHCO3
(3 ꢂ 10 mL) and finally with water (1 ꢂ 10 mL). The resulting or-
ganic phase was dried over Na2SO4 and concentrated to dryness
under vacuum. The crude reaction was dissolved in 0.5 mL of pyr-
idine and 10.0 mmol of acetic anhydride, the mixture was stirred at
room temperature during 30 min. Then the reaction mixture was
washed with 5% HCl solution (1 ꢂ 5 mL), followed by saturated
solution of NaHCO3 (3 ꢂ 10 mL), brine (3 ꢂ 10 mL), and finally with
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2.2. Crystal structure determination
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Crystals of 11a suitable for X-ray were obtained from hexane–
methylene chloride (99:1), by slow evaporation of the solvent at
room temperature. Data collection was performed at 293(2) K
Xcalibur, Atlas, Gemini diffractometer with Cu
Ka-radiation,
k = 1.54184 Å. The structure was solved by direct methods and
refined using SHELXL-97 [20]. Crystallographic data have been
deposited at the Cambridge Crystallographic Center No. CCDC
892961. In the absence of anomalous dispersion, the absolute
configuration was set according to the starting material and Friedel
pairs were merged.
Scheme 1. Structures obtained from acetolysis of botogenin (4a) and hecogenin (5a) acetates.
Please cite this article in press as: Gómez-Calvario V et al. Synthetic pathway to 22,23-dioxocholestanic chain derivatives and their usefulness for obtaining
brassinosteroid analogues. Steroids (2013), http://dx.doi.org/10.1016/j.steroids.2013.04.014

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water (30 mL). The resulting organic phase was dried over Na2SO4
and concentrated to dryness under vacuum. The residue was puri-
fied by column chromatography (4:1 hexane–EtOAc) affording 7a
(57%) [19] or 7b (51%).
2.5.2. (20S,25R)-12,22,23-Trioxo-5
diacetate 3-tosylate (10b)
a
-cholesta-3b,16b,26-triyl 16,26-
Yellow solid, mp 77–79 °C, [
a
]
D
+58° (c 0.1, CHCl3), IR mmax:
1734, 1709, 1456, 1362, 1238, 1175, 1030. 1H NMR d: 7.77 (2H,
d, J = 8.0 Hz, Hortho), 7.33 (2H, d, J = 8.4 Hz, Hmeta), 5.14 (1H, ddd,
J16–17 = J16–15a = 7.0 Hz, J16–15b = 3.4 Hz, H-16), 4.35 (1H, m, H-3),
3.95 (1H, dd, Jgem = 10.8 Hz, J26a–25 = 6.0 Hz, H-26a), 3.89 (1H, dd,
Jgem = 10.8 Hz, J26b–25 = 6.0 Hz, H-26b), 3.34 (1H, dq, J20–
17 = 11.2 Hz, J20–21 = 6.8 Hz, H-20), 2.97 (1H, dd, Jgem = 18.4 Hz,
J24a–25 = 6.8 Hz, H-24a), 2.80 (1H, dd, Jgem = 18.4 Hz, J24b–
25 = 6.8 Hz, H-24b), 2.53 (1H, m, H-15a), 2.44 (3H, s, CH3-Ts), 2.05
(3H, s, CH3CO2-16), 2.04 (3H, s, CH3CO2-26), 1.24 (3H, s, CH3-18),
1.14 (3H, d, J = 7.2 Hz, H-21), 1.01 (3H, d, J = 6.8 Hz, CH3-27), 0.84
(3H, s, CH3-19). 13C NMR d: 214.2 (C-12), 199.2 (C-22), 196.9 (C-
23), 170.9 (CH3CO2-26), 169.9 (CH3CO2-16), 144.3 (Cipso), 134.2
(Cpara), 129.6 (Cmeta), 127.4 (Cortho), 81.3 (C-3), 73.4 (C-16), 68.3
(C-26), 56.0 (C-13), 55.0 (C-9), 53.2 (C-14), 50.0 (C-17), 44.1 (C-
5), 39.3 (C-24), 37.2 (C-20 and C-11), 35.9 (C-1), 35.5 (C-10), 34.3
(C-4 and C-15), 34.0 (C-8), 30.5 (C-7), 27.9 (C-25), 27.8 (C-2),
27.6 (C-6), 21.4 (CH3-Ts), 21.0 (CH3CO2-16), 20.8 (CH3CO2-26),
16.9 (C-27), 14.7 (C-21), 12.9 (C-18), 11.4 (C-19). HRMS-FAB (m/
z): [M+H]+ calcd for C38H52O10S: 701.3359, found 701.3327.
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2.4.1. (20S,25R)-12,23-Cyclo-22-oxo-5
3b,16b,26-triyl 16,26- diacetate 3-tosylate (7b)
Colorless solid, mp 74–76 °C, [ +68° (c 0.1, CHCl3); IR mmax:
a-cholest-12(23)-ene-
a]
D
1736, 1659, 1361, 1240. 1H NMR d: 7.79 (2H, d, J = 8.4 Hz, Hortho),
7.34 (2H, d, J = 8.4 Hz, Hmeta), 5.23 (1H, ddd, J16–17 = J16–
15a = 6.6 Hz, J16–15b = 3.0 Hz, H-16), 4.41 (1H, m, H-3), 3.88 (1H,
dd, Jgem = 10.4 Hz, J26a–25 = 6.0 Hz, H-26a), 3.81 (1H, dd, Jgem = 10.4 -
Hz, J26b–25 = 6.0 Hz, H-26b), 2.45 (3H, s, CH3-Ts), 2.04 (6H, s, CH3-
CO2-16 and -26), 1.12 (3H, d, J = 6.8 Hz, H-21), 1.04 (3H, s, CH3-
18), 0.89 (3H, s, CH3-19), 0.81 (3H, d, J = 6.8 Hz, CH3-27). 13C
NMR d: 201.5 (C-22), 176.1 (CH3CO2-26), 170.4 (CH3CO2-16),
164.1 (C-12), 144.4 (Cipso), 134.5 (Cpara), 130.5 (C-23), 129.7 (Cmeta),
127.5 (Cortho), 81.5 (C-3), 73.3 (C-16), 68.9 (C-26), 55.3 (C-17), 55.0
(C-9), 53.9 (C-14), 44.7 (C-5), 43.3 (C-13), 38.9 (C-20), 36.4 (C-1),
35.7 (C-10), 34.8 (C-15), 34.7 (C-4), 33.5 (C-8), 32.6 (C-25), 31.2
(C-6), 28.1 (C-24 and C-7), 27.8 (C-2), 25.2 (C-11), 21.6 (CH3-Ts),
21.1 (CH3CO2-16), 20.9 (CH3CO2-26), 16.6 (C-27), 15.7 (C-18),
13.1 (C-21), 12.0 (C-19). HRMS-FAB (m/z): [M+H]+ calcd for
C38H52O8S: 669.3461, found 669.3461.
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2.6. (20S,25R)-12,22,23-Trioxo-5a-cholest-2-ene-16b,26-diyl
diacetate (10c)
2.5. General procedure for the oxidative cleavage of D12(23)
100 mg (0.2 mmol) of the compound 10b was dissolved in
3.0 mL of DMF; 140 mg (1.6 mmol) of LiBr and 120 mg (1.6 mmol)
of Li2CO3 were added. The mixture was refluxed throughout 1 h.
After this time, the reaction mixture was cooled to room tempera-
ture, and 3.0 mL of 10% HCl solution were added. The solid was fil-
tered off, washed with water and dried under vacuum. The crude
reaction was purified by column chromatography (9:1 hexane–
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Compound 7a or 7b (0.2 mmol) was dissolved in a solution of
3.5 mL of CH2Cl2, 1.5 mL of acetone, 1.5 mL of MeCN. Subsequently
10.0 mg (0.05 mmol) of ruthenium (III) chloride in a solution of
NaIO4 (162 mg, 0.8 mmol) in water (0.7 mL) was added. The mix-
ture was magnetically stirred at room temperature and monitored
by TLC until complete disappearance of starting material (50 min).
Then, 3.6 mL of isopropyl alcohol was added to consume the excess
of the oxidant reagent. The reaction mixture was filtered over silica
gel. The organic phase was extracted with 50.0 mL of CH2Cl2. The
EtOAc), affording 10c (82%). Colourless oil, [
a]D +90° (c 0.14, CHCl3),
IR
m
max: 1780, 1736, 1704, 1457, 1376, 1237, 1026. 1H NMR d: 5.60
(1H, m, H-3), 5.54 (1H, m, H-2), 5.16 (1H, ddd, J16–17 = J16–
15a = 7.2 Hz, J16–15b = 3.2 Hz, H-16), 3.97 (1H, dd, Jgem = 10.8 Hz,
J26a–25 = 6.0 Hz, H-26a), 3.91 (1H, dd, Jgem = 10.8 Hz, J26b–
25 = 6.0 Hz, H-26b), 3.34 (1H, dq, J20–17 = 11.6 Hz, J20–21 = 6.8 Hz, H-
20), 2.96 (1H, dd, Jgem = 18.4 Hz, J24a–25 = 6.8 Hz, H-24a), 2.83 (1H,
dd, Jgem = 18.4 Hz, J24b–25 = 7.2 Hz, H-24b), 2.57 (1H, m, H-15a),
2.31 (1H, dd, J17–20 = 11.6 Hz, J17–16 = 7.0 Hz, H-17), 2.06 (6H, s, CH3-
CO2-16 and -26), 1.27 (3H, s, CH3-18), 1.16 (3H, d, J = 6.8 Hz, H-21),
1.02 (3H, d, J = 6.8 Hz, CH3-27), 0.80 (3H, s, CH3-19). 13C NMR d:
215.0 (C-12), 199.4 (C-22), 197.0 (C-23), 171.1 (CH3CO2-26), 170.1
(CH3CO2-16), 125.7 (C-3), 125.0 (C-2), 73.7 (C-16), 68.5 (C-26),
56.0 (C-13), 55.3 (C-9), 53.5 (C-14), 50.0 (C-17), 41.0 (C-5), 39.5
(C-24), 38.8 (C-11), 37.6 (C-20), 37.2 (C-1), 35.0 (C-10), 34.5 (C-4),
34.3 (C-8), 30.6 (C-7), 29.8 (C-15), 28.1 (C-6), 28.0 (C-25), 21.1 (CH3-
CO2-16), 20.9 (CH3CO2-26), 17.0 (C-27), 14.9 (C-21), 13.0 (C-18),
11.2 (C-19). HRMS-FAB (m/z): [M+H]+ calcd for C31H44O7:
529.3165, found 529.3207.
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residue was washed with
a
solution (10%) of NaHSO3
(3 ꢂ 30 mL), saturated solution of NaHCO3 (3 ꢂ 30 mL), with brine
(2 ꢂ 30 mL) and finally with water (40 mL). The resulting organic
phase was dried over Na2SO4 and concentrated to dryness under
vacuum. The crude reaction was purified by column chromatogra-
phy (4:1 hexane–EtOAc) affording 10a (64%) or 10b (65%).
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2.5.1. (20S,25R)-12,22,23-Trioxo-5
a
-cholesta-3b,16b,26-triyl
triacetate (10a)
Yellow oil, [a]D +24.5° (c 0.13, CHCl3); IR mmax: 1785, 1732, 1707,
1368. 1H NMR d: 5.09 (1H, ddd, J16–17 = J16–15a = 7.0 Hz, J16–
15b = 3.4 Hz, H-16), 4.58 (1H, m, H-3), 3.89 (1H, dd, Jgem = 10.4 Hz,
J26a–25 = 6.0 Hz, H-26a), 3.84 (1H, dd, Jgem = 10.4 Hz, J26b–
25 = 6.0 Hz, H-26b), 3.28 (1H, dq, J20–17 = 11.6 Hz, J20–21 = 6.4 Hz,
H-20), 2.89 (1H, dd, Jgem = 18.4 Hz, J24a–25 = 6.6 Hz, H-24a), 2.75
(1H, dd, Jgem = 18.4 Hz, J24b–25 = 6.8 Hz, H-24b), 1.98 (6H, s, CH3CO2-
16 and -26), 1.94 (3H, s, CH3CO2-3), 1.18 (3H, s, CH3-18), 1.10 (3H,
d, J21–20 = 6.4 Hz, CH3-21), 0.95 (3H, d, J27–25 = 7.2 Hz, CH3-27), 0.81
(3H, s, CH3-19); 13C NMR d: 214.5 (C-12), 199.4 (C-22), 197.0 (C-
23), 171.1 (CH3CO2-26), 170.5 (CH3CO2-16), 170.1 (CH3CO2-3),
73.7 (C-16), 73.0 (C-3), 68.5 (C-26), 55.4 (C-13), 53.4 (C-9), 56.1
(C-14), 50.1 (C-17), 44.3 (C-5), 39.5 (C-24), 37.5 (C-20), 37.4 (C-
11), 36.1 (C-1), 35.9 (C-10), 34.5 (C-15), 33.6 (C-4), 34.2 (C-8),
30.7 (C-7), 28.1 (C-25), 27.1 (C-2), 27.9 (C-6), 21.3 (CH3CO2-26),
21.1 (CH3CO2-16), 20.9 (CH3CO2-3), 17.0 (C-27), 14.9 (C-21), 13.1
(C-18), 11.7 (C-19). HRMS-FAB (m/z): [M+H]+ calcd for C33H48O9:
589.3377, found 589.3377.
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201
252
2.7. General procedure for the carbonyl groups reduction
253
254
255
256
257
258
259
260
261
1 mmol of compound (10a or 10b or 10c) was dissolved in
25.0 mL of methanol, and 0.5 mmol of NaBH4 was added. The mix-
ture was magnetically stirred at room temperature for 5 min and
then 10.0 mL of water were added. The organic phase was extracted
with EtOAc (4 ꢂ 50.0 mL), washed with brine (2 ꢂ 100 mL) and fi-
nally with water (50 mL). The organic phase was dried over Na2SO4
and concentrated to dryness under vacuum. The crude reaction was
purified by column chromatography (4:1 hexane–EtOAc) affording
11a (54%), or 11b (47%), or 11c (35%).
Please cite this article in press as: Gómez-Calvario V et al. Synthetic pathway to 22,23-dioxocholestanic chain derivatives and their usefulness for obtaining
brassinosteroid analogues. Steroids (2013), http://dx.doi.org/10.1016/j.steroids.2013.04.014

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264
265
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326
327
328
329
330
2.7.1. (20R,23R,25R)-12b,23-Dihydroxy-22-oxo-5
3b,16b,26-triyl triacetate (11a)
a
-cholesta-
37.5 (C-20), 36.4 (C-24), 34.4 (C-10), 34.3 (C-15), 33.9 (C-8), 30.8
(C-4 and C-11), 29.9 (C-7), 29.4 (C-25), 28.2 (C-6), 21.2 (CH3CO2-
26), 20.8 (CH3CO2-16), 16.8 (C-21), 15.6 (C-27), 11.4 (C-19), 8.0
(C-18). HRMS-FAB (m/z): [M+H]+ calcd for C31H48O7: 533.3478,
found 533.3364.
Colourless crystal, mp 163–165 °C, [
a]D +21.5° (c 0.79, CHCl3), IR
m
max: 3497, 3448, 1729, 1369, 1240. 1H NMR d: 5.16 (1H, ddd, J16–
17 = J16–15a = 7.2 Hz, J16–15b = 3.2 Hz, H-16), 4.66 (1H, m, H-3), 4.34
(1H, m, H-23), 3.97 (2H, m, H-26), 3.67 (1H, d, J23(OH)–23 = 6.0 Hz,
23-OH), 3.23 (1H, m, H-12), 3.10 (1H, dq, J20–17 = 10.8 Hz, J20–
21 = 6.4 Hz, H-20), 2.45 (1H, m, H-15a), 2.19 (1H, m, H-25), 2.06
(3H, s, CH3CO2-26), 2.06 (3H, s, CH3CO2-3), 2.01 (3H, s, CH3CO2-
16), 1.05 (3H, d, J27–25 = 6.4 Hz, CH3-27), 0.99 (3H, d, J21–
20 = 6.8 Hz, CH3-21), 0.87 (3H, s, CH3-18), 0.80 (3H, s, CH3-19);
13C NMR d: 215.1 (C-22), 171.2 (CH3CO2-26), 170.6 (CH3CO2-3),
170.3 (CH3CO2-16), 76.4 (C-12), 75.2 (C-16), 74.8 (C-23), 73.3 (C-
3), 69.5 (C-26), 55.7 (C-17), 52.5 (C-9), 52.2 (C-14), 46.8 (C-13),
44.3 (C-5), 37.5 (C-20), 36.5 (C-24), 36.4 (C-1), 35.3 (C-10), 34.5
(C-15), 33.9 (C-8), 33.7 (C-11), 31.2 (C-4), 31.0 (C-7), 29.4 (C-25),
28.1 (C-6), 27.2 (C-2), 21.3 (CH3CO2-26), 21.2 (CH3CO2-3), 20.9
(CH3CO2-16), 16.8 (C-21), 15.7 (C-27), 12.0 (C-19), 8.1 (C-18).
HRMS-FAB (m/z): [M+H]+ calcd for C33H52O9: 593.3690, found
593.3689. Crystal data: C33H52O9, colorless prisms, temperature
293(2) K, wavelength 1.54184 Å, crystal system monoclinic, space
267
268
269
331
332
2.8. (20R,23R,25R)-2
a,3a,12b,23-Tetrahydroxy-22-oxo-5a-cholest-
3b,16b,26-triyl triacetate (11d)
270
271
272
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
100 mg (0.2 mmol) of compound 11c was dissolved in 1.6 mL of
THF and 1.0 mL of a 50% NMO aqueous solution. Then a solution of
OsO4 (3.0 mg, 0.01 mmol) in tert-butanol (2.5 mL) was added. The
mixture was magnetically stirred at room temperature and moni-
tored by TLC until complete disappearance of starting material
(16 h). After this time, 2.0 mL of a 15% sodium sulfite aqueous solu-
tion was added and was magnetically stirred at room temperature
(2 h). Then the THF was evaporated under vacuum, and the organic
phase was extracted with 100.0 mL of EtOAc. The crude reaction
was washed with brine (2 ꢂ 30 mL), and finally with water
(30 mL). The resulting organic phase was dried over Na2SO4 and
concentrated to dryness under vacuum. The crude reaction was
purified by column chromatography (1:4 hexane–EtOAc) to afford
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
gropup
b = 8.1811(2) Å, c = 15.5362(4) Å,
V = 1644.26(8) Å3. Z = 2, Dx = 1.197 g/cmꢁ3
P21,
unit
cell
dimensions
a = 13.3953(4) Å,
105.0403°, = 90°,
= 0.698 mmꢁ1
a
90°,
b
c
11d (67%). Colourless solid, mp 177–178 °C, [
a]
D
+14° (c 0.1,
,
l
,
CHCl3). IR
m
max: 3381, 1733, 1666, 1588, 1498, 1245, 1043. 1H
F(000) = 644.0. Collected reflections: 64694 with in a theta range
of 2.95–74.12° (ꢁ16 6 h 6 16, ꢁ10 6 k 6 8, ꢁ19 6 l 6 19) . Final
NMR d: 5.14 (1H, ddd, J16–17 = J16–15a = 7.2 Hz, J16–15b = 3.6 Hz, H-
16), 4.37 (1H, dd, J23–24a = 10.8 Hz, J23–24b = 2.8 Hz, H-23), 3.96
(3H, m, H-26 and H-3), 3.67 (1H, m, H-2), 3.19 (1H, dd, J12ax–
11ax = 10.8 Hz, J12ax–11eq = 4.8 Hz, H-12), 3.10 (1H, dq, J20–
17 = 10.8 Hz, J20–21 = 6.4 Hz, H-20), 2.44 (1H, m, H-15a), 2.17 (1H,
m, H-25), 2.06 (6H, s, CH3CO2-26 and -16), 1.99 (1H, m, H-17),
1.05 (3H, d, J = 6.8 Hz, CH3-27), 0.97 (3H, d, J = 6.0 Hz, H-21), 0.87
(3H, s, CH3-18), 0.78 (3H, s, CH3-19). 13C NMR d: 215.8 (C-22),
171.5 (CH3CO2-26), 170.6 (CH3CO2-16), 76.0 (C-12), 75.4 (C-16),
74.8 (C-23), 69.6 (C-26), 69.0 (C-3), 68.9 (C-2), 55.8 (C-17), 52.5
(C-9), 52.1 (C-14), 46.7 (C-13), 40.2 (C-1), 37.9 (C-5), 37.5 (C-20),
36.6 (C-10), 36.0 (C-24), 34.4 (C-15), 34.1 (C-4), 33.2 (C-8), 30.9
(C-11), 30.7 (C-7), 29.4 (C-25), 27.3 (C-6), 21.2 (CH3CO2-26), 20.8
(CH3CO2-16), 16.9 (C-21), 15.5 (C-27), 12.2 (C-19), 8.1 (C-18).
HRMS-FAB (m/z): [M+H]+ calcd for C31H50O9: 567.3533, found
567.3582.
R indices [I > 2r(I)], R1 = 0.0488, wR2 = 0.1285; R indices [all data],
R1 = 0.0639, wR2 = 0.1425. Largest diff. peak and hole, 0.248 and
ꢁ0.213 e Åꢁ3
.
352
353
354
355
356
357
358
359
360
361
362
363
291
292
293
294
295
296
297
2.7.2. (20R,23R,25R)-12b,23-Dihydroxy-22-oxo-5
a-cholesta-
3b,16b,26-triyl 16,26-diacetate 3-tosylate (11b)
Colorless oil, IR m
max: 3495, 1735, 1715, 1359, 1175, 1032. 1H
NMR d: 7.77 (2H, d, J = 8.4 Hz, Hortho), 7.33 (2H, d, J = 8.4 Hz, Hmeta),
5.14 (1H, ddd, J16–17 = J16–15a = 7.2 Hz, J16–15b = 3.6 Hz, H-16), 4.35
(2H, m, H-23 and H-3) 3.96 (2H, m, H-26), 3.63 (1H, d, J23(OH)–
23 = 6.0 Hz, 23-OH), 3.20 (H, dd, J12ax–11ax = 6.4 Hz, J12ax–11eq = 3.2 -
298
299
300
301
302
303
304
305
306
307
308
309
310
Hz, H-12), 3.10 (1H, dq, J
= 11.0 Hz, J20–21 = 6.8 Hz, H-20), 2.45
(3H, s, CH3-Ts), 2.07 (3H,2s0–,17CH3CO2-26), 2.05 (3H, s, CH3CO2-16),
1.04 (3H, d, J = 6.8 Hz, CH3-27), 0.98 (3H, d, J = 6.4 Hz, H-21), 0.85
(3H, s, CH3-18), 0.75 (3H, s, CH3-19). 13C NMR d: 215.2 (C-22),
171.2 (CH3CO2-26), 170.3 (CH3CO2-16), 144.4 (Cipso), 134.5 (Cpara),
129.7 (Cmeta), 127.5 (Corto), 81.9 (C-3), 76.4 (C-12), 75.2 (C-16),
74.9 (C-23), 69.5 (C-26), 55.7 (C-17), 52.3 (C-9), 52.1 (C-14), 46.8
(C-13), 44.4 (C-5), 37.5 (C-20), 36.6 (C-24), 36.4 (C-1), 34.9 (C-
10), 34.5 (C-15), 34.4 (C-11), 33.7 (C-8), 31.1 (C-4), 30.9 (C-7),
29.5 (C-25), 28.1 (C-2), 27.9 (C-6), 21.6 (CH3-Ts), 21.3 (CH3CO2-
26), 20.1 (CH3CO2-16), 16.8 (C-21), 15.7 (C-27), 11.9 (C-19), 8.1
(C-18). HRMS-FAB (m/z): [M]+ calcd for C38H56O10S: 704.3594,
found 704.3558.
364
3. Results and discussion
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
The compound 7a described by our research group [19] is very
stable and offers a new way to generate 12,22,23-trioxocholestanic
derivatives. In addition, in this work we report the formation of 7b
containing a tosylate group at C-3 in order to produce both the
oxocholestanic chain and the cis-2,3-diol function.
The hecogenin acetate (5a) (commercially available) was trea-
ted under basic hydrolysis conditions to afford hecogenin (5),
which was subsequently tosylated generating derivative 5b.
The treatment of 5a or 5b with BF3ꢀOEt2 in dichloromethane at
room temperature afforded the pentacyclic compounds (7a–b,
Scheme 2). The tosylate group is stable under the reaction condi-
tions providing a slightly lower yield of 7b (51%), compared with
7a (57%).
311
312
313
314
315
316
2.7.3. (20R,23R,25R)-12b,23-Dihydroxy-22-oxo-5a-cholest-2-ene-
3b,16b,26-triyl triacetate (11c)
Colorless solid, mp 136–138 °C, [
a] +66.4° (c 0.33, CHCl3). IR
D
m
max: 3414, 1732, 1725, 1657, 1470, 1236, 1056. 1H NMR d: 5.60
(1H, m, H-3), 5.54 (1H, m, H-2), 5.16 (1H, ddd, J16–17 = J16–
15a = 6.8 Hz, J16–15b = 3.6 Hz, H-16), 4.37 (1H, d, J23–24a = 8.4 Hz, H-
23), 3.97 (2H, m, H-26), 3.84 (1H, d, J23(OH)–23 = 3.6 Hz, 23-OH),
3.23 (1H, dd, J12ax–11ax = 10.8 Hz, J12ax–11eq = 4.4 Hz, H-12), 3.10
(1H, dq, J20–17 = 11.0 Hz, J20–21 = 6.4 Hz, H-20), 2.47 (1H, m, H-
15a), 2.19 (1H, m, H-25), 2.07 (3H, s, CH3CO2-26), 2.06 (3H, s, CH3-
CO2-26), 1.06 (3H, d, J = 6.4 Hz, CH3-27), 0.99 (3H, d, J = 6.4 Hz, H-
21), 0.89 (3H, s, CH3-18), 0.73 (3H, s, CH3-19). 13C NMR d: 215.1
(C-22), 171.2 (CH3CO2-26), 170.4 (CH3CO2-16), 125.8 (C-3), 125.3
(C-2), 76.4 (C-12), 75.3 (C-16), 74.8 (C-23), 69.5 (C-26), 55.7(C-
17), 52.4 (C-9), 52.3 (C-14), 46.7 (C-13), 41.1 (C-5), 39.3 (C-1),
317
318
319
320
321
322
323
324
325
The spectroscopy data of the pentacyclic compound 7a was
compared with that reported in the literature [19]. The main differ-
ences between 7a and 7b in 1H NMR are the signals of the tosyl
group as well as the nearby hydrogen of steroidal skeleton H-3
and H-19 (Table 1).
Due to the presence of the tosyl group, the 13C NMR signal for C-
3 of compound 7b appear at higher frequencies with respect to 7a
(Table 2).
Please cite this article in press as: Gómez-Calvario V et al. Synthetic pathway to 22,23-dioxocholestanic chain derivatives and their usefulness for obtaining
brassinosteroid analogues. Steroids (2013), http://dx.doi.org/10.1016/j.steroids.2013.04.014

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Scheme 2. Synthetic pathway for obtaining 12,23-dihydroxy-22-oxocholestanic compounds 11a-d from hecogenin acetate (5a).
Table 1
Table 4
Selected 1H chemical shifts d (ppm) of 7a–b in CDCl3.
Selected 13C chemical shifts (d, ppm) of 10a–c in CDCl3.
Compd.
Hortho
Hmeta
CH3-Ts
H-3
H-19
Compd.
C-2
C-3
C-12
C-22
C-23
7a
7b
–
7.79
–
7.34
–
2.45
4.68
4.41
0.93
0.89
10a
10b
10c
–
–
73.0
81.3
125.7
214.6
214.2
215.0
199.4
199.2
199.4
197.1
196.9
197.0
125.0
Table 2
Selected 13C chemical shifts d (ppm) of 7a–b in CDCl3.
Table 5
Influence of quantities of NaBH4 in the distribution of compounds 11a–c.
Compd.
C-3
Ipso
Ortho
Meta
Para
7a
7b
73.0
81.5
–
144
–
–
–
Entry Molar ratio steroid/reducing agent (10a–
c/NaBH4)
11a
(%)
11b
(%)
11c
(%)
127.5
129.7
134.5
1
2
3
4
2.0:0.5
2.0:1.0
2.0:2.0
2.0:3.0
40
54
35
32
42
47
45
46
31
35
34
31
386
387
388
389
390
391
392
393 Q2
394
395
396
397
398
The pentacyclic products 7a–b exhibit one double bond at C-12
and C-23, this characteristic gives them high potential as precur-
sors of steroids containing a 22,23-dioxocholestanic side chain as
10a–b; for this purpose we performed an oxidation reaction over
the double bond with RuO4, resulting in the opening of the fused
ring. The new compounds were obtained with retention of config-
uration of all asymmetric centers and the carbonyl group at C-12
was regenerated (Scheme 2). This new methodology is very useful
in the synthesis of C-22 and C-23 functionalized cholestanic chains.
Compounds 10a–c reacted with NaBH4 for 5 min, at room temperature, in
methanolic solution.
a
The
D
2-derivative (10c) was prepared by elimination of the tosyl-
ate group in 10b (Scheme 2).
In 1H NMR spectra of derivatives 10a–c are observed an ABX
system for the diasterotopic protons H-26a (3.95–3.97 ppm) and
Table 3
Selected 1H chemical shifts d (ppm) of 10a–c in CDCl3.
Compd.
H-26a
H-26b
H-20
H-24a
H-24b
10a
10b
10c
3.96
3.95
3.97
3.90
3.89
3.91
3.34
3.34
3.34
2.96
2.97
2.96
2.82
2.80
2.83
Fig. 3. Data from X-ray diffraction of 11a. Ellipsoids are drawn at the 30%
probability level.
Please cite this article in press as: Gómez-Calvario V et al. Synthetic pathway to 22,23-dioxocholestanic chain derivatives and their usefulness for obtaining
brassinosteroid analogues. Steroids (2013), http://dx.doi.org/10.1016/j.steroids.2013.04.014

STE 7389
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V. Gómez-Calvario et al. / Steroids xxx (2013) xxx–xxx
Table 6
Selected 1H chemical shifts (d, ppm) of 11a–c in CDCl3.
Compd.
H-2
H-16
H-3
H-23
H-26
23-OH
H-12
H-20
H-25
27
21
18
19
11a
11b
11c
11da
–
–
5.54
3.67
5.15
5.14
5.16
5.14
4.66
4.35
5.60
3.96
4.34
4.35
4.37
4.37
3.97
3.96
3.97
3.96
3.67
3.63
3.83
–
3.23
3.20
3.23
3.19
3.10
3.10
3.10
3.10
2.19
–
2.19
2.67
1.05
1.04
1.06
1.05
0.99
0.98
0.99
0.97
0.87
0.85
0.89
0.73
0.80
0.75
0.73
0.78
a
For this compound it was added a few drops of a MeOD.
Table 7
Selected 13C chemical shifts (d, ppm) of 11a–d in CDCl3.
Compd.
C-2
C-3
C-12
C-20
C-21
C-22
C-23
C-25
C-26
C-27
11a
11b
11c
27.2
–
125.3
68.9
73.3
81.9
125.8
69.0
76.4
76.4
76.4
76.3
37.5
37.5
37.5
37.5
16.8
16.8
16.8
16.9
215.1
215.2
215.1
215.8
74.8
74.9
74.8
75.4
29.4
29.5
29.4
29.4
69.5
69.5
69.5
69.6
15.7
15.7
15.6
15.5
11da
a
For this compound it was added a few drops of a MeOD.
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
H-26b (3.89–3.91 ppm), these protons exhibit a long distance cor-
relations with C-24, C-25 and C-27 as deduced by the HMBC exper-
iment. The 1H NMR spectra also show signals for H-20 and H-24
shifted to higher frequencies due a deshielding effect caused by
spectra with 11a suggested that compounds 11a–c have the same
configuration at the new chiral centers in C-12 and C-23 (12R,23R),
including the epimerization at C-20 (now R) (Tables 6 and 7).
Finally, Table 6 shows the selected 1H NMR chemical shifts of
11a–d, it is important to notice the values for the new chiral cen-
ters formed after reduction. Chemical shifts for protons H-23 are
observed at 4.35–4.37 ppm, these protons exhibit interactions at
the HMBC experiment with C-24, C-25. The signal assigned to
H-12 at 3.19–3.23 ppm, is deduced from its correlation with C-
18, C-13 and C-17. Analogously, the assignment of H-20 (at
3.10 ppm for 11a–d) was achieved from its correlations with C-
21, C-13, and C-17. IR spectroscopy confirms the presence of alco-
the a-carbonyl groups at C-22 and C-23, respectively. The H-20 is
observed as a doublet of quadruplet signal at 3.34 ppm (see Table
3), the signal of the diasterotopic protons on C-24 is displayed as an
ABM system (2.96–2.97 and 2.80–2.83 ppm, respectively).
The 13C NMR spectra of compounds 10a–c show the C-12
ketone carbonyl signals between 214.2 and 215.0 ppm, while those
of the dicarbonyl system at C-22 and C23 appear at 199.2–199.4
and 196.9–197.1 ppm, respectively (see Table 4), indicating the
existence of a conjugated
p
system. In the spectrum for 10c signals
hol groups with a broad band around 3500 cmꢁ1
.
for the vinyl carbons C-2 and C-3 are present at 125.0 and
127.7 ppm, respectively.
Compounds 10a–c were reduced using NaBH4 in a methanolic
solution in different molar ratios of steroid/NaBH4. The principal
products were those derivatives where the carbonyl groups at
C-12 and C-23 were reduced (11a–c, Scheme 2).
The relative ratio steroid/reducing agent is shown in Table 5.
For the reduction of C-12 and C-23 carbonyl groups, the best yields
observed were obtained using a small excess of NaBH4 (entry 2).
The addition of more equivalents of reducing agent did not lead
to the reduction of carbonyl group on C-22.
The attack of the hydride nucleophile on the Re or the Si faces of
each carbonyl group should produce a mixture of diasteroisomers.
The result was, in effect, a mixture of compounds with a very sim-
ilar retention factors (Rf). From these mixtures, compounds 11a–c
were isolated as major products.
In conclusion, we reported that the pentacyclic steroidal com-
pounds (7a–b) are important intermediates to obtain the
12,22,23-trioxocholestanic chain (10a–b) by oxidation of the dou-
ble bond at C-12 and C-23 with RuO4. In these derivatives the car-
bonyl group at C-12 was regenerated and retention of the
configuration of all chiral centers was observed. The compound
10c was obtained through an elimination of tosyl group. The com-
pounds 10a–c were subjected to reduction with NaBH4 to obtain
the 20-epi-12,23-dihydroxy-22-oxocholestanic derivatives (11a–
c). Finally, the selective cis-dihydroxylation of 11c generated 11d.
This methodology is a very useful new route to obtain functional-
ized cholestanic chain at C-22 and C-23.
469
4. Uncited reference
A single crystal of (20S,25R)-3b,16b,26-triacetoxy-5a-cholesta-
Q3 470
[1e].
12,22,23-trione (11a, Fig. 3) was obtained from evaporation of a
solution in a hexane–methylene chloride mixture (99:1). The X-
ray diffraction data analysis unequivocally established that the
absolute configuration of C-12, C-20, and C-23 were R. Further-
more, data confirmed the reduction of C-12 and C-23 with dis-
tances of 1.431(5) Å, 1.412(6) Å of a single bond in both carbons
and showed the epimerization of C-20; therefore we were able to
assign the absolute configuration of all stereocenters of compound
11a.
471
Acknowledgments
472
473
474
The authors thank CONACyT (Project No. 84306) for financial
support and scholarships to V.G.C. and A.V.A.G. Also we thank
VIEP-BUAP and Dr. Angel Mendoza for X-ray data collection.
The cis-hydroxylation of the double bond at C-2 in 11c was car-
ried out using a catalytic amount of osmiun tetroxide (OsO4) and
N-methylmorpholine-N-oxide (NMO), as re-oxidant, in THF, to
475
Appendix A. Supplementary data
obtain the 2a,3a-diol on the A ring of compound 11d.
476
477
478
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2013.04.
014.
Although we were not able to obtain crystals of 11b–c, the close
proximity of chemical shifts of C-12, C-20, C-23 in 1H and 13C NMR
Please cite this article in press as: Gómez-Calvario V et al. Synthetic pathway to 22,23-dioxocholestanic chain derivatives and their usefulness for obtaining
brassinosteroid analogues. Steroids (2013), http://dx.doi.org/10.1016/j.steroids.2013.04.014

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Please cite this article in press as: Gómez-Calvario V et al. Synthetic pathway to 22,23-dioxocholestanic chain derivatives and their usefulness for obtaining
brassinosteroid analogues. Steroids (2013), http://dx.doi.org/10.1016/j.steroids.2013.04.014