R. Zeferino-Diaz et al.
Steroidsxxx(xxxx)xxx–xxx
H-3), 4.31 (1H, ddd, J16,17 = 7.3 Hz, J16,15 = 4.0 and 6.1 Hz, H-16),
3.36 (1H, dd, J26eq,25 = 4.0 Hz, Jgem = 10.8 Hz, H-26eq), 3.26 (1H, dd,
J26ax,25 = 11.4 Hz, Jgem = 10.8 Hz, H-26ax), 2.27 (1H, m, H-7eq), 2.26
(1H, m, H-5), 2.16 (1H, m, H-4eq), 0.88 (3H, d, J21,20 = 6.9 Hz, CH3-
21), 0.72 (3H, d, J27,25 = 6.4 Hz, CH3-27), 0.72 (3H, s, CH3-19), 0.66
(3H, s, CH3-18). 13C NMR (δ): 39.0 (C-1), 124.6 (C-2), 124.1 (C-3), 20.6
(C-4), 53.0 (C-5), 210.7 (C-6), 46.6 (C-7), 36.8 (C-8), 53.4 (C-9), 39.6
(C-10), 21.4 (C-11), 39.2 (C-12), 40.4 (C-13), 56.2 (C-14), 31.2 (C-15),
80.0 (C-16), 61.8 (C-17), 13.2 (C-18), 16.0 (C-19), 41.3 (C-20), 14.2 (C-
21), 108.8 (C-22), 31.0 (C-23), 28.4 (C-24), 29.9 (C-25), 66.4 (C-26),
16.8 (C-27). HRMS (FAB) m/z for C27H40O3 Calcd: 412.2977. Found:
2.3. Biological activity
2.3.1. The rice lamina inclination test
Seeds of two rice (Oryza sativa) varieties (Morelos A06 and Morelos
A08) were germinated in the darkness for 7 days, and uniform seedlings
were selected. Then, leaf segments, each consisting of the second la-
mina (length 1 cm), a lamina joint, and sheath (length 1 cm), were
excised and grown in distilled water at 30 °C [32]. They were then
incubated in 1.0 mL of 2.5 mM potassium maleate solution containing a
finite amount of a test sample for 48 h at 30 °C under the dark condi-
tions, using a dim red light bath. The induced angles between laminae
and sheaths were measured after the incubation period. Untreated
leaves and a commercial sample of homobrassinolide (HB) were used as
controls.
413.2981 [M + H]+
.
2.2.5. (25R)-2α,3α-dihydroxy-5α-spirostan-6-one (9)
To a solution of 8 (2.1 g, 5.0 mmol) in 5 ml of t-BuOH, freshly
prepared CTAP (2.0 g, 5.0 mmol) in 20 ml of t-BuOH/water (8:2) was
added dropwise. The reaction mixture was stirred at rt for 24 h and then
diluted in CH2Cl2 (50.0 mL). After that, 15.0 mL of 5% NaOH were
added and the stirring was maintained for 2 h. The crude product was
extracted with CH2Cl2 (2 × 30.0 ml) and the combination of the ex-
tracts was dried over anhydrous Na2SO4, filtered, and concentrated in
vacuo. The resulting solid was purified by chromatography on a
Combiflash apparatus with a gradient of hexane/ethyl acetate 10:0 →
2:3 for 15 min to yield a white solid (1.5 g, 66%) colorless needles; mp
224–226 °C [31]. [α]D = + 3.5 (c 0.5, CHCl3). IR: 3475, 3387 (OH),
2936 (CH, aliphatic), 1721 (C]O, ketone), 980, 920, 860 (OeC–O
spiroketal). 1H NMR (δ): 4.41 (1H, ddd, J16,17 = 8.4 Hz, J16,15 = 4.2
and 6.0 Hz, H-16), 4.04 (1H, br.s, H-3), 3.75 (1H, m, H-2), 3.47 (1H,
ddd, J26eq,25 = 4.4 Hz, Jgem = 10.8 Hz, J26eq,24eq = 1.2 Hz, H-26eq),
3.36 (1H, dd, J26ax,25 = Jgem = 10.8 Hz, H-26ax), 2.68 (1H, dd,
J5,4eq = 3.2 Hz, J5,4ax = 12.6 Hz, H-5), 2.31 (1H, m, H-7eq), 0.98 (3H,
d, J21,20 = 6.8 Hz, CH3-21), 0.79 (3H, d, J27,25 = 6.4 Hz, CH3-27), 0.77
(3H, s, CH3-19), 0.77 (3H, s, CH3-18). 13C NMR (δ): 40.1 (C-1), 68.2 (C-
2), 68.3 (C-3), 26.3 (C-4), 50.7 (C-5), 211.8 (C-6), 46.8 (C-7), 37.1 (C-
8), 53.7 (C-9), 40.9 (C-10), 21.0 (C-11), 39.4 (C-12), 42.5 (C-13), 56.4
(C-14), 31.5 (C-15), 80.4 (C-16), 61.9 (C-17), 13.6 (C-18), 16.4 (C-19),
41.6 (C-20), 14.5 (C-21), 109.3 (C-22), 31.3 (C-23), 28.7 (C-24), 30.2
(C-25), 66.8 (C-26), 17.1 (C-27). HRMS (FAB) m/z for C27H42O5 Calcd:
3. Results and discussion
Recently, we reported that cholestan-22-one steroids promote plant
growth [28], showing that such a side chain could be considered as a
pharmacophore. Herein we report the synthesis of 26-hydroxychole-
stan-22-one derivatives bearing a hydroxyl function at C-16 in the
acetate form, and introducing oxygenated functional groups in rings A
and B.
The synthetic pathway is described in Scheme 1. As starting mate-
rial diosgenin (1) was chosen, since it is one of the most versatile and
commercially available steroidal sapogenins. 1 was tosylated under
standard conditions to obtain quantitatively compound 2. The latter
was immediately used in the next step without further purification.
Compound 2 was functionalized under the hydroboration-oxidation
protocol previously reported, followed by a subsequent oxidation with
PDC to obtain compound 3 [30]. The tosyl group remained unchanged
under these reductive and alkaline conditions, for which strong evi-
dence is the formation of the 6α-hydroxy derivative which is the major
compound obtained after the oxidation of the mixture of alkylboranes
[33]. The overall yield for this step is 92%. For the synthesis of the title
compounds, 3 was selected as starting point.
To synthesize 6 and 7, compound 3 reacted with the radical anion
sodium naphthalenide (SN) to reduce the tosyl group under two reac-
tion conditions, at room temperature and at -80 °C [30]. The resulting
3α,5-cyclo-5α-steroid 4 (reaction at rt) was obtained in 91% [34]. The
cleavage of the tosylate by SN produced a radical at C-3 and the 3α,5-
cyclo group was generated by the cleavage of the CeH bond at C-5.
When the reaction with SN was performed at -80 °C, the CeO bond of
the tosylate could be selectively cleaved and laxogenin (5) was obtained
in 89% [18,30,35]. The spiroketal side chains of 4 and 5 were opened
after a Lewis acid mediated acetolysis by means of Ac2O/BF3·OEt2 at
0 °C (see Scheme 1). The i-steroid 4 provided the enol ester 6 in 85%. To
explain the formation of 6, we must consider the activation of the
oxygen atom of the carbonyl at C-6 by the Lewis acid, which reacted
with acetic anhydride to trigger the nucleophilic attack of an acetate
ion at C-3β, pushing the bond between C-3 and C-5 to C-5 – C-6. Lax-
ogenin (5) furnished compound 7 in 83% under similar reaction con-
ditions, providing a different route to obtain such compound [36].
To obtain 10, the first step was the elimination of the tosylate group
of compound 3, by means of LiBr/Li2CO3 in DMF producing 8 in 70%.
An α-dihydroxylation of the double bond in 8 was performed em-
ploying cetyltrimethylammonium permanganate (CTAP) as the oxi-
dizing agent [37]. Usually this step is carried out employing OsO4, but
it is costly and extremely toxic, so CTAP represents an excellent alter-
native. Treatment with a solution of CTAP in t-BuOH/H2O (8:2), fol-
lowed by an alkaline work-up provided after 24 h the 2α,3α-diol
(compound 9) in 66% yield. Next, to settle down the 26-hydro-
xycholesta-22-one side chain, the Lewis acid mediated acetolysis re-
ferred above produced compound 10 in 88% yield.
446.3032. Found: 447.3045 [M + H]+
.
2.2.6. (25R)-26-hydroxy-6,22-dioxo-5α-cholestan-2α,3α,16β-triyl
triacetate (10)
Compound 10 was synthesized from 9, per the procedure described
for 6. The crude product was purified by chromatography on a
Combiflash apparatus with a gradient of hexane/ethyl acetate 10:0 →
6:4 for 10 min to yield 1.1 g (83%) of a pale cream powder, mp
260–262 °C. [α]D = -27.0 (c 0.5, CHCl3). IR: 3536 (OH), 2952 (CH,
aliphatic), 1732 (C]O, ketone), 1670 (C]O, acetate). 1H NMR (δ):
5.37 (1H, br-s, H-3), 4.97 (1H, m, H-16), 4.97 (1H, m, H-2), 3.42 (2H, d,
J26,25 = 5.91 Hz, H-26), 2.98 (1H, m, H-20), 2.66 (1H, m, H-23a), 2.61
(1H, dd, J5,4eq = 4.8 Hz, J5,4ax = 11.9 Hz, H-5), 2.41 (1H, m, H-23b),
2.37 (1H, m, H-15a), 2.29 (1H, dd, Jgem = 13.2 Hz, J7eq,8 = 4.5 Hz, H-
7eq), 2.10 (3H, s, CH3CO2-3), 2.06 (1H, m, H-7ax), 2.00 (3H, s,
CH3CO2-2), 1.98 (3H, s, CH3CO2-16), 1.15 (3H, d, J21,20 = 7.12 Hz,
CH3-21), 1.05 (1H, m, H-15b), 0.91 (3H, d, J27,25 = 6.7 Hz, CH3-27),
0.86 (3H, s, CH3-18), 0.83 (3H, s, CH3-19). 13C NMR (δ): 37.2 (C-1),
68.9 (C-2), 67.8 (C-3), 24.6 (C-4), 51.6 (C-5), 210.0 (C-6), 46.0 (C-7),
36.6 (C-8), 53.6 (C-9), 42.2 (C-10), 20.8 (C-11), 39.0 (C-12), 42.3 (C-
13), 53.2 (C-14), 34.2 (C-15), 74.9 (C-16), 54.8 (C-17), 13.4 (C-18),
13.2 (C-19), 43.1 (C-20), 16.6 (C-21), 213.5 (C-22), 38.5 (C-23), 26.1
(C-24), 35.2 (C-25), 67.2 (C-26), 16.4 (C-27), 170.2 (CH3CO2-2), 169.9
(CH3CO2-3), 169.7 (CH3CO2-16), 20.9 (CH3CO2-2), 21.0 (CH3CO2-3),
20.9 (CH3CO2-16). HRMS (FAB) m/z for C33H50O9 Calcd: 590.3455.
The three final 26-hydroxycholestan-22-one derivatives are quite
stable. A combination of COSY, HSQC, and HMBC experiments helped
Found: 591.3529 [M + H]+
.
3