Synthesis and plant growth promoting activity of dinorcholanic lactones bearing the 5α-hydroxy-6-oxo moiety

Article abstract of DOI:10.1016/j.jsbmb.2012.10.007

The naturally occurring dinorcholanic lactone vespertilin and two other non-natural derivatives bearing the 5α-hydroxy-6-oxo moiety were synthesized starting from the readily available steroid sapogenin diosgenin. The obtained compounds showed plant growt

Full text of DOI:10.1016/j.jsbmb.2012.10.007

Journal of Steroid Biochemistry & Molecular Biology 134 (2013) 45–50
Contents lists available at SciVerse ScienceDirect
Journal of Steroid Biochemistry and Molecular Biology
journal homepage: www.elsevier.com/locate/jsbmb
Synthesis and plant growth promoting activity of dinorcholanic lactones bearing
the 5␣-hydroxy-6-oxo moiety
a
b
a
Anielka Rosado-Abón , Guadalupe de Dios-Bravo , Rogelio Rodríguez-Sotres ,
a,∗
Martín A. Iglesias-Arteaga
Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 México D.F., Mexico
a
b
Universidad Autónoma de la Ciudad de México, Plantel San Lorenzo Texonco, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
The naturally occurring dinorcholanic lactone vespertilin and two other non-natural derivatives bearing
the 5␣-hydroxy-6-oxo moiety were synthesized starting from the readily available steroid sapogenin
diosgenin. The obtained compounds showed plant growth promoting activity in the bean’s second inter-
node elongation assay.
Received 16 August 2012
Received in revised form 2 October 2012
Accepted 8 October 2012
©
2012 Elsevier Ltd. All rights reserved.
Keywords:
Beckmann rearrangement
Dinorcholanic lactones
5␣-Hydroxy-6-oxo-steroids
Synthesis
Plant growth promoting activity
1
. Introduction
Our recent studies have shown that even BA bearing the
5
␣-hydroxy-6-oxo moiety and a cholestane side chain without oxy-
Brassinosteroids are potent plant growth promoters. In addi-
genated functions produce plant growth stimulation suggesting
that the presence of the 5␣-hydroxy-6-oxo moiety is capable to
induce such effects [19]. This has prompted us to extend our stud-
ies to other 5␣-hydroxy-6-oxo-steroids with modified side chains.
Herein we report on the synthesis of dinorcholanic lactones bear-
ing the 5␣-hydroxy-6-oxo moiety and the evaluation of their plant
growth promoting activity in the bean’s second internode elonga-
tion assay.
tion to brassinolide (1), the most active member, this family of
compounds is constituted by more than 70 naturally occurring
compounds that are in general characterized by the presence of
oxygenated functions in the rings A and B as well as in the side chain
(
see Fig. 1). The well-known fact that the structural modifications
found in the naturally occurring compounds produce moderate to
drastic differences in the plant growth stimulating activity, has led
to the establishment of generally accepted structural requirements
for a good plant growth stimulating activity [1–6].
Although the structural requirements for a good biological activ-
ity have been clearly established, a number of brassinosteroids
analogues (BA) bearing slight to drastic structural modifications
have shown low to moderate and even high plant growth pro-
moting activity [2–18]. In particular, several BA bearing the
2. Experimental
2.1. General conditions
Reactions were monitored by TLC on ALUGRAM® SIL G/UV254
plates from MACHEREY-NAGEL. Chromatographic plates were
sprayed with a 1% solution of vanillin in 50% HClO4 and heated until
color developed. Melting points were measured on a Melt-Temp
II equipment. Mass spectra were registered in a Thermo-Electron
spectrometer model DFS (Double Focus Sector). NMR spectra were
recorded in CDCl3 or DMSO-d6 solution in a Varian INOVA 400
and 300 spectrometers using the solvent signal as reference. NMR
signals assignments were made with the aid of a combination of
5
␣-hydroxy-6-oxo moiety have shown high plant growth promot-
ing activity [16,17]. As a part of our program on the synthesis of
bioactive steroids we have reported the synthesis of some BA with
furostane or cholestane side chains (i.e., 7 and 8, Fig. 2) that have
shown moderate plant growth promoting activity [18,19].
1
1
1
13
2
D homonuclear ( H– H) and heteronuclear ( H– C) correlation
1
1
1
1
∗ Corresponding author. Tel.: +52 55 56223803; fax: +52 55 56223803.
E-mail address: martin.iglesias@unam.mx (M.A. Iglesias-Arteaga).
techniques, which included H– H COSY, H– H Nuclear Over-
hauser effect (NOESY), Heteronuclear Single Quantum Correlation
0
960-0760/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jsbmb.2012.10.007

4
6
A. Rosado-Abón et al. / Journal of Steroid Biochemistry & Molecular Biology 134 (2013) 45–50
HO
HO
HO
OH
OH
HO
OH
HO
OH
HO
OH
HO
HO
HO
HO
O
O
HO
H
O
H
O
O
H
H
O
O
brassinolide(1)
homobrassinolide(2)
24-epibrassinolide(3)
castasterone(4)
Fig. 1. Some naturally occurring brassinosteroids.
(
HSQC). All 2D NMR spectra were recorded using the standard
evaporated, and the mixture was poured into H O/ice. The result-
2
pulse sequences and parameters recommended by the manu-
facturer and processed employing the NMR processing program
MestreNova.
ing solid was filtered off, washed with abundant water and dried
in the air to afford the oxime 11 (1.586 g, 3.27 mmol, 91%). M.p.
◦
◦
1
208–210 C. Lit. 208.5–211 C [20]. H NMR (400 MHz, CDCl ) ı ppm
3
8
.38 (s, 1H, C N OH), 5.35 (d, J = 4.1 Hz, 1H, H-6), 4.60 (m, 1H, H-3),
2
2
.1.1. (25R)-3ˇ-Acetoxy-spirost-5-en-23-one, namely
3-oxodiosgenin acetate (10)
NaNO2 (0.5256 g) was slowly added (45 min) to a solution of
4.46 (ddd, J = 15.2 Hz, 7.4 Hz, 1H, H-16), 3.60 (dd, J = 11.2 Hz, 11.2 Hz,
1H, H-26 ax.), 3.52 (dd, J = 11.2 Hz, 3.5 Hz, 1H, H-26 eq.), 3.31 (dd,
J = 14.0 Hz, 2.5 Hz, 1H, H-24 eq.), 2.80 (dq, J = 6.7, 6.7 Hz, 1H, H-20),
2.31 (m, 2H, H-4), 2.02 (s, 3H, CH COO-3) 1.02 (s, 3H, CH -19), 0.97
diosgenin acetate (3 g, 6.57 mmol) and BF ·Et O (2.69 mL) in acetic
3
2
3
3
acid (75 mL). After conclusion of the addition, the addition of
(d, J = 7.0 Hz, 3H, CH -21), 0.88 (d, J = 6.5 Hz, 1H, CH -27), 0.79 (s,
3 3
3H, CH -18). C NMR (100 MHz) ı ppm 36.7 C-1, 28.2 C-2, 73.9
13
BF ·Et O (2.69 mL) and NaNO (0.5256 g in 45 min) were repeated
3
2
2
3
and the resulting mixture was stirred for 1 h and poured into
ice/water. The produced solid was filtered, washed with abundant
water, dissolved in ethyl acetate (200 mL) and the organic solu-
C-3, 38.0 C-4, 139.7 C-5, 122.2 C-6, 32.0 C-7, 31.8 C-8, 49.9 C-9, 36.9
C-10, 20.8 C-11, 39.7 C-12, 40.6 C-13, 56.4 C-14, 31.6 C-15, 81.5
C-16, 61.1 C-17, 16.2 C-18, 19.3 C-19, 35.9 C-20, 14.4 C-21, 108.6
C-22, 154.5 C-23, 27.7 C-24, 31.3 C-25, 65.7 C-26, 17.0 C-27, 170.5
CH COO-3, 21.4 CH COO-3.
tion was washed with 5% aqueous solution of Na CO3 (2 × 50 mL),
2
H O (1 × 50 mL) NaCl (1 × 50 mL), dried (anh. Na SO ) and evapo-
2
2
4
3
3
rated. The resulting syrup was dissolved in the smallest amount of
1
/1 benzene/hexane solution left to stand overnight in a chromato-
graphic column packed with Al O3 (Brockman activity III). Elution
2.1.3. 3ˇ-Acetoxy-16ˇ-hydroxy-dinorchol-5-enic acid 22 → 16
lactone, namely vespertilin acetate (12)
2
with 10/1 hexane/ethyl acetate mixture afforded 1.093 (2.32 mmol,
◦
3
5%) of 23-oxodiosgenin acetate. M.p. 190–192 C (from ethyl
BF ·Et O (1.46 mL) was added to a solution of the oxime 11
3
2
◦
1
acetate/hexane). Lit. 191–192 C [20]. H NMR (400 MHz, CDCl3)
(1.362 g, 2.81 mmol) in acetic acid (28 mL) and the mixture was
◦
ı ppm 5.35 (d, J = 5.1 Hz, 1H, H-6), 4.63–4.56 (m, 2H, H-3 and H-16),
stirred at 60 C for 2.5 h before pouring into water. The result-
3
2
.77 (dd, J = 11.2 Hz, 11.2 Hz, 1H, H-26 ax.), 3.57 (m, 1H, H-26 eq.),
.87 (dq, J = 6.9, 6.9 Hz, 1H, H-20), 2.43 (m, 2H, H-24), 2.31 (m, 2H,
ing solid was filtered off, washed with water and dissolved in
ethyl acetate (100 mL) that was washed with H O (2 × 25 mL), 5%
2
H-4), 2.27 (m, 1H, H-25), 2.01 (s, 3H, CH COO-3), 1.02 (s, 3H, CH -
Na CO solution (3 × 25 mL), H O (2 × 25 mL) and brine (1 × 25),
3
3
2
3
2
1
0
7
3
9), 0.92 (d, J = 6.5 Hz, 3H, CH -21), 0.92 (d, J = 6.5 Hz, 3H, CH -27),
.78 (s, 3H, CH -18). C NMR (100 MHz) ı ppm 36.7 C-1, 27.7 C-2,
dried and evaporated to afford the lactone 12 that was purified in
a chromatographic column packed with silica gel (30 g) employing
hexane/ethyl acetate 85/15 mixture as eluent to afford the pure ves-
3
3
1
3
3
3.8 C-3, 38.1 C-4, 139.8 C-5, 122.1 C-6, 32.0 C-7, 31.3 C-8, 49.9 C-9,
6.9 C-10, 20.7 C-11, 39.5 C-12, 40.7 C-13, 56.5 C-14, 31.8 C-15, 83.3
◦
pertilin acetate 12 (1.0160 g, 2.6 mmol, 93%). M.p. 210–212 C (from
◦
1
C-16, 61.6 C-17, 16.0 C-18, 19.3 C-19, 34.7 C-20, 14.4 C-21, 109.8
C-22, 201.8 C-23, 45.2 C-24, 35.8 C-25, 65.6 C-26, 17.0 C-27, 170.5
CH COO-3, 21.4 CH COO-3.
ethyl acetate/hexane) Lit. 213–214 C [20]. H NMR (400 MHz,
CDCl ) ı ppm 5.36 (d, J = 5.1 Hz, 1H, H-6), 4.94 (ddd, J = 7.8 Hz, 4.7 Hz,
3
3
3
1H, H-16), 4.58 (tdd, J = 10.4 Hz, 6.3 Hz, 4.2 Hz, 1H, H-3), 2.58 (m, 1H,
H-20), 2.02 (s, 3H, CH COO-3) 1.31 (d, J = 7.7 Hz, 3H, CH -21), 1.02
3
3
13
2
.1.2. (25R)-23-Hydroximino-spirost-5-en-3ˇ-ol acetate (11)
(s, 3H, CH3-19), 0.76 (s, 3H, CH3-18). C NMR (100 MHz) ı ppm
36.9 C-1, 27.7 C-2, 73.7 C-3, 38.0 C-4, 139.8 C-5, 121.9 C-6, 31.8 C-7,
31.2 C-8, 50.0 C-9, 36.6 C-10, 20.3 C-11, 38.1 C-12, 41.4 C-13, 54.7
C-14, 33.1 C-15, 82.7 C-16, 58.9 C-17, 13.7 C-18, 19.3 C-19, 36.0
C-20, 18.0 C-21, 181.3 C-22, 170.5 CH3COO-3, 21.4 CH3COO-3.
Sodium acetate (0.6 g in 5 mL H O) and NH OH·HCl (0.6 g,
2
2
8
.63 mmol, in 3 mL H O) were added to a solution of the ketone 10
2
(
1.58 g, 3.59 mmol) in ethanol (100 mL) and 1,4-dioxan (5 mL) and
the mixture was stirred under reflux for 3 h. Half of the solvent was
OH
OH
OH
HO
OH
O
HO
HO
HO
HO
HO
HO
HO
OH
OH
HO
OH
5
O
6
O
7
O
8
O
Fig. 2. Synthetic BA bearing the 5␣-hydroxy-6-oxo moiety.

A. Rosado-Abón et al. / Journal of Steroid Biochemistry & Molecular Biology 134 (2013) 45–50
47
2
.1.4. 3ˇ,16ˇ-Dihydroxy-dinorchol-5-enic acid 22 → 16 lactone,
2.1.7. 5,16ˇ-Dihydroxy-6-oxo-5˛-dinorchol-2-enic acid 22 → 16
lactone (16)
namely vespertilin (13)
A suspension of vespertilin acetate (12) (68 mg, 0.18 mmol) was
Tosyl chloride (0.3694 g, 1.91 mmol) was added to a solution
of the dihydroxylated ketone 16 (0.3565 g, 0.95 mmol) in pyridine
(4 mL), the mixture was stirred overnight, poured in 3% HCl/ice. The
resulting solid was filtered off, washed with plenty of water, dis-
◦
stirred at 45 C for 40 min in a saturated solution of K CO in
2
3
methanol (2 mL) and poured into water. After drop wise addition
of HCl 6 N to adjust to pH = 1, the mixture was left to stand for
2
0 min and cooled. The produced solid was filtered off and dis-
solved in ethyl acetate (35 mL) washed with H O (2 × 20 mL), dried
2
solved in ethyl acetate diethyl ether mixture, dried (anh. Na SO )
(anh. Na SO ) and evaporated. The obtained solid was refluxed
2
4
2
4
and evaporated to vespertilin (13) (60 mg, 0.17 mmol, 97%). M.p.
for 40 min in dry DMF (10 mL) with LiBr (0.6219 g) and Li CO3
(0.5149 g) and the mixture was cooled to room temperature. Ethyl
acetate (35 mL) was added, the mixture was filtered with suction
2
◦
◦
1
2
24–226 C (from ethyl acetate hexane) Lit. 225–226 C [21].
H
NMR (400 MHz, CDCl ) ı ppm 5.33 (m, 1H, H-6), 4.94 (ddd, J = 7.8 Hz,
3
4
.7 Hz, 1H, H-16), 3.50 (m, 1H, H-3), 2.58 (qd, J = 7.6 Hz, 1.0 Hz, 1H,
and the organic layer was washed with H O (6 × 12 mL), dried
2
H-20), 1.30 (d, J = 7.6 Hz, 3H, CH -21), 1.01 (s, 3H, CH -19), 0.75 (s,
(Na SO ) and evaporated. The produced solid was purified in a
3
3
2
4
1
3
3
H, CH -18). C NMR (100 MHz) ı ppm 37.2 C-1, 31.5 C-2, 71.6
chromatographic column packed with silica gel (15 g) employing
hexane/ethyl acetate 1/1 mixture as eluent to afford the unsatu-
3
C-3, 42.1 C-4, 140.9 C-5, 120.9 C-6, 31.8 C-7, 31.2 C-8, 50.1 C-9, 36.5
C-10, 20.3 C-11, 38.2 C-12, 41.4 C-13, 54.8 C-14, 33.1 C-15, 82.7
C-16, 58.9 C-17, 13.7 C-18, 19.4 C-19, 36.0 C-20, 18.0 C-21, 181.4
C-22.
◦
1
rated ketol 16 (0.2647 g, 0.74 mmol, 78%). M.p. 242–244 C H NMR
(300 MHz, CDCl + CD OD) ı ppm 5.66–5.55 (m, 2H, H-2 and H-3),
3
3
4.93 (ddd, J = 7.7 Hz, 4.5 Hz, 1H, H-16), 2.72 (dd, J = 12.5 Hz, 12.5 Hz,
1
2
H, H-7 ax.), 2.61 (m, 1H, H-1), 2.55 (dq, J = 7.8 Hz, 3.8 Hz, 1H, H-
0), 1.28 (d, J = 7.6 Hz, 3H, CH -21), 0.70 (s, 3H, CH -19), 0.68 (s, 3H,
3
3
2
2
.1.5. 3ˇ-Acetoxy-5,16ˇ-dihydroxy-6-oxo-5˛-dinorcholanic acid
2 → 16 lactone (14)
13
CH -18). C NMR (75 MHz) ı ppm 29.7 C-1, 122.3 C-2, 125.1 C-3,
3
3
1
5
4.3 C-4, 77.5 C-5, 211.0 C-6, 42.3 C-7, 36.4 C-8, 45.0 C-9, 42.0 C-
0, 20.2 C-11, 37.8 C-12, 41.9 C-13, 54.3 C-14, 32.6 C-15, 82.4 C-16,
8.7 C-17, 13.6 C-18, 14.4 C-19, 36.0 C-20, 17.8 C-21, 181.3 C-22.
MCPBA (0.3799 g, 2.2 mmol) was added to a solution of vesper-
tilin acetate (12) (0.6052 g, 1.57 mmol) in CH Cl2 (8 mL) and the
mixture was stirred until the starting material disappeared (1 h,
2
+
+
MS (EI, 70 eV): 359 [M+1] , 358 M , 326, 325, 313, 297, 261, 223,
205, 165, 147, 133, 105, 93 (100%), 91, 79, 55. HRMS (EI, 70 eV):
observed 358.2125 calculated for C22H30O4 358.2139.
TLC). Acetone (20 mL) was added and the mixture was cooled to
◦
0
C in an ice bath before drop wise addition of a solution of CrO3
(
0.2804 g, 2.8 mmol) in water (1 mL). The ice bath is removed, the
mixture was stirred at room temperature for 20 min and cooled to
◦
2.1.8. 2˛,3˛,5,16ˇ-Tetrahydroxy-6-oxo-5˛-dinorcholanic acid
0
C in the ice bath prior to drop wise addition of a solution of CrO3
22 → 16 lactone (17)
(
0.7143 g, 7.14 mmol) in water (2.15 mL). The ice bath was removed
N-methylmorpholine N-oxide (0.4720 g) and 2.84 mL of a
2.5 mg/mL solution of OsO4 in tert-butanol were added to a solu-
and the mixture stirred for 2.5 h, before addition of water (20 mL)
1
and extraction with ethyl acetate (2 × 25 mL). The organic layer was
tion of the unsaturated ketol 16 (211.8 mg, 0.59 mmol) in THF
washed with water (10 × 20 mL), 10% NaHCO solution (5 × 20 mL),
4
(6.25 mL) and the mixture was stirred for 24 h. A solution of Na SO3
2
water (2 × 20 mL) and brine (20 mL), dried (anh. Na SO ) and evap-
2
4
(
0.2134 g) in H O (0.5 mL) was added, the mixture was stirred
2
orated to afford the desired ketol (14) (0.5543 g, 1.33 mmol, 85%).
◦
◦
1
overnight, ethyl acetate was added and the organic layer was
M.p. 280.6–282.2 C (from acetone) Lit. 280.6–282.2 C [22].
H
washed with brine (5 × 20 mL), dried (anh. Na SO ) and evap-
2
4
NMR (400 MHz, CDCl ) ı ppm 5.01 (m, 1H, H-3), 4.93 (ddd, J = 7.7 Hz,
3
orated. The produced solid was purified in a chromatographic
4
.5 Hz, 1H, H-16), 2.78 (dd, J = 12.6 Hz, 12.6 Hz, 1H, H-7 ax.), 2.56
m, 1H, H-20), 2.21 (m, 1H, H-15), 2.09 (dd, J = 12.6 Hz, 4.7 Hz, 1H,
H-7 eq.), 1.98 (s, 3H, CH COO-3), 1.30 (d, J = 7.6 Hz, 3H, CH -21),
column packed with silica gel (10 g) employing CH Cl /methanol
2
2
(
9
0
5/5 mixture to afford the trihydroxylated ketone 17 (131.4 mg,
.34 mmol, 58%). M.p. 304–306 C (from ethyl acetate hexane).
3
3
◦
1
H
0
.80 (s, 3H, CH -19), 0.71 (s, 3H, CH -18). 13C NMR (100 MHz)
3
3
NMR (400 MHz, DMSO-d ) ı ppm 5.66 (s, 1H, OH-5), 5.47 (d, J = 3.3,
6
ı ppm 26.2 C-1, 29.5 C-2, 70.6 C-3, 32.2 C-4, 80.0 C-5, 211.4 C-
1
H, OH-3), 4.96 (m, 1H, H-16), 4.55 (d, J = 6.0 Hz, 1H, OH-2), 3.93
6
4
1
, 41.5 C-7, 36.4 C-8, 44.2 C-9, 42.4 C-10, 20.6 C-11, 37.9 C-12,
2.2 C-13, 54.3 C-14, 32.7 C-15, 82.3 C-16, 58.8 C-17, 13.7 C-18,
(m, 1H, H-3), 3.65–3.58 (m, 1H, H-2), 2.60 (m, 1H, H-20), 1.20 (d,
13
J = 7.6 Hz, 3H, CH -21), 0.69 (s, 3H, CH -19), 0.63 (s, 3H, CH -18).
C
3
3
3
3.9 C-19, 36.0 C-20, 17.9 C-21, 181.0 C-22, 171.1 CH COO-3, 21.3
3
NMR (100 MHz) ı ppm 30.5 C-1, 66.4 C-2, 69.2 C-3, 34.4 C-4, 79.1 C-
, 210.0 C-6, 40.9 C-7, 35.3 C-8, 44.3 C-9, 41.7 C-10, 20.0 C-11, 37.1
CH COO-3.
3
5
C-12, 44.6 C-13, 53.6 C-14, 32.2 C-15, 81.9 C-16, 57.6 C-17, 13.2
C-18, 14.4 C-19, 35.6 C-20, 17.5 C-21, 180.6 C-22. MS (EI, 70 eV):
394 [M+2] , 393 [M+1] , 392 M , 374, 348, 331, 313, 289, 276, 275,
261, 247, 221, 205, 175, 173, 159, 174, 133, 119, 107, 93, 91, 79, 69,
67, 55(100%). HRMS (EI, 70 eV): observed 392.2175 calculated for
C22H32O6 392.2193.
2
2
.1.6. 3ˇ,5,16ˇ-Trihydroxy-6-oxo-5˛-dinorcholanic acid
2 → 16 lactone (15)
+
+
+
◦
The ketol 14 (0.5324 g, 1.27 mmol) was stirred at 45 C for 40 min
in a saturated solution of K CO3 in methanol (95 mL) and poured
2
into water. After drop wise addition of HCl 6 N to adjust to pH = 1,
the solution was left to stand for 20 min and cooled. The produced
solid was filtered off and dissolved in ethyl acetate diethyl ether
mixture, dried (anh. Na SO ) and evaporated to afford the dihy-
droxylated ketone 15 (0.4074 g 1.08 mmol, 85%). M.p. 300–302 C
from acetone, Lit. 300 C [22]. H NMR (400 MHz, DMSO-d ) ı ppm
2.2. Biological test
2
4
◦
2.2.1. Bean’s second internode elongation assay
◦
1
6
The details of this test have been described elsewhere [19]. Bean
seeds were germinated in sterile wet paper towels, and seedlings
were planted in small pots with agrolite. Plants were watered for
two weeks with Hoghland solution. Compounds were dissolved
in a mixture of lanolin and ethanol (1:1), and an aliquot of 2 ␮L
containing the indicated dose was applied to the base of the sec-
ond internode. After 48 h, the length of the second internode (IL)
was measured. Groups of 15 plants were used for each treatment,
and three independent experiments were performed. The overall
5
3
.34 (s, 1H, OH-5), 4.96 (m, 1H, H-16), 4.36 (d, J = 5.5 Hz, 1H, OH-3),
.69 (m, 1H, H-3), 2.70 (dd, J = 12.4 Hz, 12.4 Hz, 1H, H-7 ax.), 2.58
(
dq, J = 7.4, 7.4 Hz, 1H, H-20), 1.20 (d, J = 7.5 Hz, 3H, CH -21), 0.67 (s,
3
13
3
H, CH -19), 0.62 (s, 3H, CH -18). C NMR (100 MHz) ı ppm 29.6
3 3
C-1, 30.4 C-2, 65.2 C-3, 35.7 C-4, 79.0 C-5, 211.9 C-6, 41.3 C-7, 36.0
C-8, 43.9 C-9, 41.9 C-10, 20.3 C-11, 37.2 C-12, 41.7 C-13, 53.7 C-14,
3
2.2 C-15, 82.0 C-16, 57.7 C-17, 13.3 C-18, 13.6 C-19, 35.3 C-20, 17.5
C-21, 180.6 C-22.

4
8
A. Rosado-Abón et al. / Journal of Steroid Biochemistry & Molecular Biology 134 (2013) 45–50
O
O
O
O
N
OH
O
O
O
i
ii
3
CH COO
3
CH COO
3
CH COO
9
10
11
iii
O
O
i) a: NaNO
II) H NOH·HCl/NaOAc/C
iii) BF ·Et O/CH COOH 60 ºC
iv) a: K CO /CH OH. b: HCl;
2
/BF
3
·Et
2
O/CH
3
H
COOH, b: Al
5
2 3
O 2 h.
2
2
OH reflux
O
O
3
2
3
3
2
3
iv
HO
3
CH COO
13
12
Scheme 1. Synthesis of vestertilin (13).
statistical significance of the differences between the treatments
different dosages) was tested using the distribution-independent
prepared by Mola-Gárate and coworkers starting from solaso-
dine [21] and following the lengthy protocol described by Sato
in the early 1960s [27,28]. Our shorter synthetic protocol to ves-
pertilin acetate (12) started with treatment of diosgenin acetate
(9) with BF ·Et O and NaNO in acetic acid followed by chro-
(
test of Kruskal–Wallis one-way analysis of variance (ANOVA on
ranks). When found, the significance of IL differences vs. the con-
trol was tested using the Dunn’s method [23]. Histograms of the
internode length data were found to fit to the skew-normal distri-
bution and estimates of the IL mean and the standard error of the IL
mean were obtained using the “SN” package [24] for the R software
3
2
2
matography in neutral alumina (Brockmann activity III) to afford
23-oxodiosgenin acetate (10). Conversion of the ketone 10 into
23-hydroxiiminodiosgenin acetate (11) was achieved by refluxing
[
25]. From these data, estimates were obtained for the maximal
with NH OH·HCl and sodium acetate in ethanol–dioxane mix-
2
elongation response (ILmax) of the second internode, produced by
each compound, and the dose of compound producing 50% of ILmax
(
ture. BF ·Et O-induced Beckmann rearrangement of the oxime
3
2
◦
11 at 60 C for 2.5 h afforded vespertilin acetate 12 in 93%
yield. Finally saponification of 12 afforded vespertilin (13) (see
Scheme 1).
SD50). The estimates were obtained by fitting data to Eq. (1).
IL = (
ILmax − IL ) [Compound]
+ IL0
(1)
0
Vespertilin acetate (12) was converted into the ketol 14 by
SD + [Compound]
5
0
consecutive one-pot treatment with MCPBA and CrO . Saponifica-
3
where IL is the observed internode length at each compound con-
centration, IL0 is the internode length observed in the control
treatment (vehicle only), and ILmax and SD50 have the meaning
defined above.
tion 14 employing K CO in methanol afforded the dihydroxylated
keto-lactone 15 that was treated with tosyl chloride and pyri-
2
3
dine followed by reflux with LiBr and Li CO3 in DMF to afford
2
the unsaturated keto lactone 16. Treatment of 16 with OsO4
and N-methylmorpholine N-oxide (NMMNO) in THF afforded the
2␣,3␣,5␣-trihydroxylated keto-lactone 17 (see Scheme 2). The ␣
orientation of the 2,3-diol moiety in compound 17 can be easily
confirmed by observation of the H-2 ↔ H-19 and H-2 ↔ H-3 NOE
effects (see Fig. 3).
3
. Results and discussion
Vespertilin (13), a naturally occurring dinorcholanic lactone,
was originally isolated by Suárez and coworkers [26] and later
O
O
O
O
O
O
i
ii
3
CH COO
3
CH COO
HO
OH
OH
iii
12
14
15
16
O
O
O
O
i) a: MCPBA/CH
ii) K CO /CH OH
III) a: TsCl/pyr, b: LiBr/Li
iv) OsO /NMMNO/THF
2
Cl
2
, b: CrO
3
/H
2 2 2
O/acetone/CH Cl
2
3
3
2
CO
3
/DMF reflux
O
O
4
HO
HO
v
OH
OH
17
O
O
Scheme 2. Synthesis of vestertilin derivatives 15 and 17.

A. Rosado-Abón et al. / Journal of Steroid Biochemistry & Molecular Biology 134 (2013) 45–50
49
Fig. 3. Selected 1H NMR signals and NOE correlations in compound 17.
Table 1
3.1. Biological activity
Estimated maximal elongation response (ILmax) and half-stimulatory dose (SD50) of
the observed second internode elongation in bean plants treated with the studied
compounds.
The biological activity of the compounds 13, 15 and 17 was
a
determined with the bean’s second internode elongation assay
vide supra), employing homobrassinolide (2) as reference. Con-
Compound
ILmax (mm)
SD50 (pg)
(
2
5.7 ± 0.2
11.2 ± 1.1
7.5 ± 1.3
9.0 ± 0.9
<1b
trol plants were treated with vehicle only. The compounds were
tested at concentrations from 1 pg to 1 ng per plant, using the bean
second internode assay. The IL data did not conform to the normal
distribution; therefore, the statistical significance of the differences
was assessed using non-parametric statistics. From the statistical
tests, homobrassinolide (2) and the studied dinorcholanic lactones
exhibited growth promotion activity (p < 0.05). To compare the rel-
ative potency of the lactones with that from homobrassinolide (2),
an estimation of mean and standard errors employing the skew-
normal distribution was performed and the resulting values were
plotted against the applied dose of compound (see Fig. 4). These
data was then fitted to a classical saturation curve (Eq. (1), see
experimental part) to provide a more quantitative description of
the observed effects (see Table 1).
13
15
17
11.3 ± 6.8
b
<1
7.0 ± 4.5
a
The estimates were obtained by fitting the data in Fig. 4 to Eq. (1). Values are
given as mean ± the standard error of the mean.
b
Since the estimate was below the smallest dose applied, the exact value is
uncertain.
induced a stronger ILmax. According to the second internode bioas-
say, 13, 15 and 17 appear as good plant growth promoters and their
effects on plants deserve further investigation.
4
. Conclusion
Given the complexity of the plant transduction pathways
mediating plant responses to phytohormones [29], the estimated
parameters are only useful for comparative purposes. From the
data in Table 1 15 and 2 exhibited similar activity, though the cor-
responding SD50 estimates are somehow uncertain because they
were below the lowest dose tested. In addition, 13 and 17 were
effective at doses one order of magnitude higher, at least, but they
The naturally occurring dinorcholanic lactone vespertilin (13)
and two other non-natural partners (15 and 17) bearing the 5␣-
hydroxy-6-oxo moiety were prepared starting from diosgenin
acetate (9). All the obtained dinorcholanic lactones produced plant
growth promotion in the bean’s second internode elongation assay.
In particular 15 showed plant growth promoting activity com-
parable to that of homobrassinolide (2). The intriguing fact that
vespertilin 13, which lacks the 5␣-hydroxy-6-oxo moiety, also pro-
duced plant growth promotion, suggests that the lactone moiety
in side chain may be able to produce such effect and opens an
interesting subject that should be further investigated.
Acknowledgements
The authors thank to Dirección General de Asuntos del Per-
sonal Académico (DGAPA-UNAM) for financial support via project
IN221911-3 and CONACyT (México) for the scholarship granted to
A.R.-A. Additional financial support from Instituto de Ciencia y Tec-
nología del Distrito Federal (ICyT DF) is also acknowledged. We
are indebted to Professor Vladimir Khripach of the Academy of
Sciences of Byelorussia for the kind gift of homobrassinolide (2).
Thanks are due to Nuria Esturau-Escofet (USAI-UNAM) for regis-
tering NMR spectra and to Georgina Duarte Lisci and Margarita
Guzmán Villanueva (USAI-UNAM) for registering Mass spectra. We
want to express our gratitude to Dr. Carlos Cobas from Mestrelab®
for assistance with the MestreNova NMR processing program.
Fig. 4. Plant growth stimulating activity of studied compounds.

5
0
A. Rosado-Abón et al. / Journal of Steroid Biochemistry & Molecular Biology 134 (2013) 45–50
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