Synthesis and bioactivity evaluation of brassinosteroid analogs

Article abstract of DOI:10.1016/S0039-128X(00)00093-3

Four new analogs of 28-homocastasterone have been synthesized and completely characterized for the first time from stigmasterol. (22R,23R,24S)- 3β-acetoxy-22,23-dihydroxy-5α-stigmastan-6-one (17), (22R,23R,24S)-3β- bromo-22,23-dihydroxy-5α-stigmastan-6-on

Full text of DOI:10.1016/S0039-128X(00)00093-3

Steroids 65 (2000) 329–337
Synthesis and bioactivity evaluation of brassinosteroid analogs
Javier A. Ram´ırez, Osvaldo M. Teme Centurio´n, Eduardo G. Gros, Lydia R. Galagovsky*
Departamento de Qu´ımica Orga´nica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabello´n 2, Ciudad Universitaria
1428 Buenos Aires, Argentina
Received 12 July 1999; received in revised form 12 November 1999; accepted 15 December 1999
Abstract
Four new analogs of 28-homocastasterone have been synthesized and completely characterized for the first time from stigmasterol.
(22R,23R,24S)-3␤-acetoxy-22,23-dihydroxy-5␣-stigmastan-6-one (17), (22R,23R,24S)-3␤-bromo-22,23-dihydroxy-5␣-stigmastan-6-one
(18), (22R,23R,24S)-3␤-acetoxy-5,22,23-trihydroxy-5␣-stigmastan-6-one (20), and (22R,23R,24S)-3␤-bromo-5,22,23-trihydroxy-5␣-stig-
mastan-6-one (21), were obtained through a synthetic route based on regioselective ⌬5 epoxidation. Compounds 17 and 18, bearing a 5␣H
moiety, were prepared through a reductive opening of the 5␤,6␤ epoxy precursor, and compounds 20 and 21, analogs with a 5␣OH moiety
were obtained by hydrolytic opening of a mixture of 5␣,6␣ and 5␤,6␤ epoxy precursors. Known compounds 19 and 22 were also obtained
following the described synthetic routes, respectively. The new compounds were tested with the traditional auxin-like bioassay for
brassinosteroids with 19 and 22 as standards. All compounds were comparatively evaluated for their inhibitory effect on the replication of
DNA (HSV-1) virus. © 2000 Elsevier Science Inc. All rights reserved.
Keywords: Brassinosteroid; Synthesis; Bioactive; Phytohormone; Antiviral
1. Introduction
5␣-stigmastan-6-one (17), (22R,23R,24S)-3␤-bromo-22,23-
dihydroxy-5␣-stigmastan-6-one (18), and (22R,23R,24S)-2␣,
3␣,22,23-tetrahydroxy-5␣-stigmastan-6-one (19) (28-homo-
ethylcastasterone).
Scheme 2 describes the hydrolytic opening of a mix-
ture of 5␣,6␣ and 5␤,6␤ epoxy precursors to obtain analogs
with a 5␣OH moiety: (22R,23R,24S)-3␤-acetoxy-5,22,23-
trihydroxy-5␣-stigmastan-6-one (20), (22R,23R,24S)-3␤-
bromo-5,22,23-trihydroxy-5␣-stigmastan-6-one (21), and
(22R,23R,24S)-2␣,3␣,5␣,22,23-pentahydroxy-5␣-
stigmastan-6-one (22).
Brassinosteroids represent a new group of steroidal phy-
tohormones with high growth-promoting activity and anti-
stress effects [1–3]. Since the discovery of the first member
named brassinolide in 1979, about 40 other native brassi-
nosteroids have been identified from a broad variety of
plants [4]. Research on brassinosteroids has recently been
encouraged as studies have suggested that they, like other
phytohormones, act in part by regulating gene expression
[5,6].
Brassinosteroids synthesis was based on relatively few
strategies involving the A-B steroidal ring junction [7,8]. In
this paper, we report the synthesis of six analogs (see Fig. 1),
four of which are new, through an alternative synthetic strategy
that involves regioselective 5,6 epoxidation of the ⌬5 double
bond of stigmasterol and subsequent reductive or hydrolytic
opening of that epoxide. Scheme 1 depicts the reductive open-
ing of the 5␤,6␤ epoxy precursor to achieve analogs with a
5␣H moiety: (22R,23R,24S)-3␤-acetoxy-22,23-dihydroxy-
Auxin-like bioactivity of the synthetic compounds was
evaluated by the rice lamina inclination bioassay. Though
larger effects were detected for known compounds 19 and
22 (see Table 1), further studies should be done to determine
possible practical applications of the new compounds. Other
brassinosteroid analogs have shown bioactivity at field trials
notwithstanding they were not active at the bioassay level
[3].
Prompted by results indicating antiviral effects for oxy-
sterols [9] and steroidal marine compounds [10], we intro-
duce in this paper some new interesting data concerning
inhibition of HSV-1 virus yield in infected Vero cells by
synthetic brassinosteroids 17–22 (see Table 1).
* Corresponding author. Tel.: ϩ54-11-4576-3346; fax: ϩ54-11-4576-
3385.
E-mail address: lyrgala@qo.fcen.uba.ar (L.R. Galagovsky).
0039-128X/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(00)00093-3

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J.A. Ram´ırez et al. / Steroids 65 (2000) 329–337
Fig. 1. Target compounds.
2. Experimental
commercial grade. Reactions were monitored by TLC on
precoated plates with silicagel F254 0.2 mm (Merck, West
Point, PA, USA). Column chromatography was carried out
on silica gel 60, 0.04–0.063 mm (Merck).
2.1. General
Stereochemistry at C-22 and C-23 was defined by spec-
troscopic data correspondence with already described com-
pounds.
Melting points (m.p.) were determined on a Fisher Johns
1
apparatus and are uncorrected. H and 13C NMR spectra
were recorded on a Brucker AC-200 at 200.1 and 50.3 MHz,
respectively. Chemical shifts (␦) are given in ppm down-
field from TMS as the internal standard, and coupling con-
stants ( J) values are in Hz. High-resolution mass spectra
(EI or FAB) were obtained for all new compounds on a
ZAB BEqQ instrument (VG-Micromass). Low resolution
mass spectra were recorded on a Varian-MAT CH-7 A, on
a VG-TRIO-2, or on a Shimadzu QP-5000 at 70 eV. Unless
otherwise indicated, all solvents and reagents used were of
2.2. Syntheses of compounds
2.2.1. (22E)-5␣-Stigmast-22-en-3␤,6␤-diol (1a)
Lithium aluminum hydride (151 mg, 3.9 mmol) was
added to a solution of aluminum chloride (971 mg, 7.3
mmol) in dry THF (10 ml). After 5 min, a solution of
(22E)-3␤-acetoxy-5␤,6␤-epoxystigmast-22-ene (300 mg,
Scheme 1. Reagents and conditions: (a) LiAlH4/AlCl3/THF/r.t. (b) TBDMSCl/imidazole/DMF/r.t. (c) PCC/CH2Cl2/0°C. (d) nBu4NF/THF/r.t. (e) AcCl/Py/r.t.
(f) MsCl/Py/r.t. (g) LiBr/MeOH/reflux. (h) LiBr/DMF/reflux. (i) K2OsO4. 2H2O/(DHQD)2Phal/methanesulfonamide/K3Fe(CN)6/K2CO3/t-BuOH/H2O.

J.A. Ram´ırez et al. / Steroids 65 (2000) 329–337
331
Scheme 2. Reagents and conditions: (a) HClO4/dioxane/H2O/r.t. (b) PCC/CH2Cl2/0°C. (c) K2OsO4. 2H2O/(DHQD)2Phal/methanesulfonamide/K3Fe(CN)6/
K2CO3/t-BuOH/H2O. (d) LiBr/acetone/reflux. (e) KI/acetone/reflux.
0.64 mmol) in dry THF (10 ml) was added dropwise, and
the reaction mixture was stirred for 3 h. Then acetone (5 ml)
and water (5 ml) were added carefully. Extraction with ethyl
ether and purification by chromatography (hexane/EtOAc)
at room temperature. The solvent was evaporated off, and
the residue was extracted with methylene chloride/water.
The organic layer was dried (MgSO4) and evaporated,
yielding 100% of (22E)-3␤-(t-butyldimethylsilyloxy)-5␣-
stigmast-22-en-6␤-ol (2). m.p.: 170–171°C. 1H NMR
(CDCl3): 0.70 (3H, s, 18-H3), 1.03 (3H, s, 19-H3), 3.60 (1H,
m, 3␣-H), 3.75 (1H, m, 6␣-H), 0.40 (3H, s, CH3Si-), 0.88
(9H, s, (CH3)3CSi), 5.10 (2H, m, 22-H and 23-H). 13C NMR
(CDCl3): Ϫ4.6, 12.2, 12.3, 15.8, 18.2, 19.0, 21.1, 21.1, 21.2,
24.3, 25.4, 25.9, 28.9, 30.4, 31.9, 32.0, 34.0, 35.8, 38.7,
39.5, 39.9, 40.5, 42.6, 47.6, 51.3, 54.4, 56.1, 56.4, 72.5,
72.3, 129.3, 138.3. MS (EI): m/z (%), 487 (Mϩ-[(CH3)3C-],
1), 469 (3), 255 (5), 83 (100).
1
gave compound 1a in 44% yield. m.p.: 200–201°C. H
NMR (CDCl3): 0.68 (3H, s, 18-H3), 1.00 (3H, s, 19-H3),
3.60 (1H, m, 3␣-H), 3.75 (1H, m, 6␣-H), 5.10 (2H, m, 22-H
and 23-H). 13C NMR (CDCl3): 12.0, 12.1, 15.5, 21.1, 21.2,
24.1, 18.8, 25.2, 20.9, 31.0, 30.3, 28.8, 35.2, 31.7, 38.4,
34.8, 39.3, 40.3, 39.7, 42.4, 47.4, 51.1, 54.2, 56.0, 56.2,
71.2, 71.6, 129.2, 138.2. MS (EI) m/z (%): 430 (Mϩ, 14),
273 (12), 255 (18), 55 (100). HRMS (EI): Calculated for
C29H50O2: 430.3811. Found: 430.3812.
2.2.2. (22E)-3␤-(t-Butyldimethylsilyloxy)-5␣-stigmast-22-
en-6␤-ol (2)
2.2.3. (22E)-3␤-(t-Butyldimethylsilyloxy)-5␣-stigmast-22-
en-6-one (3)
(22E)-5␣-Stigmast-22-en-3␤,6␤-diol (1a) (522 mg, 1.21
mmol), dissolved in dry dimethylformamide (DMF) (40
ml), was treated with imidazole (210 mg, 3.08 mmol) and
t-butyldimethylsilyl chloride (220 mg, 1.45 mmol) for 24 h
A solution of 2 (150 mg, 0.27 mmol) in methylene
chloride (5 ml) was added to a suspension of pyridinium
chlorochromate (260 mg, 1.20 mmol) in methylene chloride
(10 ml) at 0°C. The reaction mixture was stirred for 20 min
and then allowed to reach room temperature. The suspen-
sion was filtered through a pad of silicagel; removal of the
solvent from the filtrate gave a crude solid, which was
purified by chromatography (hexane/EtOAc) to afford com-
Table 1
Bioactivities of synthetic compounds 17–22
1
pound 3 (95%). m.p.: 151–152°C. H NMR (CDCl3): 0.68
Compound
Auxin-like effect
Average angle
Antiviral effect
(3H, s, 18-H3), 0.75 (3H, s, 19-H3), 3.52 (1H, m, 3␣-H),
0.40 (3H, s, CH3Si), 0.88 (9H, s, (CH3)3CSi), 5.10 (2H, m,
22-H and 23-H). 13C NMR (CDCl3): Ϫ4.6, 12.2, 13.2, 18.2,
18.2, 19.0, 21.1, 21.2, 21.5, 24.1, 25.4, 25.9, 28.7, 30.3,
31.3, 31.9, 36.9, 37.9, 39.5, 40.4, 41.0, 42.9, 46.8, 51.2,
54.1, 56.0, 57.0, 57.0, 71.5, 129.6, 138.0, 211.1, MS (EI):
m/z 485 (Mϩ-(CH3)3C-, 100), 345 (2), 83 (49), 43 (19).
HRMS (EI): Calculated for C31H53SiO [M-[(CH3)3C-]]ϩ:
485.3815. Found 485.3813.
%
IC50
*
CC50
*
IS50
17
18
19
20
21
22
41 Ϯ 4
32 Ϯ 2
70 Ϯ 3
20 Ϯ 2
34.5 Ϯ 3
52 Ϯ 4
—
58.6
22.4
53.3
41.3
57.6
17.7
40.4
48.8
238
248
209
230
23
10.6
4.7
5.1
4
1.3
1
45.7
100.0
28.6
49.3
85.0
—
40
479
Stigmasterol
10
* Concentration expressed in ␮M.

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J.A. Ram´ırez et al. / Steroids 65 (2000) 329–337
2.2.4. (22E)-3␤-Hydroxy-5␣-stigmast-22-en-6-one (4)
Tetrabutylammonium fluoride (0.85 ml of 1 M in THF)
was added to a solution of compound 3 (200 mg, 0.37
mmol) in anhydrous THF (10 ml), and the reaction mixture
was stirred for 4 h at room temperature. The solvent was
evaporated off, and the crude residue was partitioned be-
tween ether/water. The ethereal layer was dried and evap-
orated to give compound 4 in quantitative yield. m.p.: 145–
layer was washed with water, dried (MgSO4), and evapo-
rated. Compounds 3␣ and 3␤-bromo-5␣-cholest-22-en-6-
one (1:2) were separated and purified by flash chromatog-
raphy (methylene chloride), yielding 64% of 7. m.p.: 140–
141°C. 1H NMR (CDCl3): 0.67 (3H, s, 18-H3), 0.69 (3H, s,
19-H3), 3.92 (1H, m, 3␣-H). 13C NMR (CDCl3): 12.1, 12.1,
12.6, 18.9, 21.0, 21.1, 21.2, 23.9, 25.2, 28.6, 31.7, 32.3,
33.3, 37.6, 39.0, 39.2, 40.2, 40.5, 42.7, 46.4, 50.5, 51.1,
53.7, 55.8, 56.7, 58.8, 129.5, 137.8, 209.3. MS (EI): m/z 492
(Mϩ, 81Br, 5), 490 (Mϩ, 79Br, 5), 410 (Mϩ-Br, 1), 449 (4),
351 (12), 55 (100).
1
146°C (lit. 146–147°C [11]). H NMR (CDCl3): 0.67 (3H,
s, 18-H3), 0.77 (3H, s, 19-H3), 4.54 (1H, m, 3␣-H), 5.10
(2H, m, 22-H and 23-H). 13C NMR (CDCl3): 12.3, 13.2,
12.3, 19.0, 21.1, 21.2, 24.1, 21.6, 25.4, 30.1, 28.8, 30.8,
38.0, 31.9, 41.0, 43.0, 36.8, 39.5, 40.4, 46.8, 54.0, 51.3,
56.0, 57.0, 56.9, 70.8, 129.7, 138.0, 206.0. MS (EI): m/z 428
(Mϩ, 32), 410 (2), 286 (49), 55 (100).
2.2.8. (22E,24S)-5␣-Stigmasta-2,22-dien-6-one (8)
Compound 6 (421 mg, 0.83 mmol) in dimethylform-
amide (5 ml) was treated with lithium bromide under reflux
for 1 h. The solvent was evaporated and the crude solid was
dissolved in methylene chloride. This solution was washed
with water, and then the organic layer was dried and evap-
orated. After purification by column chromatography, com-
pound 8 was obtained (72% yield). m.p.: 133–134°C (lit.
2.2.5. (22E)-3␤-Acetoxy-5␣-stigmast-22-en-6-one (5)
Compound 4 (500 mg, 1.14 mmol) was dissolved in
pyridine (5 ml) and acetic anhydride (2.5 ml) was added.
The mixture was allowed to react overnight at room tem-
perature. Then, the solution was poured over 100 ml of
ice-cold 5% aq. HCl. After extraction with methylene chlo-
ride, the crude product was purified by flash chromatogra-
phy (hexane/EtOAc), yielding 5 almost quantitatively. m.p.:
1
133–135°C [13]). H NMR (CDCl3): 0.70 (3H, s, 18-H3),
0.72 (3H, s, 19-H3), 5.10 (2H, m, 22-H and 23-H), 5.63 (2H,
m, 2-H and 3-H). 13C NMR (CDCl3): 12.1, 12.2, 13.4, 21.1,
21.1, 21.2, 24.0, 18.9, 21.7, 25.3, 28.7, 31.8, 39.4, 40.3,
39.4, 37.7, 47.0, 39.9, 42.7, 51.2, 53.4, 53.8, 55.9, 56.9,
124.4, 124.9, 129.6, 138.0, 211.7. MS: m/z 410 (Mϩ, 5), 395
(2), 271 (10), 55 (100).
1
146–147°C (lit. 146–147°C [12]). H NMR (CDCl3): 0.68
(3H, s, 18-H3), 0.77 (3H, s, 19-H3), 4.67 (1H, m, 3␣-H),
2.40 (3H, s, CH3CO), 5.10 (2H, m, 22-H and 23-H). 13C
NMR (CDCl3): 11.9, 12.0, 12.7, 18.7, 20.8, 20.9, 21.2, 21.2,
23.7, 25.1, 25.8, 26.6, 28.4, 31.5, 36.1, 37.6, 39.1, 40.1,
40.6, 42.6, 46.3, 50.9, 53.5, 56.1, 56.5, 56.6, 72.5, 129.3,
137.6, 170.5, 209.6. MS (EI): m/z 470 (Mϩ, 20), 427 (3),
410 (Mϩ-AcOH, 3), 367 (16), 358 (23), 271 (20), 43 (100).
2.2.9. (22R,23R)-3␤-Acetoxy-22,23-dihydroxy-5␣-
stigmastan-6-one (17)
A mixture of 5 (760 mg, 1.61 mmol), THF (8 ml),
t-butanol/water (1:1) (32 ml), (DHQD)2-PHAL (252 mg,
0.32 mmol), methansulfonamide (308 mg, 2.77 mmol),
potassium ferricyanide (1.60 g, 4.80 mmol), potassium
carbonate (672 mg, 2.63 mmol), and potassium osmate
dihydrate (29 mg, 0.08 mmol) was stirred at room tem-
perature for 9 days. An excess of sodium bisulfite
(NaHSO3) was added until no evolution of bubbles was
observed. Layers were separated and the aqueous phase
was thoroughly extracted with methylene chloride/meth-
anol (5%). Combined organic layers were washed with
0.25 M sulfuric acid and 2% sodium hydroxide. Purifi-
cation by column chromatography (methylene chloride/
acetonitrile gradient) allowed to separate (22R,23R)-3␤-
acetoxy-22,23-dihydroxy-5␣-stigmastan-6-one (17) from
its 22S, 23S diasteromer, with 22% yield. m.p.: 274–
2.2.6. (22E)-3␤-Mesyloxy-5␣-stigmast-22-en-6-one (6)
Compound 4 (500 mg, 1.14 mmol) was dissolved in
pyridine (5 ml), and methanesulfonyl chloride (0.2 ml) was
added. The reaction mixture was stirred at room temperature
for 1 h. Then, the solution was poured over 100 ml of ice
cold 5% aq. HCl. After extraction with methylene chloride,
the organic layer was dried (MgSO4) and evaporated off,
yielding 6 quantitatively. m.p.: 148–149°C. 1H NMR
(CDCl3): 0.69 (3H, s, 18-H3), 0.79 (3H, s, 19-H3), 4.65 (1H,
m, 3␣-H), 2.98 (3H, s, CH3SO2), 5.10 (2H, m, 22-H and
23-H), 13C NMR (CDCl3): 11.9, 12.0, 12.6, 18.7, 20.8, 20.9,
21.1, 23.7, 25.1, 27.0, 27.9, 28.4, 31.5, 36.0, 37.5, 38.5,
39.0, 40.1, 40.2, 42.5, 46.1, 50.9, 53.2, 55.6, 55.9, 56.4,
80.9, 129.3, 137.6, 208.8. MS (EI): m/z 506 (Mϩ, 12), 463
(3), 410 (Mϩ-MsOH, 21), 394 (34), 367 (28), 271 (39), 55
(100). HRMS (EI): Calculated for C30H50O4S, Mϩ:
506.3430. Found 506.3424.
1
275°C (d). H NMR (CDCl3/CD3OD 9:1): 0.68 (3H, s,
18-H3), 0.70 (3H, s, 19-H3), 4.68 (1H, m, 3␣-H), 3.60
(1H, d, J ϭ 8 Hz, 22-H), 3.73 (1H, d, J ϭ 8 Hz, 23-H),
2.40 (3H, s, CH3CO). 13C NMR (CDCl3/CD3OD 9:1):
11.7, 11.7, 12.9, 21.4, 23.7, 25.9, 26.7, 27.4, 36.2, 36.8,
38.0, 39.3, 40.9, 42.7, 46.4, 52.4, 53.6, 56.3, 56.4, 72.9,
210.9. HRMS (FAB): Calculated for C31H53O5 [M ϩ
H]ϩ: 505.3893. Found 505.3902.
2.2.7. (22E)-3␤-Bromo-5␣-stigmast-22-en-6-one (7)
Compound 6 (635 mg, 1.25 mmol) dissolved in methanol
(50 ml) was treated with lithium bromide (715 mg, 8.31
mmol) under reflux for 6 h. After solvent evaporation, the
residue was dissolved in methylene chloride, the organic

J.A. Ram´ırez et al. / Steroids 65 (2000) 329–337
333
2.2.10. (22R,23R)-3␤-Bromo-22,23-dihydroxy-5␣-
stigmastan-6-one (18)
The reaction mixture was stirred for 2 h at room tempera-
ture, and then neutralized with sodium bicarbonate (saturat-
ed solution). The solvent was evaporated, and the residue,
dissolved in methylene chloride, was thoroughly washed
with water. The crude product was purified by chromatog-
raphy (hexane/EtOAc), yielding 90% of compound 11.
Compound 7 (795 mg, 1.60 mmol) was treated similarly
to compound 5. Purification by silica column chromatogra-
phy (methylene chloride/acetonitrile gradient) allowed to
separate compound 18 from its 22S, 23S diasteromer with
1
1
18.3% yield. m.p.: 193–194°C. H NMR (CDCl3/CD3OD
m.p.: 237–238°C. H NMR (CDCl3): 0.70 (3H, s, H-183),
9:1): 0.70 (3H, s, 18-H3), 0.82 (3H, s, 19-H3), 3.93 (1H, m,
3␣-H), 3.60 (1H, d, J ϭ 8 Hz, 22-H), 3.73 (1H, d, J ϭ 8
Hz, 23-H). 13C NMR (CDCl3/CD3OD 9:1): 11.9, 11.9, 13.1,
21.3, 23.8, 27.6, 32.3, 33.4, 37.0, 37.8, 39.2, 39.4, 40.7,
42.8, 46.5, 50.5, 52.5, 53.8, 56.6, 59.0, 206.6. HRMS
(FAB): Calculated for C29H50O3Br [M ϩ H]ϩ: 525.2943.
Found 525.2947.
1.19 (3H, s, H-193), 2.40 (3H, s, CH3CO), 3.35 (1H, bs,
6-␣H), 5.02 (1H, m, 3-␣H), 5.10 (2H, m, 22-H and 23-H).
13C NMR (CDCl3): 12.2, 12.3, 16.7, 19.0, 21.1, 21.1, 21.2,
21.2, 24.2, 25.4, 26.7, 28.9, 30.2, 31.9, 32.1, 34.6, 37.0,
38.3, 39.8, 40.5, 42.6, 45.5, 51.2, 55.9, 56.0, 71.2, 75.7,
76.2, 129.3, 138.3, 170.5. MS (EI): m/z (%), 488 (Mϩ, 3),
470 (Mϩ-H2O, 2), 410 (7), 367 (6), 271 (9), 253 (30), 55
(100).
2.2.11. (22R,23R)-2␣,3␣,22,23-Tetrahydroxy-5␣-
stigmastan-6-one (24-homocastasterone) (19)
Compound 8 (768 mg, 1.87 mmol) was treated as de-
scribed for compound 5. After the usual work-up, com-
pound 19 was obtained in 34% yield. All physical and
spectral data for 18 were consistent with those previously
reported [14].
2.2.14. (22E)-3␤-Tosyloxystigmast-22-en-5␣,6␤-diol (12)
(22E)-3␤-Tosyloxy-5,6-epoxystigmast-22-ene
(10)
(500 mg, 0.85 mmol) was dissolved in a solution of
dioxane (60 ml), water (15 ml), and 70% perchloric acid
(10 ml). The mixture was allowed to react for 2 h at room
temperature and then neutralized with sodium bicarbon-
ate (s.s.). The solvent was evaporated, and the residue,
dissolved in methylene chloride, was thoroughly washed
with water. Purification by chromatography (hexane/
EtOAc) yielded 94% of 12. m.p.: 142–143°C (d). 1H
NMR (CDCl3): 0.68 (3H, s, H-183), 1.16 (3H, s, H-193),
3.52 (1H, bs, 6␣-H), 4.90 (1H, m, 3␣-H), 5.10 (2H, m,
22-H and 23-H). 13C NMR (CDCl3): 12.3, 12.4, 16.7,
19.1, 21.1, 21.2, 21.2, 21.5, 24.2, 25.4, 27.7, 28.9, 30.2,
31.9, 32.3, 34.6, 37.1, 38.2, 39.8, 40.5, 42.5, 45.7, 51.3,
56.0, 56.1, 75.3, 75.8, 80.5, 127.5, 129.4, 129.6, 134.7,
138.3, 144.2. MS (EI): m/z 428 (Mϩ-TsOH, 3), 410 (17),
392 (41), 43 (100).
2.2.12. (22E)-5,6-Epoxy-3␤-tosyloxystigmast-22-ene (10)
(22E)-3␤-Tosyloxystigmasta-2,22-diene (800 mg, 1.41
mmol) in methylene chloride (250 ml) was treated with
m-chloroperbenzoic acid (430 mg, 2.10 mmol) at Ϫ78°C.
Work-up gave a mixture of 5␣,6␣ and 5␤,6␤-epoxides
(5:1). Purification by chromatography (methylene chloride/
hexane) yielded (22E)-5␣,6␣-epoxy-3␤-tosyloxystigmast-
22-ene (65%) and (22E)-5␤,6␤-epoxy-3␤-tosyloxystig-
mast-22-ene (15%).
First eluted compound (5␣,6␣-epoxide): m.p.: 171–
173°C. 1H NMR (CDCl3): 0.63 (3H, s, 18-H3), 1.00 (3H, s,
19-H3), 4.60 (1H, m, 3␣-H), 2.80 (1H, m, 6␤-H), 1.44 (3H,
s, CH3-Ph), 5.10 (2H, m, 22-H and 23-H), 7.26 and 7.72
(4H, d, J ϭ 9 Hz, CH3-Ph). MS (EI): m/z 410 (Mϩ-TsOH,
12), 392 (11), 367 (5), 298 (6), 271 (9), 55 (100).
2.2.15. (22E)-3␤-Acetoxy-5␣-hydroxystigmast-22-en-6-one
(13)
Second eluted compound (5␤,6␤-epoxide): m.p.: 96°C.
1H NMR (CDCl3): 0.61 (3H, s, 18-H3), 1.03 (3H, s, 19-H3),
4.60 (1H, m, 3␣-H), 1.44 (3H, s, CH3-Ph), 2.95 (1H, m,
6␣-H), 5.10 (2H, m, 22-H and 23-H) 7.26 and 7.72 (4H, d,
J ϭ 9 Hz, CH3-Ph). 13C NMR (CDCl3): 11.8, 12.2, 16.8,
18.9, 21.0, 21.1, 21.5, 21.8, 24.1, 25.3, 27.9, 28.7, 29.5,
31.8, 32.2, 34.7, 36.1, 38.5, 39.5, 40.4, 42.0, 51.0, 51.1,
55.8, 56.1, 62.2, 63.3, 79.3, 127.5, 129.3, 129.6, 134.7,
138.1, 144.2. MS (EI): m/z (%): 410 (Mϩ-TsOH, 4), 392
(8), 367 (2), 298 (3), 271 (6), 55 (100).
Compound 11 (300 mg; 0.51 mmol) dissolved in meth-
ylene chloride (10 ml) was added to a suspension of pyri-
dinium chlorocromate (170 mg, 0.78 mmol) in methylene
chloride (25 ml) at 0°C. After 20 min the ice bath was
removed and the reaction was allowed to stand for 4 h at
room temperature. The suspension was then filtered off
through a pad of silica gel D. The solvent was evaporated,
and the crude solid was purified by column chromatogra-
phy. Elution with hexane/ethyl acetate (8:2) provided 13
1
[15] in 82% yield. m.p.: 253–254°C. H NMR (CDCl3):
0.66 (3H, s, 18-H3), 0.80 (3H, s, 19-H3), 2.40 (3H, s,
CH3CO), 5.00 (1H, m, 3␣-H), 5.10 (2H, m, 22-H and 23-H).
13C NMR (CDCl3): 12.2, 12.2, 13.9, 19.0, 21.1, 21.2, 21.2,
21.3, 24.0, 25.4, 26.3, 28.8, 29.5, 31.8, 32.3, 37.3, 39.5,
40.4, 41.7, 42.5, 43.0, 44.3, 51.2, 56.0, 56.4, 70.8, 80.2,
129.6, 138.1, 170.5, 212.4. MS (EI): m/z 486 (Mϩ, 3), 426
(3), 408 (4), 365 (13), 269 (14), 55 (100).
2.2.13. (22E)-3␤-Acetoxystigmast-22-en-5␣,6␤-diol (11)
A mixture of 5␣,6␣ and 5␤,6␤ epoxides (9) was obtained
by epoxidation of (22E)-3␤-acetoxystigmasta-5,22-diene
with m-chloroperbenzoic acid using the same procedure
described for compound 10. The mixture (500 mg, 1.06
mmol) of compound 9 was dissolved in a solution of diox-
ane (70 ml), water (17 ml), and 70% perchloric acid (6 ml).

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J.A. Ram´ırez et al. / Steroids 65 (2000) 329–337
2.2.16. (22E)-5␣-Hydroxy-3␤-tosyloxystigmast-22-en-6-
one (14)
2.2.19. (22R,23R)-3␤-Acetoxy-5␣,22,23-trihydroxy-
stigmastan-6-one (20)
Compound 13 (779 mg, 1.60 mmol) was treated in a
similar way as described for compound 5. Crude solid was
purified from starting material and 22S, 23S diasteromer by
column chromatography (methylene chloride/acetonitrile)
Compound 12 (300 mg, 0.50 mmol) was as described
for compound 11. Purification of crude product by flash
column chromatography with hexane/ethyl acetate (4:1)
as the eluent, yielded 94% of 14. m.p.: 88–90°C. 1H
NMR (CDCl3): 0.67 (3H, s, 18-H3), 0.73 (3H, s, 19-H3),
4.42 (1H, m, 3␣-H), 1.44 (3H, s, CH3-Ph), 5.10 (2H, m,
22-H and 23-H), 7.26 and 7.72 (4H, d, J ϭ 9 Hz, CH3-
Ph). 13C NMR (CDCl3): 12.3, 12.3, 13.9, 19.1, 21.3,
21.4, 21.3, 21.5, 24.1, 25.5, 27.4, 28.9, 29.8, 32.0, 33.6,
37.4, 39.5, 40.5, 41.7, 42.4, 43.1, 44.2, 51.3, 56.4, 56.0,
79.9, 80.4, 127.5, 129.5, 129.6, 134.7, 138.0, 144.2,
212.2. MS (EI): m/z 426 (Mϩ-TsOH, 2), 408 (8), 392 (2),
43 (100).
1
to give compound 20 (12% yield). m.p.: 197–199°C. H
NMR (CDCl3/CD3OD 9:1): 0.71 (3H, s, 18-H3), 0.77 (3H,
s, 19-H3), 2.40 (3H, s, CH3CO), 4.68 (1H, m, 3␣-H), 3.60
(1H, d, J ϭ 8.1 Hz, 22-H), 3.75 (1H, d, J ϭ 8.1 Hz, 23-H).
13C NMR (CDCl3/CD3OD 9:1): 11.7, 11.7, 12.9, 21.2, 21.4,
23.7, 25.9, 26.7, 27.4, 36.2, 36.8, 38.0, 39.3, 40.9, 42.7,
46.4, 52.4, 53.6, 56.3, 56.4, 72.9, 170.5, 210.9. HRMS
(FAB): Calculated for C31H53O6 [M ϩ H]ϩ: 521.3842.
Found 521.3849.
2.2.20. (22R,23R)-3␤-Bromo-5␣,22,23-trihydroxy-
stigmastan-6-one (21)
2.2.17. (22E)-3␤-Acetoxy-5␣-hydroxycholest-22-en-6-one
(15)
Compound 15 (816 mg, 1.60 mmol) (816 mg, 1.60
mmol) was treated in a similar way as described for com-
pound 5. Crude solid was purified from starting material and
22S, 23S diasteromer by column chromatography (methyl-
ene chloride/acetonitrile gradient) to give compound 21
Compound 14 (750 mg, 1.14 mmol), dissolved in
acetone (50 ml), was treated with lithium bromide (715
mg, 8.31 mmol) under reflux for 6 h. After solvent
evaporation, the residue was dissolved in methylene
chloride, the organic layer was washed with water, dried
(MgSO4), and evaporated. A mixture of 3␤ and 3␣-
bromides (2.3:1) was obtained. Isolation was performed
by column chromatography (methylene chloride), yield-
ing 54% of 15. m.p.: 200–201°C. 1H NMR (CDCl3): 0.68
(3H, s, 18-H3), 0.86 (3H, s, 19-H3), 4.32 (1H, m, 3␣-H),
5.10 (2H, m, 22-H and 23-H). 13C NMR (CDCl3): 12.3,
12.3, 14.1, 19.1, 21.1, 21.2, 21.3, 24.0, 25.4, 28.7, 32.0,
32.2, 32.9, 37.4, 38.8, 39.5, 40.4, 42.0, 42.3, 43.1, 44.7,
48.0, 51.3, 56.0, 56.5, 81.0, 129.6, 138.9, 211.7. MS (EI):
m/z 508 (Mϩ, 81Br, 2); 506 (Mϩ, 79Br, 2), 427 (1), 43
(100). HRMS (EI): Calculated: 506.2759. Found:
506.2754.
1
(18.7% yield). m.p.: 205°C (d). H NMR (CDCl3/CD3OD
9:1): 0.66 (3H, s, 18-H3), 0.85 (3H, s, 19-H3), 4.35 (1H, m,
3␣-H), 3.57 (1H, d, J ϭ 8.1 Hz, 22-H), 3.73 (1H, d, J ϭ
8.1 Hz, 23-H). 13C NMR (CDCl3/CD3OD 9:1): 11.6, 11.6,
13.4, 13.6, 21.0, 19.2, 18.7, 23.6, 20.9, 27.3, 28.8, 32.7,
31.9, 38.2, 37.3, 42.0, 39.4, 36.8, 44.1, 41.5, 42.7, 46.3,
48.4, 52.3, 56.0, 72.2, 74.2, 80.2, 213.4. HRMS (FAB):
Calculated for C31H53O3Br [M ϩ H]ϩ: 541.2892. Found
541.2891.
2.2.21. (22R,23R)-2␣,3␣,5␣,22,23-Pentahydroxy-
stigmastan-6-one (22)
Compound 16 (735 mg, 1.7 mmol) was treated in a
similar way as described for 5. After the usual work-up,
compound 22 was obtained in 12% yield. All physical and
spectral data for 22 were consistent with those previously
reported [16].
2.2.18. (22E)-5␣-Hydroxystigmasta-2,22-dien-6-one (16)
Compound 14 (500 mg, 0.83 mmol), dissolved in ace-
tone (100 ml) was treated with potassium iodide (350 mg,
2.10 mmol) under reflux for 2 h. Then, the solvent was
evaporated off, and the crude residue was dissolved in
dimethylsulfoxide (aprox. 20 ml). Lithium carbonate (700
mg, 3.81 mmol) was added, and this new solution was
stirred for 2 h at 120°C. The solvent was evaporated again,
and the residue was extracted with ethyl ether/water. The
organic phase was dried (MgSO4) and purified by chroma-
tography (hexane/ethyl acetate) to afford 16 in 74% yield.
2.3. Rice lamina inclination test
The activities of the compounds as plant growth promot-
ers were evaluated by means of a highly sensitive, modified
[16] rice lamina inclination test based on the procedure
described by Wada et al. [17]. Seedlings of rice (Oryza
sativa, Chuy variety) were washed with ethanol and water
and then were left in water at 30°C for 2 days (with a 16 h
photoperiod). Germinated seeds were cultivated on agar
under the same growing conditions for 4 days. Intact seed-
lings (4–5 cm long) were inoculated with 0.5 ␮l of the test
compound solution (1 mg/ml in ethanol) just under the 2nd
leaf joint. After 48 h of growing in the dark (at 30°C), the
magnitude of the induced angle between the leaf and the
sheath was measured.
1
m.p.: 154–155°C. H NMR (CDCl3): 0.67 (3H, s, 18-H3),
0.71 (3H, s, 19-H3), 5.10 (2H, m, 22-H and 23-H), 5.65 (2H,
m, 2-H and 3-H). 13C NMR (CDCl3): 12.4, 12.4, 14.6, 19.1,
21.1, 21.3, 21.3, 24.1, 25.5, 28.8, 32.0, 34.6, 34.6, 37.5,
39.5, 40.5, 42.4, 42.7, 42.9, 45.3, 51.3, 56.6, 56.9, 78.1,
122.4, 125.4, 129.5, 137.9, 211.2. MS (EI): m/z 426 (Mϩ,
10), 408 (3), 83 (23), 55 (100).

J.A. Ram´ırez et al. / Steroids 65 (2000) 329–337
335
Fig. 2. Reductive opening of 5␤,6␤ epoxide.
2.4. Antiviral assays
adsorption at 4°C residual inoculum was replaced by MM
containing 0.7% of methylcellulose and the different con-
centrations of the brassinosteroid. After 48 h of incubation
at 37°C, virus plaques were counted. The 50% antiviral
effective concentration (IC50) was calculated as the concen-
tration required to reduce virus plaques by 50%. All deter-
minations were performed in duplicate.
2.4.1. Cells and virus
Monkey kidney Vero cells were grown as monolayers in
minimum essential medium (MEM) supplemented with 6%
inactivated calf serum and 50 ␮g/ml gentamycin. Mainte-
nance medium (MM) consisted of MEM containing 2%
inactivated serum. The virus used was herpes simplex virus
type 1 (HSV-1) F strain (American Type Culture Collec-
tion; Rockville, MD, USA).
Selectivity indexes were calculated as the rate between
CC50 and IC50.
2.4.2. Cytotoxicity assay
3. Results and discussion
Cytotoxicity was determinated by the MTT colorimetric
assay [18]: cleavage of the tetrazolium salt MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
(Sigma Chemical Co., St. Louis, MO, USA) by the mito-
chondrial enzyme succinate dehydrogenase to give a blue
product (formazan) was used to assay growth and cell sur-
vival.
Vero cells grown in 96-well culture plates for 24 h were
treated with serial dilutions of the test brassinosteroid for
24, 48, or 72 h. After this time, cell monolayers were
washed with Hanks solution, and 50 ␮l of MTT (0.1 ␮g/ml)
were added to each well. After 2 h of incubation at 37°C,
4% CO2 and 100% relative humidity, supernatants were
removed and 200 ␮l of 96% ethanol were added to solubi-
lize the formazan. Optical density of each well was mea-
sured on an Eurogenetics MPR-A 4i microplate reader,
using a test wavelength of 570 nm and a reference wave-
length of 630 nm.
Synthesis of compounds 17–19 with a 5␣H moiety re-
quired (22E)-3␤-acetoxy-5␤,6␤-epoxystigmast-22-ene as
the starting compound (see Scheme 1), that was obtained by
regio and stereoselective epoxidation of stigmasteryl acetate
[19]. The reductive opening of the 5␤,6␤-epoxide to
achieve a 6␤-hydroxy derivative required the use of alane
[20] because other regular reductive reagents produced
large amounts of the 5␤-hydroxy derivative. This alane
reagent ensured a predominant trans diaxial attack of the
hydride on the more hindered C-5 position [21] of the
epoxide (see Fig. 2), leading to (22E)-3␤,6␤-dihydroxy-5␣-
stigmastan-22-ene (1a) and to (22E)-3␤,5␤-dihydroxy-5␤-
stigmastan-22-ene (1b) in a 3:1 ratio [22].
Lack of selectivity on direct oxidation [23] of compound
1a required silyl ether protection for the hydroxyl group at
C-3. Compound 2 was then oxidized with pyridine chloro-
chromate (PCC) to give compound 3. Deprotection of 3
with t-butylammonium fluoride led to (22E)-3␤-hydroxy-
5␣-stigmastan-6-one (4), which was acetylated to yield
compound 5. Sharpless’ catalytic, asymmetric dihydroxyla-
tion (CAD) [24] of compound 5 furnished target compound
17 (22R,23R)-3␤-acetoxy-22,23-dihydroxy-5␣-stigmastan-
6-one. CAD also yielded not natural occurring diastero-
meric 22S,23S compounds, in minor proportion (rate 3:1).
Target compounds 18 and 19 were obtained through a
bifurcation of this synthetic route. Mesylation of 4 led to
compound 6, which depending on the temperature of reflux
conditions, yielded either 7 or 8 when treated with lithium
bromide in methanol or dimethylformamide, respectively.
CAD performed on compound 7 yielded (22R,23R)-3␤-
bromo-22,23-dihydroxy-5␣-stigmastan-6-one (18), and
CAD performed on compound 8 yielded (24S)-homoethyl-
castasterone (19).
Results were expressed as the ratio between the optical
density in treated cultures and the optical density in the
untreated control cultures. The 50% cytotoxic concentration
(CC50) was defined as the concentration that caused a 50%
reduction in optical density.
2.4.3. Antiviral assay: virus yield inhibition method
The virus used was herpes simplex virus type 1 (HSV-1)
F strain. Vero cells were grown at 37°C with Eagle Mini-
mum Essential Medium (MEM) containing 5% of bovine
serum. Maintenance medium (MM) consisted of MEM with
1.5% of bovine serum. Compound solutions were prepared
in ethanol at a concentration of 1 mg/ml and diluted with
MM to a final concentration of 20 ␮g/ml.
Antiviral activity was evaluated by plaque reduction
method: Vero cell monolayers grown in 24-well plates were
infected with about 100 PFU of virus/well. After 1 h of
Brassinosteroid analogs bearing a 5␣-hydroxyl group (10-

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J.A. Ram´ırez et al. / Steroids 65 (2000) 329–337
22) were obtained by the synthetic pathway diagrammed in
Scheme 2. Compounds 11 and 12 were obtained as single
products by hydrolytic opening of the mixture of the 5␣,6␣ and
5␤,6␤ epoxides of (22E)-5,6-epoxystigmast-22-en-3␤-yl ace-
tate (9) or tosylate (10), respectively. Oxidation of 11 and 12
with PCC yielded 13 and 14, respectively. Compound 13 was
dihydroxylated (CAD) to yield (22R,23R)-3␤-acetoxy-5␣,22,
23-trihydroxystigmastan-6-one (20).
Compound 14 was treated with lithium bromide in ace-
tone to give the 3␤-bromo derivative (15), which was di-
hydroxylated to yield (22R,23R)-3␤-bromo-5␣,22,23-trihy-
droxy-stigmastan-6-one (21). Compound 14 was also
subjected to elimination to give (22E)-5␣-hydroxystig-
masta-2,22-dien-6-one (16) which, in turn, was tetrahy-
droxylated to obtain (22R,23R)-2␣,3␣,5␣,22,23-pentahy-
droxystigmastan-6-one (22).
recent studies have demonstrated that these compounds
are not virucides, but interesting antivirals [29].
Acknowledgments
We thank Universidad de Buenos Aires for financial
support and UMYMFOR (CONICET-FCEN) for spectro-
scopic determinations. We also thank Dr Celia Coto and Dr
Mo´nica Wachsman (FCEN-UBA) for the antiviral studies
and Lic. Marie Christine Aguayo for the auxin-like studies.
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