Pregnane-type brassinosteroids with a four-carbon ester functionality in position 20

Article abstract of DOI:10.1135/cccc19960930

Analogues of brassinolide and castasterone, containing an ester functionality with four carbon atoms in the position 20 of the pregnane skeleton, have been prepared. In the bean second internode assay the most effective compounds were (20R)-2α,3α,20-trihydroxy-5α-pregnan-6-one 20-isobutyrate (6d) and (20R)-2α,3α,20-trihydroxy-B-homo-7-oxa-5α-pregnan-6-one 20-isobutyrate (7d). On the other hand, (20R)-2α,3α,20-trihydroxy-B-homo-6-oxa-5α-pregnan-7-one 20-heptafluorobutyrate (8) was the first compound with the retarding effect in the same test.

Full text of DOI:10.1135/cccc19960930

930
Kohout, Kasal, Strnad:
PREGNANE-TYPE BRASSINOSTEROIDS WITH A FOUR-CARBON ESTER
FUNCTIONALITY IN POSITION 20*
a1
b
Ladislav KOHOUT , Alexander KASALa2 and Miroslav STRNAD
a Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
166 10 Prague 6, Czech Republic; e-mail: 1 kohout@uochb.cas.cz, 2 kasal@marylin.uochb.cas.cz
b Institute of Experimental Botany, Academy of Sciences of the Czech Republic, 772 00 Olomouc,
Czech Republic
Received January 5, 1996
Accepted February 15, 1996
Analogues of brassinolide and castasterone, containing an ester functionality with four carbon atoms
in the position 20 of the pregnane skeleton, have been prepared. In the bean second internode assay
the most effective compounds were (20R)-2α,3α,20-trihydroxy-5α-pregnan-6-one 20-isobutyrate (6d)
and (20R)-2α,3α,20-trihydroxy-B-homo-7-oxa-5α-pregnan-6-one 20-isobutyrate (7d). On the other
hand, (20R)-2α,3α,20-trihydroxy-B-homo-6-oxa-5α-pregnan-7-one 20-heptafluorobutyrate (8) was
the first compound with the retarding effect in the same test.
Key words: Brassinosteroids; PGH; Synthesis; Antibrassinolide activity.
Recent studies on brassinosteroids have focused attention on the relationship between
molecular structure and biological activity2. In one of our previous communications on
this topic3 we reported androstane derivatives of brassinolide with ester functionalities
in the position 17. Some of these compounds exhibited high brassinolide activity in the
bean second internode assay4. Recently, some other brassinosteroid esters have been
described5 the activity of which is higher than that of the parent compounds. In the
present communication we describe some pregnane type derivatives with four-carbon
ester functionalities in position 20.
Their synthesis (Scheme 1) started from (20R)-pregn-5-ene-3β,20-diol 3-tosylate6 (1)
which on reaction with chloride or anhydride of the appropriate acid in pyridine af-
forded the corresponding esters 2a–2d. Thus, reaction of compound 1 with heptafluoro-
butyric anhydride gave the heptafluorobutyrate 2a, with succinic anhydride the
hemisuccinate 2b, with butyryl chloride the butyrate 2c, and with isobutyryl chloride
the isobutyrate 2d. Reaction with potassium acetate converted the obtained esters 2a–2d
into the 3α,5α-cyclo-6-hydroxy derivatives 3a–3d which on oxidation with Jones rea-
* Part CCCLXXXIII in the series On Steroids; Part CCCLXXXII: see ref.1.
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

On Steroids
931
HO
H
RO
H
TosO
TosO
1
2
RO
H
RO
H
OH
O
3
4
RO
H
RO
H
HO
HO
H
H
O
O
6
5
RO
H
RO
H
HO
HO
HO
O
HO
O
H
H
O
O
7
8, R = OCC H
3 7
R
R
a
b
CO(CF ) CF
c
d
CO(CH ) CH
3
2 2
3
2 2
CO(CH ) COOH
COCH(CH )
2 2
3 2
SCHEME 1
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

932
Kohout, Kasal, Strnad:
gent afforded ketones 4a–4d. The ketones were treated with lithium bromide in N,N-di-
methylacetamide in the presence of pyridinium tosylate to give 2,3-olefins 5a–5d. Hy-
droxylation of these ketones with osmium tetroxide in the presence of
N-methylmorpholine N-oxide gave the corresponding castasterone analogues, diols 6a–6d.
The obtained diols 6a–6d were subjected to the Baeyer–Villiger oxidation with
trifluoroperacetic acid in dichloromethane to give the brassinolide analogues, lactones
7a–7d. The reaction with diol 6a gave also the isomeric lactone, (20R)-2α,3α, 20-trihy-
droxy-6-oxa-B-homo-5α-pregn-7-one 20-heptafluorobutyrate (8). Also in this case we
confirmed the known observation7 that in the Baeyer–Villiger reaction castasterone
analogues with 2α,3α-diol grouping give predominantly the 7-oxa lactones of the type 7,
contrary to theoretical expectation.
The biological activity was tested in the bean second internode bioassay8. Of all the
substances tested, the highest activity was found for compounds 6d and 7d; however,
even these compounds were less active than 24-epibrassinolide ((22R,23R,24R)-
2α,3α,22,23-tetrahydroxy-24-methyl-B-homo-7-oxa-5α-cholestan-6-one).
TABLE I
Bean second internode assays with compounds 5a–5d, 6a–6d, 7a–7d and 8
Compound
24-epiBRc
Lengthening of the second internode, mma
Amount applied, molb
32.3
6.7
10–10
10–8
10–10
10–7
10–10
10–11
10–7
10–7
10–9
10–9
10–8
10–7
10–11
10–9
5a
6a
7a
8a
5b
6b
7b
5c
6c
7c
5d
6d
7d
7.9
5.6
–5.4
2.9
6.7
3.6
9.9
7.2
8.6
5.8
14.0
15.1
a
b
Lengthening, compared with that of the reference plant for the amount applied. Amount causing
the maximum lengthening of the second internode (all compounds were applied in amounts 1 . 10–11
to 1 . 10–6 mol per plant). 24-epiBR: 24-epibrassinolide [(22R,23R,24R)-2α,3α,22,23-tetrahydroxy-
c
24-methyl-B-homo-7-oxa-5α-cholestan-6-one].
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

On Steroids
933
To our surprise, the compound 8 retarded substantially the growth at all doses ap-
plied, i.e, from 1 . 10–11 to 1 . 10–6 mol per plant, the highest retardation being observed
with doses 1 . 10–7 mol per plant. This is the first example of a brassinolide analogue
that exhibits antibrassinolide effect.
EXPERIMENTAL
The melting points were determined on a Kofler block and are uncorrected. Infrared spectra were
recorded on a Bruker IFS 88 spectrometer in tetrachloromethane (unless otherwise stated), wavenum-
bers are given in cm–1. 1H NMR spectra were taken in deuteriochloroform on a Varian XL-200 (FT
mode, 200 MHz) instrument with tetramethylsilane as internal reference, unless stated otherwise.
Chemical shifts are given in ppm (δ-scale), coupling constants (J) and multiplet half-widths (W1/2) in Hz.
Mass spectra were obtained with a ZAB-EG spectrometer at 70 eV. The identity of the prepared
samples was checked by mixture melting points, thin-layer chromatography (TLC), IR and proton
NMR spectra. Preparative TLC was carried out on 200 × 200 mm plates coated with 0.7 mm thick
layer of silica gel Woelm DC. Column chromatography was performed on neutral silica gel 60–120 µm.
“Usual work-up” of the solution denotes successive washing with water, 10% HCl, 5% aqueous po-
tassium hydrogen carbonate, water, drying over sodium sulfate, filtration and evaparation of the sol-
vent in vacuo to dryness. Light petroleum was a fraction boiling at 40–62 °C.
(20R)-Pregn-5-ene-3β,20-diol 3-Tosylate 20-Heptafluorobutyrate (2a)
Hydroxy derivative6 1 (841 mg, 1.78 mmol) was dissolved in pyridine (10 ml) and heptafluorobutyric
anhydride (945 mg, 2.30 mmol) was added at room temperature. After standing overnight, the reac-
tion mixture was poured into water and the product was taken up in chloroform and worked up as
usual. After standing overnight in a refrigerator, the crystalline material was triturated with ether and
collected. Crystallization from chloroform–methanol afforded 620 mg (52%) of heptafluorobutyrate
2a, m.p. 92–96 °C. IR spectrum: 1 779 (ester); 1 379, 1 192, 1 180 (tosylate). 1H NMR spectrum:
0.64 s, 3 H (3 × H-18); 0.96 s, 3 H (3 × H-19); 1.27 d, 3 H, J = 6 (3 × H-21); 2.44 s, 3 H (tosylate CH3);
4.32 m, 1 H, W1/2 = 23 (H-3α ); 5.06 m, 1 H, W1/2 = 19 (H-20); 5.28 d, 1 H, J = 5 (H-6); 7.31 d,
2 H, J = 8 (tosylate H-3 and H-5); 7.80 d, 2 H, J = 8 (tosylate H-2 and H-6). For C32H39F7O5S
(668.7) calculated: 57.48% C, 5.88% H, 19.89% F, 4.79% S; found: 57.14% C, 6.01% H, 21.50% F,
5.09% S.
(20R)-Pregn-5-ene-3β,20-diol 3-Tosylate 20-Hemisuccinate (2b)
Triethylamine (300 ml) and succinic anhydride (900 mg, 8.99 mmol) were added to a solution of
hydroxy derivative6 1 (300 mg, 0.63 mmol) in dichloromethane (18 ml). After standing for 48 h, the
mixture was poured into water and worked up in the usual manner. The chloroform solution afforded
115 mg (33%) of noncrystalline title compound. IR spectrum: 3 034 (double bond); 1 733, 1 177
1
(OCO); 1 716, 942 (COOH); 1 370, 1 189, 1 177 (tosylate). H NMR spectrum: 0.62 s, 3 H (3 × H-18);
0.96 s, 3 H (3 × H-19); 1.14 d, 3 H, J = 6 (3 × H-21); 2.44 s, 3 H (tosylate CH3); 4.29 m, 1 H, W1/2 = 27
(H-3α); 4.84 m, 1 H, W1/2 = 19 (H-20); 5.28 d, 1 H, J = 6 (H-6); 7.30 d, 2 H, J = 8 (tosylate H-3
and H-5); 7.78 d, 2 H, J = 8 (tosylate H-2 and H-6). For C31H44O7S (560.8) calculated: 66.40% C,
7.91% H, 5.72% S; found: 66.66% C, 8.11% H, 5.55% S.
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

934
Kohout, Kasal, Strnad:
(20R)-Pregn-5-ene-3β,20-diol 3-Tosylate 20-Butyrate (2c)
Butyryl chloride (1.45 g, 13.61 mmol) was added at room temperature to a solution of hydroxy deri-
vative6 1 (1.25 g, 4.64 mmol) in pyridine (6 ml). After standing overnight, the mixture was poured
into water and worked up as usual. The obtained chloroform solution was concentrated and the
residue (2.3 g) was purified by chromatography on a column of silica gel (200 g, elution with light
petroleum–benzene 1 : 1); yield 0.9 g (63%) of the product 2c, m.p. 103–104 °C. IR spectrum: 3 070,
3 035, 1 651 (C=C); 1 729, 1 160 (ester); 1 601, 1 445, 1 345, 1 189 (tosylate). 1H NMR spectrum:
0.62 s, 3 H (3 × H-18); 0.96 s, 3 H (3 × H-19); 1.14 d, 3 H, J = 5.8 (3 × H-21); 0.94 t, 3 H, J = 7.0
(ester CH3); 2.45 s, 3 H (tosylate CH3); 4.31 m, 1 H, W1/2 = 23 (H-3α); 4.82 m, 1 H, W1/2 = 20
(H-20); 5.28 d, 1 H, J = 5 (H-6); 7.32 d, 2 H, J = 8 (tosylate H-3 and H-5); 7.72 d, 2 H, J = 8
(tosylate H-2 and H-6). For C32H46O5S (542.8) calculated: 70.81% C, 8.54% H, 5.91% S; found:
70.99% C, 8.55% H, 6.11% S.
(20R)-Pregn-5-ene-3β,20-diol 3-Tosylate 20-Isobutyrate (2d)
Isobutyric anhydride (7.4 g, 52 mmol) was added at room temperature to a solution of hydroxy deri-
vative6 1 (7 g, 14.81 mmol) in pyridine (42 ml). After standing overnight, the mixture was poured
into water and worked up as usual. The obtained chloroform solution was concentrated and the
residue (8.5 g) was chromatographed on a column of silica gel (159 g, elution with light petroleum–
ether 9 : 1); yield 7.2 g (89%) of product which was crystalized from aqueous acetone; m.p. 103–108 °C.
IR spectrum: 3 070, 3 030, 1 652 (C=C); 1 728, 1 162 (ester); 1 600, 1 448, 1 344, 1 189, (tosylate).
1H NMR spectrum: 0.62 s, 3 H (3 × H-18); 0.96 s, 3 H (3 × H-19); 1.12 d, 3 H, J = 6.1 (3 × H-21);
1.144 d and 1.151 d, 2 × 3 H, J = 7.9 ((CH3)2CHCO); 2.45 s, 3 H (tosylate CH3); 4.31 m, 1 H, W1/2 = 18
(H-3α); 4.82 m, 1 H, W1/2 = 18 (H-20); 5.29 bd, 1 H, J ≈ 6 (H-6); 7.32 d, 2 H, J = 8 (tosylate H-3
and H-5); 7.80 d, 2 H, J = 8 (tosylate H-2 and H-6). For C32H46O5S (542.8) calculated: 70.81% C,
8.54% H, 5.91% S; found: 70.93% C, 8.54% H, 6.13% S.
(20R)-3α,5-Cyclo-5α-pregnane-6β,20-diol 20-Heptafluorobutyrate (3a)
Potassium acetate (1.4 g, 14.3 mmol) and water (7 ml) were added to a solution of tosylate 2a (0.79 g,
1.18 mmol) in acetone (21 ml). After boiling for 4 h, the mixture was cooled, poured into water and
extracted with chloroform. The chloroform phase was washed with water and dried over sodium sul-
fate. The solvent was evaporated and the residue (0.7 g) was chromatographed on a column of silica
gel (100 g, gradient elution with light petroleum–ether 83 : 17 to 90 : 20) to give 511 mg (84%) of
1
hydroxy derivative 3a. IR spectrum: 3 620 (hydroxyl); 3 070 (cyclopropane); 1 777, 1 239 (ester). H
NMR spectrum: 0.20–0.38 m, 1 H and 0.52 t, 1 H, J = 4.5 (two cyclopropane protons); 0.73 s, 3 H
(3 × H-18); 1.08 s, 3 H (3 × H-19); 1.29 d, 3 H, J = 6 (3 × H-21); 3.27 m, 1 H, W1/2 = 6 (H-6α);
5.11 m, 1 H, W1/2 = 18 (H-20). For C25H33F7O3 (514.5) calculated: 58.36% C. 6.47% H, 25.85% F;
found: 58.40% C, 6.44% H, 23.99% F.
(20R)-3α,5-Cyclo-5α-pregnane-6β,20-diol 20-Hemisuccinate (3b)
Potassium acetate (1.44 g, 14.67 mmol) and water (7 ml) were added to a solution of tosylate 2b
(0.72 g, 1.28 mmol) in acetone (21.4 ml). After boiling for 4 h, the mixture was cooled, poured into
water and extracted with chloroform. The chloroform phase was washed with water and dried over
sodium sulfate. The solvent was evaporated and the residue (0.6 g) was chromatographed on a column
of silica gel (80 g, gradient elution with light petroleum–ether–acetic acid 70 : 30 : 1 via 60 : 40 : 1
to 60 : 40 : 2) to give 235 mg (44%) of hydroxy derivative 3b, m.p. 203–205 °C. IR spectrum: 3 615,
1 074 (hydroxyl); 3 059, 3 016 (cyclopropane); 3 400, 1 716, 1 225, 1 216 (carboxyl); 1 732, 1 175
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

On Steroids
935
(ester). 1H NMR spectrum: 0.18–0.36 mt, 1 H and 0.44–0.58 m, 1 H (two cyclopropane protons);
0.67 s, 3 H (3 × H-18); 1.04 s, 3 H (3 × H-19); 1.12 d, 3 H, J = 6 (3 × H-21); 2.58 m, 4 H
(OOCCH2CH2COO); 3.23 m, 1 H, W1/2 = 4 (H-6α); 4.84 m, 1 H, W1/2 = 24 (H-20). For C25H38O5
(418.6) calculated: 71.74% C, 9.15% H; found: 73.27% C, 9.16% H.
(20R)-3α,5-Cyclo-5α-pregnane-6β,20-diol 20-Butyrate (3c)
Potassium acetate (0.54 mg, 5.5 mmol) and water (2.6 ml) were added to a solution of tosylate 2c
(270 mg, 0.5 mmol) in acetone (8 ml). After boiling for 6 h, the mixture was cooled, set aside in a
refrigerator overnight, poured into water and extracted with ether. The ethereal phase was washed
with water and dried over sodium sulfate. The solvent was evaporated and the residue (229 mg) was
chromatographed on 8 preparative TLC plates, elution with light petroleum–ether 7 : 3. Yield 185 mg
(96%) of oily hydroxy derivative 3c. IR spectrum: 3 615, 3 500, 970 (hydroxyl); 3 058, 3 016 (cy-
1
clopropane); 1 731, 1 187, 1 073 (ester). H NMR spectrum: 0.29 dd, 1 H, J = 5, J′ = 8.2, and 0.52 t,
1 H, J = 4.2 (two cyclopropane protons); 0.69 s, 3 H (3 × H-18); 0.94 t, 3 H, J = 7.3 (ester CH3);
1.05 s, 3 H (3 × H-19); 1.14 d, 3 H, J = 6.1 (3 × H-21); 2.24 m, 2 H, W1/2 = 14 (ester CH2–CO);
3.26 t, 1 H, J = 4 (H-6α); 4.86 m, 1 H, W1/2 = 16 (H-20). For C25H40O3 (388.6) calculated: 77.27% C,
10.38% H; found: 77.55% C, 10.44% H.
(20R)-3α,5-Cyclo-5α-pregnane-6β,20-diol 20-Isobutyrate (3d)
Potassium acetate (16.2 g, 16.51 mmol) and water (70 ml) were added to a solution of tosylate 2d (7 g,
12.90 mmol) in acetone (210 ml). After standing overnight at room temperature, the mixture was
refluxed for 1 h, cooled, poured into water and extracted with ether. The ethereal phase was washed
with water, dried over sodium sulfate and the solvent was evaporated. A part (100 mg) of the residue
(5.6 g; pure according to TLC) was purified by chromatography on 4 preparative TLC plates in light
petroleum–ether (7 : 3) to give 70 mg (78%) of hydroxy derivative 3d. IR spectrum: 3 615, 3 500
1
(hydroxyl); 3 058, 3 014 (cyclopropane); 1 727, 1 194, 1 162 (ester). H NMR spectrum: 0.29 dd, 1 H,
J = 5, J′ = 8.2, and 0.52 t, 1 H, J = 4 (two cyclopropane protons); 0.69 s, 3 H (3 × H-18); 1.05 s, 3 H
(3 × H-19); 1.13 d, 3 H, J = 5.8 (3 × H-21); 1.147 d and 1.153 d, 2 × H, J = 7 ((CH3)2CHCO); 2.47 m,
1 H, W1/2 = 14 (1 × H-7); 3.26 m, 1 H, W1/2 = 6 (H-6α); 4.83 mt, 1 H, W1/2 = 16 (H-20). For
C25H40O3 (388.6) calculated: 77.27% C, 10.38% H; found: 77.44% C, 10.33% H.
(20R)-20-Hydroxy-3α,5-cyclo-5α-pregnan-6-one Heptafluorobutyrate (4a)
Jones reagent was added to a solution of alcohol 3a (200 mg, 0.39 mmol) in acetone (10 ml) to
constant brown coloration. After standing for 10 min at room temperature, methanol (0.5 ml) was
added, the mixture was set aside for 5 min, poured into water and extracted with chloroform. The
chloroform extract was washed with water, saturated solution of potassium hydrogen carbonate, again
water, and dried over sodium sulfate. The solvent was evaporated and the residue (200 mg) was puri-
fied by chromatography on a column of silica gel (40 g) in light petroleum–ether (4 : 1) to give 169 mg
(85%) of noncrystalline keto derivative 4a. IR spectrum: 3 070 (cyclopropane); 1 776, 1 238 (ester);
1
1 691 (ketone). H NMR spectrum: 0.72 s, 3 H (3 × H-18); 1.00 s, 3 H (3 × H-19); 1.29 d, 3 H, J = 6
(3 × H-21); 2.71–2.64 m, 2 H (2 × H-7); 5.07 m, 1 H, W1/2 = 18 (H-20). For C25H31F7O3 (512.5)
calculated: 58.59% C, 6.09% H, 25.95% F; found: 58.66% C, 6.11% H, 26.33% F.
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

936
Kohout, Kasal, Strnad:
(20R)-20-Hydroxy-3α,5-cyclo-5α-pregnan-6-one Hemisuccinate (4b)
Alcohol 3b (250 mg, 0.60 mmol) was oxidized in acetone (2.5 ml) with Jones reagent in the same
manner as described in the preceding experiment. Analogous work-up gave 160 mg (63%) of keto
derivative 4b, m.p. 138–139 °C (ethanol). IR spectrum: 3 531, 3 500–2 400, 1 714, 1 379 (COOH);
1
3 024, 3 008 (cyclopropane); 1 732, 1 170 (ester); 1 690 (6-ketone). H NMR spectrum: 0.62–0.79 m,
1 H (cyclopropane proton); 0.67 s, 3 H (3 × H-18); 1.00 s, 3 H (3 × H-19); 1.14 d, 3 H, J = 6 (3 × H-21);
2.60 m, 4 H, W1/2 = 9 (OOCCH2CH2COO); 4.88 m, 1 H, W1/2 = 19 (H-20). For C25H36O5 (416.6)
calculated: 72.08% C, 8.71% H; found: 71.99% C, 9.11% H.
(20R)-20-Hydroxy-3α,5-cyclo-5α-pregnan-6-one Butyrate (4c)
Alcohol 3c (160 mg, 0.41 mmol) was oxidized with Jones reagent in acetone (5 ml) in the same
manner as described for the preparation of compound 4a. Analogous work-up gave 150 mg of the
crude product which was purified on 4 preparative silica gel plates (elution with light petroleum–ether
7 : 3) to give 90 mg (56%) of keto derivative 4c. IR spectrum: 3 124, 3 007 (cyclopropane); 1 731,
1 187, 1 058 (ester); 1 690 (ketone). 1H NMR spectrum: 0.72 t, 1 H, J = 4.6 (cyclopropane proton);
0.68 s, 3 H (3 × H-18); 0.95 t, 3 H, J = 7.3 (ester CH3); 1.00 s, 3 H (3 × H-19); 1.15 d, 3 H, J = 6.1
(3 × H-21); 2.17–2.31 m, 1 H and 2.32–2.54 m, 1 H (2 × H-7); 4.86 m, 1 H, W1/2 = 16 (H-20). For
C25H38O3 (386.6) calculated: 77.68% C, 9.91% H; found: 77.83% C, 9.90% H.
(20R)-20-Hydroxy-3α,5-cyclo-5α-pregnan-6-one Isobutyrate (4d)
Alcohol 3d (5 g, 12.87 mmol) was oxidized with Jones reagent in acetone (100 ml) in the same
manner as described for the preparation of compound 4a. Analogous work-up (extraction with ether)
gave 5 g of the crude product which was purified by column chromatography on silica gel in light
petroleum–ether (9 : 1) to give 4.2 g (84%) of keto derivative 4d. IR spectrum: 3 024, 3 007 (cyclo-
propane); 1 731, 1 192, 1 160 (ester); 1 690 (ketone). 1H NMR spectrum: 0.72 t, 1 H, J = 4.2 (cy-
clopropane proton); 0.68 s, 3 H (3 × H-18); 1.00 s, 3 H (3 × H-19); 1.14 d, 3 H, J = 6.1 (3 × H-21);
1.154 d and 1.162 d, 2 × 3 H, J = 7 ((CH3)2CHCO); 2.60–2.31 m, 2 H (2 × H-7); 4.85 m, 1 H, W1/2 = 16
(H-20). For C25H38O3 (386.6) calculated: 77.68% C, 9.91% H; found: 77.88% C, 9.77% H.
(20R)-20-Hydroxy-5α-pregn-2-en-6-one Heptafluorobutyrate (5a)
4-Toluenesulfonic acid monohydrate (340 mg, 2.29 mmol) was added to a solution of derivative 4a
(170 mg, 0.33 mmol) in sulfolane (3 ml). The mixture was heated at 155 °C for 1 h under nitrogen,
cooled, poured into water and the product was taken up in ether. The ethereal extract was washed
successively with water, saturated solution of potassium hydrogen carbonate and water, dried, and the
solvent was evaporated. The residue (140 mg) was chromatographed on a column of silica gel (20 g)
in light petroleum–ether (9 : 1). Yield 69 mg (39%) of oily olefin 5a. IR spectrum: 3 030, 1 659
1
(C=C); 1 777, 1 238 (ester); 1 714 (ketone). H NMR spectrum: 0.68 s, 3 H (3 × H-19); 0.72 s, 3 H
(3 × H-18); 1.29 d, 3 H, J = 7 (3 × H-21); 5.07 m, 1 H, W1/2 = 18 (H-20); 5.39–5.82 m, 2 H (H-2
and H-3). For C25H31F7O3 (512.5) calculated: 58.59% C, 6.09% H, 25.95% F; found: 58.31% C,
5.92% H, 24.98% F.
(20R)-20-Hydroxy-5α-pregn-2-en-6-one Hemisuccinate (5b)
Pyridinium 4-toluenesulfonate (9.4 mg) and lithium bromide (10.6 mg, 0.12 mmol) were added to a
solution of derivative 4b (100 mg, 0.24 mmol) in N,N-dimethylacetamide (1.0 ml). After heating at
160 °C for 6 h under nitrogen, the reaction mixture was cooled, poured into water and the product
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

On Steroids
937
was worked up in the usual manner (extraction with chloroform). After evaporation of chloroform,
the residue (100 mg) was purified by chromatography on 4 preparative silica gel plates (elution with
light petroleum–ether–acetic acid 60 : 40 : 1). The corresponding zones gave 86 mg (87%) of oily
olefin 5b, m.p. 137–138 °C (acetone–heptane). IR spectrum: 3 500–2 400, 1 313, 1 227 (COOH); 3 026,
1
1 227 (COOH); 3 026, 1 657 (C=C); 1 733, 1 174 (ester); 1 713 (ketone). H NMR spectrum: 0.71 s,
3 H (3 × H-19); 0.63 s, 3 H (3 × H-18); 1.15 d, 3 H, J = 6.5 (3 × H-21); 2.62 m, 4 H, W1/2 = 9
(OOCCH2CH2COO); 4.89 m, 1 H, W1/2 = 19 (H-20); 5.61 m, 2 H, W1/2 = 9 (H-2 and H-3). For
C25H36O5 (416.6) calculated: 72.08% C, 8.71% H; found: 71.93% C, 8.52% H.
(20R)-20-Hydroxy-5α-pregn-2-en-6-one Butyrate (5c)
Derivative 4c (100 mg, 0.26 mmol) in dimethylacetamide (3 ml) was treated with pyridinium 4-to-
luenesulfonate (9.4 mg, 0.05 mmol) and lithium bromide (10.6 mg, 0.12 mmol) as described in the
preceding experiment. The same work-up (extraction with ether) and purification on two preparative
plates (elution with light petroleum–ether 7 : 3) afforded 45 mg (46%) of noncrystalline olefin 5c. IR
1
spectrum: 3 080, 1 693, 1 698 (C=C); 1 731, 1 185 (ester); 1 713 (ketone). H NMR spectrum: 0.64 s,
3 H (3 × H-19); 0.71 s, 3 H (3 × H-18); 0.95 t, 3 H, J = 7.3 (ester CH3); 1.14 d, 3 H, J = 6.1 (3 × H-21);
4.86 m, 1 H, W1/2 = 15 (H-20); 5.48–5.76 m, 2 H (H-2 and H-3). For C25H38O3 (386.6) calculated:
77.68% C, 9.91% H; found: 77.51% C. 9.63% H.
(20R)-20-Hydroxy-5α-pregn-2-en-6-one Isobutyrate (5d)
Derivative 4d (1.5 g, 3.88 mmol) in N,N-dimethylacetamide (3 ml) was treated with pyridinium
4-toluenesulfonate (140 mg, 0.73 mmol) and lithium bromide (160 mg, 1.84 mmol) as described in
the preceding experiment. An analogous work-up (extraction with ether) afforded 1.2 g of residue
which was purified on a column of silica gel (300 g, elution with light petroleum–ether 9 : 1). Yield
995 mg (66%) of olefin 5d, m.p. 85–87 °C (acetone–heptane). IR spectrum: 3 080, 1 695, 1 699
1
(C=C); 1 731, 1 186 (ester); 1 714 (ketone). H NMR spectrum: 0.65 s, 3 H (3 × H-19); 0.71 s, 3 H
(3 × H-18); 1.14 d, 3 H, J = 6.1 (3 × H-21); 1.161 d and 1.170 d, 2 × 3 H, J = 7 ((CH3)2CHCO);
4.85 m, 1 H, W1/2 = 18 (H-20); 5.30–5.75 m, 2 H (H-2 and H-3). For C25H38O3 (386.6) calculated:
77.68% C, 9.91% H; found: 77.44% C. 9.70% H.
(20R)-2α,3α,20-Trihydroxy-5α-pregnan-6-one 20-Heptafluorobutyrate (6a)
A solution of OsO4 (24 mg, 0.09 mmol) in 2-methyl-2-propanol (0.24 ml), followed by N-methyl-
morpholine N-oxide (480 mg, 4.10 mmol) in water (0.5 ml), was added to a solution of olefin 5a
(480 mg, 0.94 mmol) in acetone (24 ml) and the mixture was stirred at room temperature for 5 h.
Then 10% aqueous solution of sodium sulfite (12 ml) was added and stirring was continued for 1 h.
The mixture was poured into water and the product was taken up in chloroform in the usual manner to
give 550 mg of an oily product. Purification by chromatography on silica gel (200 g) in chloroform–
ether (1 : 1) afforded 329 mg (40%) of dihydroxy derivative 6a, subliming at 200 °C after change of
modification at 142 °C. IR spectrum: 3 526, 3 415, 3 303 (OH); 1 773, 1 236, 1 216 (ester); 1 711
(ketone). 1H NMR spectrum: 0.67 s, 3 H (3 × H-18); 0.76 s, 3 H (3 × H-19); 1.30 d, 3 H, J = 6 (3 × H-21);
3.74 m, 1 H, W1/2 = 23 (H-2β); 4.02 m, 1 H, W1/2 = 9 (H-3β); 5.07 m, 1 H, W1/2 = 20 (H-20). Mass
spectrum (m/z): 546 (M+); 531 (M – CH3); 528 (M – H2O); 513 (M – CH3 – H2O). For C25H33F7O5
(546.5) calculated: 54.94% C, 6.09% H, 24.33% F; found: 55.45% C, 6.69% H, 24.18% F.
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

938
Kohout, Kasal, Strnad:
(20R)-2α,3α,20-Trihydroxy-5α-pregnan-6-one 20-Hemisuccinate (6b)
Olefin 5b (130 mg, 0.31 mmol) in acetone (6.5 ml) was treated with a solution of OsO4 (6.5 mg, 0.03
mmol) in 2-methyl-2-propanol (0.065 ml) and N-methylmorpholine N-oxide (130 mg, 1.11 mmol) in
water (0.13 ml) as described in the preceding experiment. Analogous work-up afforded 150 mg of an
oily product which was purified by chromatography on 4 preparative plates of silica gel in chlorofom–
2-propanol–acetic acid (90 : 10 : 1) to give 48 mg (35%) of dihydroxy derivative 6b, m.p. 248–255 °C
(acetone–light petroleum). IR spectrum: 3 619, 3 569, 3 518 (OH); 3 348, 2 452, 1 713 (COOH); 1 730
1
(ester); 1 713 (ketone). H NMR spectrum: 0.63 s, 3 H (3 × H-18) and 0.75 s, 3 H (3 × H-19); 1.16 d,
3 H, J = 6 (3 × H-21); 2.60 m, 4 H, W1/2 = 3 (OOCCH2CH2COO); 3.40–3.82 m, 1 H (H-2β); 3.98 m,
1 H, W1/2 = 7 (H-3β); 4.86 m, 1 H, W1/2 = 18 (H-20). Mass spectrum (m/z): 450 (M); 432 (M – H2O);
414 (M – 2 × H2O). For C25H38O7 (450.6) calculated: 66.64% C, 8.50% H; found: 66.41% C, 8.32% H.
(20R)-2α,3α,20-Trihydroxy-5α-pregnan-6-one 20-Butyrate (6c)
Olefin 5c (600 mg, 1.55 mmol) in acetone (30 ml) was treated with a solution of OsO4 (30 mg, 0.12 mmol)
in 2-methyl-2-propanol (0.3 ml) and N-methylmorpholine N-oxide (600 mg, 5.12 mmol) in water (0.6 ml)
as described in the preceding experiment. Analogous work-up afforded 0.7 g of an oily product
which was purified by chromatography on a column of silica gel in chlorofom–2-propanol (9 : 1).
Crystallization from aqueous acetone afforded 27 mg (41%) of dihydroxy derivative 6c, m.p. 98–101 °C.
1
IR spectrum: 3 423 (OH); 1 730, 1 186 (ester); 1 713 (ketone). H NMR spectrum: 0.63 s, 3 H (3 × H-18);
0.75 s, 3 H (3 × H-19); 0.97 t, 3 H, J = 7.3 (ester CH3); 1.15 d, 3 H, J = 5.8 (3 × H-21); 2.69 dd, 1 H,
J = 4, J′ = 12 (H-5α); 3.76 m, 1 H, W1/2 = 24 (H-2β); 4.04 m, 1 H, W1/2 = 7 (H-3β); 4.84 m, 1 H,
W1/2 = 18 (H-20). Mass spectrum (FAB, m/z): 421 (M + 1); 403 (M + 1 – H2O); 385 (M + 1 – 2 × H2O);
333 (M + 1 – CH3CH2CH2COOH). For C25H40O5 (420.6) calculated: 71.39% C, 9.59% H; found:
71.04% C, 9.42% H.
(20R)-2α,3α,20-Trihydroxy-5α-pregnan-6-one 20-Isobutyrate (6d)
Olefin 5d (1.2 g, 3.10 mmol) in acetone (60 ml) was treated with a solution of OsO4 (60 mg, 0.24 mmol)
in 2-methyl-2-propanol (0.6 ml) and N-methylmorpholine N-oxide (1.2 g, 10.24 mmol) in water (1.2 ml)
as described in the preceding experiment. Analogous work-up afforded 1.2 g of an oily product
which was purified by chromatography on a column of silica gel in chlorofom–2-propanol (9 : 1).
Crystallization from aqueous acetone afforded 250 mg (19%) of dihydroxy derivative 6d, m.p. 115–120 °C.
1
IR spectrum: 3 619 (OH); 1 709 (ketone and ester). H NMR spectrum: 0.65 s, 3 H (3 × H-18); 0.73 s,
3 H (3 × H-19); 1.10 d, 3 H, J = 5.8 (3 × H-21); 1.11 d, 3 H and 1.12 d, 3 H, J = 7.0 ((CH3)2CHCO);
2.67 dd, 1 H, J = 4, J′ = 12 (H-5α); 3.54–3.72 m, 1 H (H-2β); 3.90 m, 1 H, W1/2 = 6.5 (H-3β); 4.80 m,
1 H, W1/2 = 18 (H-20). Mass spectrum (FAB, m/z): 421 (M + 1); 403 (M + 1 – H2O); 385 (M + 1 –
2 × H2O); 333 (M + 1 – (CH3)2CHCOOH). For C25H40O5 (420.6) calculated: 71.39% C, 9.59% H;
found: 71.59% C, 9.30% H.
(20R)-2α,3α,20-Trihydroxy-B-homo-7-oxa-5α-pregnan-6-one 20-Heptafluorobutyrate (7a)
To a solution of ketone 6a (210 mg, 0.38 mmol) in dichloromethane (3 ml) was added a solution of
trifluoroperacetic acid, freshly prepared from trifluoroacetic anhydride (646 mg, 3.08 mmol) and hy-
drogen peroxide (30%, 105 mg) in dichloromethane (3 ml). After standing at room temperature for 1 h,
the reaction mixture was poured into water and taken up in chloroform. The chloroform extract was
washed with water, saturated solution of potassium hydrogen carbonate, water, and dried over so-
dium sulfate. Removal of the solvent in vacuo gave 112 mg of a residue which contained two pro-
ducts (TCL). The residue was chromatographed on 6 preparative plates in chloroform–2-propanol (9 : 1).
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

On Steroids
939
Work-up of the lipophilic zones gave 56 mg (26%) of lactone 7a, m.p. 160–165 °C (aqueous ethanol).
1
IR spectrum: 3 412, 1 065 (OH); 1 773, 1 236, 1 218 (ester); 1 728, 1 711, 1 186 (lactone). H NMR
spectrum: 0.71 s, 3 H (3 × H-18); 0.91 s, 3 H (3 × H-19); 1.28 d, 3 H, J = 6 (3 × H-21); 2.97–3.26 m,
1 H (H-5α); 3.75 m, 1 H, W1/2 = 22 (H-2β); 4.07 m, 3 H, W1/2 = 8 (H-3β and 2 × H-7a); 5.087 m, 1 H,
W1/2 = 18 (H-20). Mass spectrum (m/z): 562 (M+); 544 (M – H2O). For C25H33F7O6 (562.5) calcu-
lated: 53.38% C, 5.91% H, 23.64% F; found: 53.18% C, 6.05% H, 23.10% F.
(20R)-2α,3α,20-Trihydroxy-B-homo-7-oxa-5α-pregnan-6-one 20-Hemisuccinate (7b)
Ketone 6b (65 mg, 0.14 mmol) in dichloromethane (3 ml) was treated with a solution of trifluoroper-
acetic acid for 5 h as described in the preceding experiment. Analogous work-up and thin-layer chro-
matography on 4 preparative plates in chloroform–2-propanol–acetic acid (90 : 10 : 1) afforded 9.5 mg
(14%) of pure lactone 7b. IR spectrum (CHCl3): 3 620, 3 570, 3 520 (OH); 3 400–2 400, 1 714
(COOH); 1 733 (ester); 1 728 (lactone). 1H NMR spectrum: 0.64 s, 3 H (3 × H-18); 0.92 s, 3 H
(3 × H-19); 1.16 d, 3 H, J = 6 (3 × H-21); 3.70 m, 1 H, W1/2 = 21 (H-2β); 4.02 m, 3 H, W1/2 = 7
(H-3β and 2 × H-7a); 4.84 m, 1 H, W1/2 = 18 (H-20). For C25H38O8 (466.6) calculated: 64.36% C,
8.21% H; found: 65.77% C, 8.44% H.
(20R)-2α,3α,20-Trihydroxy-B-homo-7-oxa-5α-pregnan-6-one 20-Butyrate (7c)
Ketone 6c (120 mg, 0.29 mmol) in dichloromethane (2.4 ml) was treated with a solution of trifluo-
roperacetic acid as described in the preceding experiment. Analogous work-up and chromatography
on 6 preparative plates in chloroform–acetone (4 : 1) afforded 72 mg (55%) of lactone 7c. IR spec-
1
trum: 3 429 (OH); 1 731 (ester and lactone). H NMR spectrum: 0.66 s, 3 H (3 × H-18); 0.92 s, 3 H
(3 × H-19); 0.95 t, 3 H, J = 7 (ester CH3); 1.14 s, 3 H, J = 6 (3 × H-21); 3.14 dd, 1 H, J = 4, J′ = 12
(H-5α); 3.75 dm, 1 H, J = 12, W1/2 = 11 (H-2β); 4.06 m, 3 H, W1/2 = 9 (H-3β); 4.09 m, 2 H, J = 5
(2 × H-7a); 4.87 m, 1 H, W1/2 = 18.5 (H-20). Mass spectrum (FAB, DMSO, m/z): 459 (M + Na); 437
(M + 1); 419 (M + 1 – H2O); 401 (M + 1 – 2 × H2O); 349 (M + 1 – CH3CH2CH2COOH). For
C25H40O6 (436.6) calculated: 68.78% C, 9.23% H; found: 68.88% C, 9.22% H.
(20R)-2α,3α,20-Trihydroxy-B-homo-7-oxa-5α-pregnan-6-one 20-Isobutyrate (7d)
Ketone 6d (170 mg, 0.40 mmol) in dichloromethane (5.1 ml) was treated with a solution of trifluoro-
peracetic acid as described in the preceding experiment. Analogous work-up and chromatography on
7 preparative plates in chloroform–acetone (4 : 1) afforded 89 mg (50%) of lactone 7d. IR spectrum
(CHCl3): 3 500 (OH); 1 720 (ester and lactone). 1H NMR spectrum: 0.66 s, 3 H (3 × H-18); 0.90 s,
3 H (3 × H-19); 1.13 d, 3 H, J = 6 (3 × H-21); 1.14 d, 3 H and 1.15 d, 3 H, J = 7 ((CH3)2CHCO);
3.12 dd, 1 H, J = 4, J′ = 12 (H-5α); 3.71 m, 1 H, W1/2 = 22 (H-2β); 4.01 m, 3 H, W1/2 = 8 (H-3β);
4.07 m, 2 H, J = 5 (2 × H-7a); 4.84 m, 1 H, W1/2 = 18.5 (H-20). Mass spectrum (FAB, DMSO, m/z):
459 (M + Na); 437 (M + 1); 419 (M + 1 – H2O); 401 (M + 1 – 2 × H2O); 349 (M + 1 –
(CH3)2CHCOOH). For C25H40O6 (436.6) calculated: 68.78% C, 9.23% H; found: 68.99% C, 9.11% H.
(20R)-2α,3α,20-Trihydroxy-B-homo-6-oxa-5α-pregnan-7-one 20-Heptafluorobutyrate (8)
Work-up of the polar zones on preparative plates in the preparation of lactone 7a afforded 37 mg
(18%) of lactone 8. IR spectrum: 3 408 (OH); 1 774, 1 236, 1 218 (ester); 1 729, 1 709 (lactone).
1H NMR spectrum: 0.95 s, 6 H (3 × H-18 and 3 × H-19); 1.45 d, 3 H, J = 5 (3 × H-21); 3.73 m, 1 H,
W1/2 = 21 (H-2β); 4.03 m, 1 H, W1/2 = 11 (H-3β); 4.54 m, 1 H, W1/2 = 15 (H-5α); 5.01 m, 1 H, W1/2 = 18
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

940
Kohout, Kasal, Strnad:
(H-20). Mass spectrum (m/z): 562 (M+); 544 (M – H2O). For C25H33F7O6 (562.5) calculated: 53.38% C,
5.91% H, 23.64% F; found: 53.10% C, 5.51% H, 23.17% F.
The authors are indebted to Dr P. Fiedler for interpretation of IR spectra measured by Mrs K.
Matouskova. Proton NMR spectra were recorded by Mrs M. Snopkova, mass spectra by Dr J.
Kohoutova. The technical assistance of Mrs J. Neumannova and Miss L. Dalibova is gratefully
acknowledged. This work was supported in part by Grant No. 203/95/1309 of the Grant Agency of the
Czech Republic.
REFERENCES
1. Pouzar V., Slavikova T., Cerny I.: Collect. Czech. Chem. Commun. 61, 404 (1996).
2. Yokota T., Mori K. in: Molecular Structure and Biological Activity of Steroids, Uniscience
report series (W. L. Duax and M. Bohl, Eds), p. 317. CRC Press, Boca Raton 1991.
3. Kohout L.: Collect. Czech. Chem. Commun. 54, 3348 (1989).
4. Kohout L., Strnad M., Kaminek M. in: Brassinosteroids: Chemistry, Bioactivity and Applications
(H. G. Cutler, T. Yokota and G. Adam, Eds), p. 57. American Chemical Society, Washington,
DC 1991.
5. Japan 06-25281 (1994); Chem. Abstr. 120, 317801 (1994).
6. Kohout L., Strnad M.: Collect. Czech. Chem. Commun. 57, 1731 (1992).
7. Takatsuto S., Ikekawa N.: Tetrahedron Lett. 24, 917 (1983).
8. Mitchell J. W., Livingstone G. A.: Methods of Studying Plant Hormones and Growth Regulating
Substances, Agricultural Handbook No. 336, p. 26. U.S. Government Printing Office,
Washington, D.C. 1968.
Collect. Czech. Chem. Commun. (Vol. 61) (1996)