Synthesis of (20S)-2α,3α-dihydroxy-6-oxo-7-oxa-7a-homo-5α-pregnane-20- carboxylic acid as a brassinosteroid part of ligand for binding to affinity chromatography carriers

Article abstract of DOI:10.1135/cccc20001754

(20S)-2α,3α-Dihydroxy-6-oxo-7-oxa-7a-homo-5α-pregnane- 20- carboxylic acid (12) as a brassinosteroid ligand for affinity chromatography carriers was synthesized from bisnorcholenic acid. The brassinolide activity in the bean second internode bioassay of t

Full text of DOI:10.1135/cccc20001754

1
754
Kohout, Chodounská, Macek, Strnad:
SYNTHESIS OF (20S)-2α,3α-DIHYDROXY-6-OXO-7-OXA-7a-HOMO-
α-PREGNANE-20-CARBOXYLIC ACID AS A BRASSINOSTEROID
5
PART OF LIGAND FOR BINDING TO AFFINITY
+
CHROMATOGRAPHY CARRIERS
Ladislav KOHOUTa1,*, Hana CHODOUNSKÁ , Tomáš MACEK
a2
a3
b
and Miroslav STRNAD
a
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
Flemingovo nám. 2, CZ-166 10 Prague 6, Czech Republic; e-mail: kohout@uochb.cas.cz,
hchod@uochb.cas.cz, tom.macek@uochb.cas.cz
Laboratory of Growth Regulators, Institute of Experimental Botany, Academy of Sciences
of the Czech Republic and Faculty of Natural Sciences of Palacký University, Šlechtitelů 11,
CZ-783 71 Olomouc, Czech Republic; e-mail: strnad@risc.upol.cz
1
2
3
b
Received October 2, 2000
Accepted Novem ber 27, 2000
(
20S)-2α,3α-Dih ydroxy-6-oxo-7-oxa-7a-h om o-5α-pregn an e-20-carboxylic acid (12) as a
brassin osteroid ligan d for affin ity ch rom atograph y carriers was syn th esized from
bisn orch olen ic acid. Th e brassin olide activity in th e bean secon d in tern ode bioassay of th e
syn th esized (20S)-5α-pregn an e-20-carboxylic acid derivatives is also described.
Key words: Steroids; Brassin olide; Brassin osteroids; NMR spectroscopy; Plan t growth activity;
Affin ity carrier ligan d.
In th e course of our in vestigation 2–4 on structure–activity relation sh ips of
brassin osteroids, we turn ed our atten tion to brassin olide an alogues of
(
20S)-5α-pregn an e-20-carboxylic acid. Th ese com poun ds with con ven ien t
spacers m ay fin d use as ligan ds for covalen t bin din g to affin ity ch rom ato-
graph y carriers poten tially useful for protein receptor isolation from plan t
extracts.
Th e ch eapest startin g m aterial for th e syn th esis is com m ercially available
(
20S)-3β-acetoxy-5-pregn en -20-carboxylic acid5 (1; i.e. bisn orch olen ic acid
acetate). Acid 1 was con verted with diazom eth an e in to its m eth yl ester 2,
wh ich on sapon ification afforded th e kn own 3β-alcoh ol 3. To obtain
6
tosylate 4, we used a procedure sim ilar to th at described previously . Th e
+
Part CDX in th e series On Steroids. Part CDIX see ref.1
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

On Steroids
1755
subsequen t steps of th e syn th esis were m odified in th e followin g way:
tosylate 4 was tran sform ed in to m eth yl (20S)-6β-h ydroxy-3α,5-cyclo-
5
α-pregn an e-20-carboxylate (5) on treatm en t with potassium acetate an d de-
6
rivative 5 was im m ediately oxidized with th e Jon es reagen t to th e kn own
m eth yl (20S)-6-oxo-3α,5-cyclo-5α-pregn an e-20-carboxylate (6). To open th e
cyclopropan e rin g, we treated com poun d 6 with lith ium brom ide in
N,N-dim eth ylacetam ide in th e presen ce of pyridin ium tosylate at 160 °C.
Th is reaction directly afforded m eth yl (20S)-6-oxo-5α-pregn -2-en e-20-carb-
oxylate (8). Th e sequen ce proved to be sim pler an d afforded h igh er yields
COOR2
COOCH3
1
R O
1
2
OH
1
2
3
4
, R = Ac; R = H
1
2
, R = Ac; R = CH3
5
1
2
, R = H; R = CH3
1
2
, R = Tos; R = CH3
COOR
COOR
H
O
O
6
7
, R= CH3
, R= H
8, R= CH3
9, R= H
COOCH3
COOR
HO
HO
HO
HO
O
H
O
H
O
10
11, R= CH3
2, R= H
1
COOR
HO
HO
O
H
1
1
3, R= CH3
4, R= H
O
SCHEME 1
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

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756
Kohout, Chodounská, Macek, Strnad:
6
th an th e route described in ref. (yield 68% versus 16%). Th e reaction was
also applied to (20S)-6-oxo-3α,5-cyclo-5α-pregn an e-20-carboxylic acid (7)
obtain ed by alkalin e h ydrolysis of m eth yl ester 6, sim ply affordin g
(
20S)-6-oxo-5α-pregn -2-en e-20-carboxylic acid (9). Osm ium tetroxide cata-
lyzed h ydroxylation of olefin 8 with 4-m eth ylm orph olin e N-oxide resulted
in th e form ation of 2α,3α-diol 10. Th is diol was directly treated with
7
trifluoroperoxyacetic acid in dich lorom eth an e (as we described previously )
with out an y protection of th e diol groups to yield th e products of
Baeyer–Villiger oxidation as a m ixture of m eth yl (20S)-2α,3α-dih ydroxy-
6
-oxo-7-oxa-7a-h om o-5α-pregn an e-20-carboxylate (11) an d m eth yl
(
(
20S)-2α,3α-dih ydroxy-6-oxa-7-oxo-7a-h om o-5α-pregn an e-20-carboxylate
13) (Sch em e 1). Th e latter com poun d could n ot be isolated at th is step but
it was detected by th e correspon din g sign als in th e NMR spectra of m oth er
liquors from th e crystallization of lacton e 11. Alkalin e h ydrolysis of th is
m ixture afforded (20S)-2α,3α-dih ydroxy-6-oxo-7-oxa-7a-h om o-5α-pregn an e-
2
5
7
0-carboxylic acid (12) an d (20S)-2α,3α-dih ydroxy-6-oxa-7-oxo-7a-h om o-
α-pregn an e- 20-carboxylic acid (14). (20S)-2α,3α-Dih ydroxy-6-oxo-7-oxa-
a-hom o-5α-pregnane-20-carboxylic acid (12) was obtain ed in 25% overall
yield based on th e startin g (20S)-3β-acetoxypregn -5-en e-20-carboxylic acid
8
(
1). Derivative 12 was used as a brassin osteroid ligan d with a con ven ien t
spacer an d boun d covalen tly to an affin ity ch rom atograph y carrier for pro-
tein isolation from plan t extracts. Th e results on protein receptor isolation
will be publish ed in due course.
TABLE I
a
Len gth en in g of th e secon d in tern ode (m m ) in th e bean secon d in tern ode assay of lacton e
2 an d 24-epiBR
b
1
Am oun t applied, m ole
Com poun d
1
· 10–7
1 · 10–8
1 · 10–9
1 · 10–10
1 · 10–11
1 · 10–12
2
4-EpiBR
NTc
6.6
20.4
7.6
30.8
8.9
32.3
12.0
18.6
9.0
12.1
8.0
Lacton e 12
a
Th e len gth en in g of th e secon d in tern ode m ean s its len gth en in g in m m as com pared with
b
th at of th e referen ce plan t for th e applied am oun t given .
24-epiBR m ean s 24-epi-
brassin olide, i.e. (22R,23R,24R)-2α,3α,22,23-tetrah ydroxy-24-m eth yl-7-oxa-7a-h om o-5α-
c
ch olestan -6-on e. Not tested.
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

On Steroids
1757
Th e brassin olide activities of th e com poun d syn th esized were m easured
by a m odified bean secon d in tern ode bioassay as described in Experim en -
tal. Of th e tested com poun ds 10–14, th e h igh est activity was foun d for
lacton e 12; h owever, even th is com poun d exh ibited a m uch lower activity
th an th e n atural 24-epibrassin olide [(22R,23R,24R)-2α,3α,22,23-tetra-
h ydroxy-24-m eth yl-7-oxa-7a-h om o-5α-ch olestan -6-on e] (Table I).
EXPERIMENTAL
Th e m eltin g poin ts were determ in ed on a m icro m eltin g poin t Electroth erm al (U.S.A.). Opti-
2
5
cal rotation s were m easured at 25 °C in ch loroform (un less oth erwise stated) an d [α] values are
D
–
1
2
–1
given in 10 deg cm
g . In frared spectra were recorded on a Bruker IFS 88 spectrom eter in
–
1
1
tetrach lorom eth an e (un less oth erwise stated), waven um bers are given in cm
.
H NMR
spectra were taken in deuterioch loroform on a Varian XL-200 (FT m ode, 200.04 MHz) in -
strum en t in deuterioch loroform solution s with tetram eth ylsilan e as an in tern al referen ce
un less oth erwise stated. Ch em ical sh ifts are given in ppm (δ-scale), couplin g con stan ts (J)
an d m ultiplet h alf-width (W1/2) in Hz. All values were obtain ed by first-order an alysis. Mass
spectra were obtain ed with a ZAB-EG spectrom eter at 70 eV. Th e iden tity of th e prepared
sam ples was ch ecked by m eltin g poin ts, th in -layer ch rom atograph y (TLC) perform ed on sil-
ica gel G (ICN Bioch em icals, th e detection was carried out by sprayin g with sulfuric acid
1
an d h eatin g), IR an d H NMR spectra. Preparative TLC was carried out on 200 × 200 m m
plates coated with 0.7 m m layer of silica gel Woelm DC, th e detection by sprayin g th e
plates with a 0.2% m eth an olic m orin e solution , by UV detection or by sprayin g with sulfu-
ric acid an d h eatin g th e side strips of th e plates. For colum n ch rom atograph y, n eutral silica
gel 60–120 µm was used (Service Laboratories of th e In stitute).
“
Usual” work-up m ean s extraction with a given organ ic solven t, wash in g th e organ ic
ph ase with 5% h ydroch loric acid, water, 5% aqueous potassium h ydrogen carbon ate, water,
dryin g over an h ydrous sodium sulfate, filterin g an d evaporation of th e solven t to dryn ess
un der reduced pressure.
Ligh t petroleum refers to a fraction boilin g at 40–62 °C.
Bean Secon d In tern ode Bioassay
Seeds of bean (Phaseolus vulgaris L., var. Pin to) were germ in ated for two days an d selected
seeds were plan ted in to pots con tain in g perlite an d m odified Hoaglan d’s solution (h alf con -
cen tration , pH 5.7). Th e pots were placed in ligh t-con trolled cultivation room (25–27 °C,
2
ligh t 48 W/m , ligh t/dark period 16 h /8 h ). Groups of eigh t 7-day-old bean seedlin gs with
1
–2 m m lon g secon d in tern odes were treated with differen t am m oun ts of th e tested com -
poun ds in 2 µl of lan olin . Con trol plan ts were treated on ly with lan olin . Th e m easurem en ts
were don e after 5 days. Th e differen ce in th e len gth of th e secon d in tern ode of treated an d
con trol plan ts was used as a m easure of th e activity.
Meth yl (20S)-3β-Acetoxypregn -5-en e-20-carboxylate (2)
(
(
20S)-3β-Acetoxypregn -5-en e-20-carboxylic acid5 (1; 3.9 g, 10 m m ol) was dissolved in eth er
100 m l). A solution of diazom eth an e (1.26 g, 30 m m ol) in eth er (48 m l) was added. Th e re-
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

1
758
Kohout, Chodounská, Macek, Strnad:
action m ixture was poured in to water after 10 m in an d th en worked up as usual. Pure
m eth yl ester 2 (4.25 g; 100%) was obtain ed. An alytical sam ple was crystallized from aceton e:
2
D
5
9
m .p. 144–146 °C, [α] –42 (c 1.14) in accordan ce with literature . IR: 3 060, 1 668 (C=C), 1 735
(
C=O acetate an d ester), 1 434 (MeO), 1 215, 1 033 (C–O acetate), 1 196, 1 160 (C–O m eth yl
1
ester). H NMR: 0.69 s, 3 H (3 × H-18); 1.02 s, 3 H (3 × H-19); 1.19 d, 2 H, J = 6.7 (3 × H-21);
.03 s, 3 H (CH COO); 3.645 s, 3 H (CH OOC); 4.60 m , 1 H, W = 24 (H-3α); 5.37 br d,
2
3
3
1/2
W1/2 = 8, J = 4.6 (H-6).
Meth yl (20S)-3β-Hydroxypregn -5-en e-20-carboxylate (3)
A solution of m eth yl (20S)-3β-acetoxypregn -5-en e-20-carboxylate (2; 2.01 g, 5 m m ol) an d
potassium h ydrogen carbon ate (1.8 g, 18 m m ol) in m eth an ol (450 m l) an d water (23 m l) was
h eated at reflux for 90 m in . After evaporation of th e solven t, th e m ixture was diluted with
water an d eth er, th e eth ereal layer was separated, wash ed with water an d dried (Na SO ).
2
4
Evaporation in vacuo afforded pure product 3 in 99% yield (1.8 g). An alytical sam ple was
2
5
crystallized from m eth an ol: m .p. 141–142 °C, [α] –45 (c 1.25), givin g iden tical ph ysical
D
con stan ts as described in th e literature10. IR: 3 622, 3 496 (OH), 3 028, 1 668 (C=CH), 1 737
(
C=O), 1 435 (OMe), 1 194 (C–OH), 1 162, 1 054 (C–O ester). 1H NMR: 0.70 s, 3 H (3 ×
H-18); 1.01 s, 3 H (3 × H-19); 1.19 d, 3 H, J = 7.0 (3 × H-21); 2.44 m , 1 H, W1/2 = 8 (H-20);
3
.52 m , 1 H, W1/2 = 24 (H-3α); 3.65 s, 3 H (CH OOC); 5.36 br d, J = 5.5, W1/2 = 8.5 (H-6).
3
Meth yl (20S)-3β-(4-Toluen sulfon yloxy)pregn -5-en e-20-carboxylate (4)
Meth yl (20S)-3β-h ydroxypregn -5-en e-20-carboxylate (3; 2.3 g, 6.4 m m ol) was dissolved in
pyridin e (23 m l) an d th e solution was treated with tosyl ch loride (2.3 g, 12 m ol). After six
days, th e m ixture was worked up as usual in to ch loroform . Evaporation in vacuo afforded
2
.72 g (83%) of product 4. An alytical sam ple was dissolved in ch loroform an d eth er was
added. Crystals obtain ed on stan din g m elted at 140–142 °C (dec.), wh ich is in agreem en t
6
with th e literature value . IR: 3 065, 3 033 (Tos, C=C), 1 736 (C=O), 1 600, 1 495, 1 395
1
(
C H -ring), 1 379, 1 178, 568, 556 (SO ), 1 161 (C–O ester). H NMR: 0.67 s, 3 H (3 × H-18);
6
4
2
0
.97 s, 3 H (3 × H-19); 1.18 d, 3 H, J = 6.7 (3 × H-21); 2.49 s, 3 H (CH3 tosylate); 3.64 s, 3 H
(
CH OOC); 4.32 m , 1 H, W1/2 = 24 (H-3α); 5.32 br d, 1 H, J = 6, W1/2 = 8.5 (H-6); 7.34 d, 2 H
3
an d 7.80 d, 2 H, J = 8.0 (C H of th e tosyl group).
6
4
Meth yl (20S)-6-Oxo-3α,5-cyclo-5α-pregn an e-20-carboxylate (6)
A m ixture of tosylate 4 (935 m g, 1.8 m m ol) an d potassium acetate (2 g, 20 m m ol) in ace-
ton e (30 m l) an d water (9 m l) were h eated at reflux for 6.5 h . After coolin g, th e reaction
m ixture was poured in to water an d worked up as usual by extraction with eth er. Th e crude
m eth yl (20S)-6β-h ydroxy-3α,5-cyclo-5α-pregn an e-20-carboxylate (5) [IR: 3 625 (OH), 3 070
1
(
(
cyclopropan e), 1 737, 1 160 (COOCH ). H NMR: 0.19–0.39 m , 1 H an d 0.39–0.60 m , 1 H
3
cyclopropan e proton s); 0.73 s, 3 H (3 × H-18); 1.05 s, 3 H (3 × H-19); 1.22 d, 3 H, J = 7.0 (3 ×
H-21); 2.46 m , 1 H, W1/2 = 6.2 (20-H); 3.26 m , 1 H, W1/2 = 8 (H-6α); 3.67 s, 3 H (CH OOC)]
3
was dissolved in aceton e (20 m l) an d treated with th e Jon es reagen t at 5 °C un til perm an en t
oran ge colour persisted. Th e excess reagen t was destroyed by th e addition of propan -2-ol.
After filtration , th e solution was diluted with eth er an d worked up as usual. Evaporation of
th e solven ts in vacuo afforded 860 m g of a solid residue. Th e residue was ch rom atograph ed
on a colum n of silica gel (100 g); elution with ligh t petroleum –eth er (7 : 3) gave 589 m g
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

On Steroids
1759
2
D
5
(
91%) of pure keton e 6. M.p. 112–113 °C (m eth an ol–water), [α] +34 (c 1.19) in accordan ce
6
with th e literature . IR: 3 070 (cyclopropan e), 1 738 (C=O ester), 1 690 (6-C=O), 1 434,
1
1
379, 1 374 (COOCH ), 1 162 (C–O). H NMR: 0.73 s, 3 H (3 × H-18); 1.01 s, 3 H (3 ×
3
H-19); 1.205 d, 3 H, J = 7.0 (3 × H-21); 2.44 m , 1 H, W 1/ 2 = 6.0 (20-H); 3.65 s, 3 H
CH OOC).
(
3
(
20S)-6-Oxo-3α,5-cyclo-5α-pregn an e-20-carboxylic Acid (7)
A solution of m eth yl ester 6 (50 m g, 0.14 m m ol) an d potassium h ydroxide (100 m g, 1.8
m m ol) in m eth an ol (10 m l) was h eated at reflux un der n itrogen for 4 h . Th e solution was
th en n eutralized with 5% aqueous HCl, evaporated to dryn ess in vacuo, water was added an d
th e product extracted with ch loroform . Th e ch loroform layer was wash ed with water, dried
with sodium sulfate an d th e solven t evaporated in vacuo. Th e oily residue (50 m g) was puri-
fied by preparative TLC on silica gel (2 plates) in ligh t petroleum –eth er (1 : 1). Extraction of
th e zon es con tain in g th e product afforded 42 m g (87%) of acid 7, m .p. 177–179 °C (m eth a-
n ol–water). IR (KBr): 3 070 (cyclopropan e), 2 639, 2 573 (dim er COOH), 1 739 (dim er
1
COOH), 1 714 (m on om er COOH), 1 678 (6-C=O). H NMR: 0.75 s, 3 H (3 × H-18); 1.01 s, 3 H
(
(
3 × H-19); 1.26 d, 3 H, J = 6.7 (3 × H-21); 2.44 m , 1 H, W1/2 = 6.0 (20-H). For C22H32O3
344.5) calculated: 76.70% C, 9.36% H; foun d: 76.34% C, 9.42% H.
Meth yl (20S)-6-Oxo-5α-pregn -2-en e-20-carboxylate (8)
Pyridin ium tosylate (50 m g, 0.27 m m ol) an d lith ium brom ide (50 m g, 0.57 m m ol) were
added to a solution of cyclopropan e derivative 6 (500 m g, 1.4 m m ol) in N,N-dim eth yl-
acetam ide (15 m l). Th e reaction m ixture was h eated at 160 °C un der n itrogen for 4 h . Fur-
th er portion s of pyridin ium tosylate (100 m g, 0.55 m m ol) an d lith ium brom ide (100 m g,
1
.13 m m ol) were added to th e reaction m ixture an d h eatin g con tin ued at 160 °C for an -
oth er 6 h . After coolin g, th e m ixture was poured in to water, th e product extracted with
eth er an d th e eth ereal solution worked up as usual. Th e crude product (500 m g) was
ch rom atograph ed on a colum n of silica gel (ligh t petroleum –eth er, 4 : 1) to afford 462 m g
2
D
5
(
92%) of olefin 8, m .p. 149–150 °C (m eth an ol) an d [α] +26 (c 1.21) are in accordan ce with
6
th e literature . IR: 1 738 (C=O ester), 1 712 (6-C=O), 1 687 (C=C), 1 435 (COOMe), 1 162
C–O). H NMR: 0.69 s, 3 H (3 × H-18); 0.71 s, 3 H (3 × H-19); 1.20 d, 3 H, J = 7.0 (3 ×
1
(
H-21); 3.65 s, 3 H (CH OOC); 5.63 dm , 2 H (H-2 an d H-3). For C23H34O3 (358.5) calculated:
3
7
7.05% C, 9.56% H; foun d: 77.19% C, 9.31% H.
(
20S)-6-Oxo-5α-pregn -2-en e-20-carboxylic Acid (9)
Meth yl ester 8 (40 m g, 0.11 m m ol) in m eth an ol (4 m l) was h ydrolyzed in th e sam e way as
described for th e preparation of com poun d 7. After a sim ilar work-up, th e residue (40 m g)
was purified by preparative TLC on silica gel (2 plates) in ligh t petroleum –eth er (1 : 1). Th e
zon es con tain in g th e product afforded 29 m g (77%) of acid 9, m .p. 168–169 °C (eth an ol),
2
5
1
[
α] +23 (c 1.11). IR (KBr): 1 742 (COOH), 1 713 (6-C=O). H NMR: 0.70 s, 3 H an d 0.71 s,
D
3
(
7
H (3 × H-18 an d 3 × H-19); 1.24 d, 3 H, J = 7.5 (3 × H-21); 5.55 d, 1 H an d 5.68 m , 1 H
AB system , H-2 an d H-3). For C22H32O3 (344.5) calculated: 76.70% C, 9.36% H; foun d:
6.41% C, 9.31% H.
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

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Kohout, Chodounská, Macek, Strnad:
Meth yl (20S)-2α,3α-Dih ydroxy-6-oxo-5α-pregn an e-20-carboxylate (10)
A solution of olefin 8 (249 m g, 0.89 m m ol) in aceton e (12.5 m l) an d tetrah ydrofuran
(
(
12.5 m l) was m ixed with a solution of osm ium tetroxide (25 m g) in tert-butyl alcoh ol
0.25 m l), N-m eth ylm orfolin e N-oxide (0.4 g) an d water (0.6 m l). After stan din g at room
tem perature un der argon for 36 h , 10% solution of sodium sulfite (3.0 m l) was added an d
th e m ixture was stirred at room tem perature for 30 m in . Th e product was extracted with
ch loroform an d processed in th e usual m an n er to give 280 m g of crude m aterial.
Ch rom atograph y on a colum n of silica gel (eth er–eth yl acetate–ch loroform , 1 : 1 : 2) afforded
2
D
5
1
79 m g (51%) of diol 10, m .p. 197–198 °C (m eth an ol–water), [α] –3 (c 0.91) in accordan ce
6
with th e literature . IR: 3 610, 3 585, 1 050 (OH), 1 730 (C=O ester), 1 714 (6-C=O), 1 175
C–O). H NMR: 0.68 s, 3 H (3 × H-18); 0.71 s, 3 H (3 × H-19); 1.19 d, 3 H, J = 7.0 (3 × H-21);
1
(
3
3
.65 s, 3 H (COOCH ); 3.77 m , 1 H, W
= 26 (H-2β); 4.07 m , 1 H, W1/2 = 8 (H-3β). MS, m/z:
1/2
3
+
92 (M ), 377 (M – CH ), 374 (M – H O). For C23H36O5 (392.5) calculated: 70.38% C, 9.24%
3
2
H; foun d: 70.31% C, 9.11% H.
Meth yl (20S)-2α,3α-Dih ydroxy-6-oxo-7-oxa-7a-h om o-5α-pregn an e-20-carboxylate (11)
A solution of trifluoroperoxyacetic acid in dich lorom eth an e (38 m l), prepared from 5 g
(
13 m m ol) of trifluoroacetic an h ydride an d 0.5 m l (8 m m ol) of 50% h ydrogen peroxide, was
added to a solution of keton e 10 (785 m g, 2.0 m m ol) in dich lorom eth an e (16 m l). After
stan din g at room tem perature for 20 h , th e reaction m ixture was poured in to a 10% potas-
sium h ydrogen carbon ate solution . Th e product was extracted with ch loroform , th e organ ic
extract was wash ed with water, dried over sodium sulfate an d th e solven t was evaporated in
vacuo. Th e residue (845 m g) was ch rom atograph ed on a colum n of silica gel (150 g) in a
ch loroform –eth er–ligh t petroleum m ixture (1 : 1 : 1) to give 701 m g (86%) of a product
2
5
wh ich con tain ed m ain ly com poun d 11, m .p. 187–188 °C (eth an ol–water), [α] +35 (c 1.16)
D
6
in accordan ce with th e literature . IR (ch loroform ): 3 620 (OH), 1 730 (C=O ester), 1 723
1
(
C=O lacton e). H NMR: 0.71 s, 3 H (3 × H-18); 0.94 s, 3 H (3 × H-19); 1.19 d, 3 H, J = 6.7 (3 ×
H-21); 2.55 m , 1 H (20-H); 3.65 s, 3 H (COOCH ); 3.74 m , 1 H, W
W1/2 = 8 (H-3β); 4.08–4.12 m , 2 H (H-7a). MS, m/z: 408 (M ), 393 (M – CH ), 390 (M – H O).
= 25 (H-2β); 4.15 m , 1 H,
1/2
3
+
3
2
For C23H36O6 (408.5) calculated: 67.62% C, 8.88% H; foun d: 67.61% C, 8.91% H.
Meth yl (20S)-2α,3α-Dih ydroxy-6-oxa-7-oxo-7a-h om o-5α-pregn an e-20-carboxylate (13)
Th is 6-oxa-7-oxo-isom er 13 of com poun d 11 was in separable from th is substan ce an d was
1
iden tified by H NMR: 0.73 s, 3 H (3 × H-18); 0.96 s, 3 H (3 × H-19); 3.44 dd, 1 H (H-5) as a
m in or com pon en t in th e m oth er liquor after th e crystallization of lacton e 11.
(
20S)-2α,3α-Dih ydroxy-6-oxo-7-oxa-7a-h om o-5α-pregn an e-20-carboxylic Acid (12)
Meth yl ester 11 (820 m g, 2.01 m m ol) in m eth an ol (80 m l) with potassium h ydroxide (1.2 g,
2
5
1 m m ol) was h eated at reflux un der n itrogen for 10 h . Th e solution was n eutralized with
% aqueous HCl, 37% HCl (0.5 m l) in m eth an ol (4.5 m l) was subsequen tly added an d th e
reaction m ixture was left to stan d at room tem perature for 30 m in . Th e m ixture was evapo-
rated to dryn ess, water (100 m l) was added, an d th e product was extracted with ch loroform
(
3 × 60 m l). Th e ch loroform solution was dried with sodium sulfate an d evaporated un der
reduced pressure. Th e residue (690 m g) was crystallized from eth an ol–water to afford 317
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

On Steroids
1761
m g of th e product. Th e m oth er liquor was evaporated un der reduced pressure to dryn ess
an d th e residue was again crystallized from eth an ol an d water to afford 155 m g of product
1
2. On e m ore crystallization of th e m oth er liquor afforded a furth er portion of th e product,
givin g a total of 557 m g (70.3%) of com poun d 12, m .p. 321–322 °C (eth an ol–water) in ac-
6
25
cordan ce with th e literature , [α]D +45 (c 1.23, m eth an ol). IR (KBr): 3 367, 3 142 (OH), 2 622
dim er COOH), 1 746 (C=O lacton e), 1 705 (C=O carboxyl), 1 281 (C=O of dim er COOH),
(
1
1
1
1
062, 1 039, 966 (C–OH). H NMR (CD OD): 0.95 s, 3 H (3 × H-18); 1.09 s, 3 H (3 × H-19);
3
.38 d, 3 H, J = 6.9 (3 × H-21); 2.53 m , 1 H, W1/2 = 6 (20-H); 3.79 dm , 1 H (H-2β); 4.13 m ,
H (H-3β); 4.20–4.44 m , 2 H (2 × H-7a); (DMSO-d ): 0.68 s, 3 H (3 × H-18); 0.77 s, 3 H (3 ×
6
H-19); 1.07 d, 3 H, J = 7.0 (3 × H-21). For C22H34O6 (394.5) calculated: 66.98% C, 8.69% H;
foun d: 66.91% C, 8.51% H.
(
20S)-2α,3α-Dih ydroxy-6-oxo-7-oxa-7a-h om o-5α-pregn an e-20-carboxylic acid (14)
Careful separation of a part (40 m g) of th e evaporated m oth er liquor from th e crystallization
of isom er 12 on preparative TLC plates on silica gel (8 plates) in ch loroform –m eth an ol
1
(
(
4 : 1) afforded 1.6 m g of th e m ore polar isom er, lacton e 14. H NMR (CD OD): 0.94 s, 3 H
3
+
3 × H-18); 1.13 s, 3 H (3 × H-19); 3.44 d, 1 H (H-5). FAB-MS, m/z: 395 (M + 1) .
The authors are indebted to Dr P. Fiedler for recording and interpretation of IR spectra. Our thanks
1
are due to Ms M. Snopková and Dr R. Pohl for the measurements of H NMR spectra. Mass spectra were
recorded by Dr J. Kohoutová of the Laboratory of Mass Spectrometry. The elemental analyses were car-
ried out in the Analytical Laboratory (Dr L. Válková, head). Technical assistance of Dr I. Paulová and
Ms J. Neumannová is gratefully acknowledged. This work was supported in part by the Grant Agency
of the Academy of Sciences of the Czech Republic (grant No. A4055803).
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Collect. Czech. Chem. Commun. (Vol. 65) (2000)