Synthesis and biological activity of 7a-homo- and 7a,7b-dihomo-5α- cholestane analogues of brassinolide

Article abstract of DOI:10.1135/cccc20032171

New analogues of castasterone, (20R)-2α,3α-dihydroxy-7a-homo- 5α-cholestan-7-one (12) and (20R)-3α,4α-dihydroxy-7a-homo- 5α-cholestan-7-one (14), and brassinolide, (20R)-2α,3α- dihydroxy-7a,7b-dihomo-7a-oxa-5α-cholestan-7-one (15), (20R)-3α,4α-dihydroxy-7a,7b-dihomo-7a-oxa-5α-cholestan-7-one (16) and (20R)-7-oxa-7a-homo-5α-cholestan-2α,3α-diol (24), were synthesized and their biological and spectral properties studied.

Full text of DOI:10.1135/cccc20032171

On Steroids
2171
SYNTHESIS AND BIOLOGICAL ACTIVITY OF 7a-HOMO- AND
7a,7b-DIHOMO-5α-CHOLESTANE ANALOGUES OF BRASSINOLIDE+
1
2
3,
Miroslav ŠÍŠA , Miloš BUDĚŠÍNSKÝ and Ladislav KOHOUT *
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
1
Flemingovo nám. 2, CZ-166 10 Prague 6, Czech Republic; e-mail: sisa@uochb.cas.cz,
2
3
budesinsky@uochb.cas.cz, kohout@uochb.cas.cz
Received Jan uary 15, 2003
Accepted Septem ber 1, 2003
New an alogues of castasteron e, (20R)-2α,3α-dih ydroxy-7a-h om o-5α-ch olestan -7-on e (12) an d
(20R)-3α,4α-dih ydroxy-7a-h om o-5α-ch olestan -7-on e (14), an d brassin olide, (20R)-2α,3α-di-
h ydroxy-7a,7b-dih om o-7a-oxa-5α-ch olestan -7-on e (15), (20R)-3α,4α-dih ydroxy-7a,7b-di-
h om o-7a-oxa-5α-ch olestan -7-on e (16) an d (20R)-7-oxa-7a-h om o-5α-ch olestan -2α,3α-diol
(24), were syn th esized an d th eir biological an d spectral properties studied.
Keyw ord s: Steroids; Brassin osteroids; NMR spectroscopy; Hom och olestan es; Dih om o-
ch olestan es; Plan t growth regulators; Bayer–Villiger oxidation .
Brassin osteroids, a class of poten t plan t growth regulators, h ave a prom ise
of poten tial use in agriculture due to th eir ability to im prove crop yields,
overcom e en viron m en tal stress2 an d bein g en viron m en tally frien dly. Sin ce
th e ch em ical structure of brassin olide3 was establish ed, structurally related
com poun ds h ave been syn th esized an d th eir biological activities evaluated.
In th is con n ection , two problem s sh ould be addressed. Th e first is th e struc-
ture–activity relation sh ips of brassin osteroids, th e secon d is th eir activity in
differen t bioassays. Havin g studied4–8 th e structure–activity relation sh ips of
brassin osteroids, we n ow turn our atten tion to brassin olide an alogues of
7a-h om o- an d 7a,7b-dih om o-5α-ch olestan es9.
To elaborate th e syn th esis, (20R)-7-oxo-ch olest-5-en -3β-yl acetate (1) was
used as a startin g m aterial (Sch em e 1). Th is com poun d was h ydrogen ated
over th e Adam s catalyst to afford (20R)-7-oxo-5α-ch olestan -3β-yl acetate
(2). Th e reaction of keton e 2 with powdered potassium cyan ide an d acetic
acid provided a m ixture of epim eric cyan oh ydrin s 3a an d 3b; pure isom er
+ Part CDXV in th e Series On Steroids; Part CDXIV see ref.1
Collect. Czech. Chem. Commun. (Vol. 68) (2003)
doi:10.1135/cccc20032171

2172
Šíša, Buděšínský, Kohout:
(i)
AcO
O
O
AcO
H
1
2
(ii)
(iii)
R1
RO
AcO
(iv)
H
R2
H
O
3
4, R = Ac
5, R = H
6, R = Ts
7, R = Ms
3a, R1 = CN, R2 = OH
3b, R1 = OH, R2 = CN
H
H
O
O
8
9
(v)
Cl
Cl
H
H
O
O
10
11
(i) H2/PtO2; (ii) KCN; (iii) NaNO2; (iv) collidine, ∆; (v) LiBr
SCHEME 1
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

On Steroids
2173
3a was obtain ed on crystallization . Th e secon d epim er 3b was isolated from
th e m oth er liquor usin g preparative th in layer ch rom atograph y (TLC). Th e
determ in ation of con figuration at C(7) in both epim ers was n ot straigh tfor-
ward due to th e absen ce of a h ydrogen atom at C(7). All altern ative at-
tem pts – (i) in situ TAI-acylation m eth od10, (ii) NOE m easurem en ts, an d/or
(iii) couplin g con stan ts J(C,H) – required a com plete structural assign m en t
of proton an d carbon sign als. Th is was ach ieved for both epim ers 3a an d 3b
by th e com bin ation of 2D h om o- an d h eteron uclear correlation NMR tech -
n iques; th e results are given in Table I. Th e observed in duced TAI-acylation
sh ifts (∆δ) of th e proton s in th e vicin ity of th e acylated OH group sup-
ported th e con figuration assign m en t sh own in Fig. 1a, with som e n egative
∆δ values for th e proton s in 1,3-diaxial orien tation to th e OTAC group: th is
effect can be explain ed by th e reduction of van der Waals effect of th e
acylated OH group. Furth er argum en ts could be obtain ed from in terproton
NOEs, wh ere differen t NOE con tacts (ch aracteristic for each epim er) were
expected, as sh own in Fig. 1b. Un fortun ately, th e sign al of th e OH proton
was clearly visible on ly for 3b. In th e ROESY spectrum of 3b, th e ch aracter-
istic NOE con tacts of OH to H-6α, H-6β, H-8, H-15α an d H-15β were ob-
served, in dicatin g th e structure 3b (in agreem en t with th e TAI-acylation
results). Fin al eviden ce of th e con figuration at C(7) was derived from th e
observed J(C,H) couplin gs of th e C≡N carbon . In accordan ce with th e situa-
tion illustrated in Fig. 1c, th e C≡N carbon sign al appears as a quartet with
th ree sm all J(C,H) ≈ 2 Hz in epim er 3a (C≡N carbon in gauche-orien tation to
H-6α, H-6β an d H-8), wh ile in th e secon d epim er 3b, th is sign al sh ows as a
doublet of triplets with two large J(C,H) ≈ 9 Hz an d on e sm aller J(C,H) ≈ 5
Hz couplin g (C≡N carbon in trans-orien tation to H-6β, H-8 an d gauche-ori-
en tation to H-6α).
Cyan oh ydrin s 3a an d 3b were con verted to (20R)-7-oxo-7a-h om o-5α-
ch olestan -3β-yl acetate (4), as described in th e literature11. Hydrolysis of
acetate 4 with potassium carbon ate in m eth an ol afforded (20R)-3β-
h ydroxy-7a-h om o-5α-ch olestan -7-on e (5), wh ich furn ish ed th e correspon d-
in g sulfon ates 6 an d 7 on treatm en t with 4-toluen esulfon yl ch loride or
m eth an esulfon yl ch loride, respectively. Ch lorides 10 an d 11 were also ob-
tain ed from th is reaction as side products. On reaction 12 with sym -
collidin e, both sulfon ates 6 an d 7 gave a m ixture of two isom eric olefin s
(2,3- an d 3,4-olefin s) 8 an d 9. Th e sam e m ixture was also obtain ed on treat-
m en t of ch lorides 10 an d 11 with lith ium brom ide. Th e position of th e
1
double bon d was eviden t from th e an alysis of th eir H NMR spectra as well
as from subsequen t reaction s. Th ese olefin s were h ydroxylated with os-
m ium tetroxide5 in th e presen ce of N-m eth ylm orph olin e N-oxide. Th e
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

2174
Šíša, Buděšínský, Kohout:
TABLE I
1H an d 13C NMR ch em ical sh ifts of cyan oh ydrin s 3a an d 3b in CDCl3
Proton
3a
3b
Carbon
3a
3b
1α
1β
2α
2β
3
1.04
1.76
1.08
1.77
1
36.30
27.21
72.94
32.70
36.85
41.93
69.03
42.32
47.54
34.96
21.16
39.36
43.70
50.32
24.88
28.32
55.51
12.05
11.35
35.68
18.70
36.05
23.75
39.45
28.00
22.78
22.54
123.42
170.72
21.37
36.18
27.17
72.71
32.82
40.39
40.77
74.39
44.60
50.33
36.08
21.16
39.10
43.78
52.42
26.07
28.58
54.78
12.07
12.26
35.56
18.73
35.06
23.72
39.47
28.01
22.79
22.55
120.51
170.48
21.34
2
1.83
1.87
3
1.48
1.47
4
4.68
4.72
5
4α
4β
5
1.58
1.68
6
1.35
1.40
7
1.67
1.54
8
6α
6β
8
1.80
1.83
9
1.90
1.64
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
CN
C=O (Ac)
CH3
1.79
1.64
9
1.11
0.98
11α
11β
12α
12β
14
1.58
1.60
1.31
1.35
1.12
1.19
1.98
2.00
1.40
1.48
15α
15β
16α
16β
17
1.92
1.87
1.62
1.44
1.33
1.33
1.91
1.96
1.12
1.15
18
0.714
0.856
1.36
0.688
0.860
1.37
19
20
21
0.910
1.33; 1.02
1.33; 1.13
≈1.13 (2 H)
1.52
0.920
1.35; 1.05
1.34; 1.13
≈1.12 (2 H)
1.53
22
23
24
25
26
0.867
0.872
0.865
2.57
27
0.863
a
OH
OAc
2.03
2.03
a
Th e position of th e OH sign al was n ot determ in ed.
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

On Steroids
2175
2,3-olefin 8 gave (20R)-2α,3α-dih ydroxy-7a-h om o-5α-ch olestan -7-on e (12),
accom pan ied with a sm all am oun t of (20R)-2β,3β-dih ydroxy-7a-h om o-5α-
ch olestan -7-on e (13). Th e 3,4-olefin 9 yielded a sin gle diol, (20R)-3α,4α-
dih ydroxy-7a-h om o-5α-ch olestan -7-on e (14) (Sch em e 2). Diols 12 an d 14
can be con sidered as ch olestan e h om o-an alogues of castasteron e.
Baeyer–Villiger oxidation of castasteron e an alogues 12 an d 14 produced
ch olestan e h om o-an alogues of brassin olide. Keton e 12 was oxidized to
yield on e product, (20R)-2α,3α-dih ydroxy-7a-oxa-7a,7b-dih om o-5α-ch olest-
an -7-on e (15) an d, sim ilarly, keton e 14 gave a sin gle product, (20R)-3α,4α-
dih ydroxy-7a-oxa-7a,7b-dih om o-5α-ch olestan -7-on e (16). Th e structures of
1
dih ydroxylacton es 15 an d 16 were con firm ed by H an d 13C NMR spectros-
a
3a
3b
b
3a
3b
c
3a
3b
FIG. 1
Th e in duced TAI-acylation sh ifts of proton s (a) in epim ers 3a an d 3b; ch aracteristic NOE con -
tacts (b), expected for com poun ds 3a an d 3b; th e vicin al J(C,H) couplin gs (c), observable in
epim ers 3a an d 3b
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

2176
Šíša, Buděšínský, Kohout:
copy. A com plete structural assign m en t of proton an d carbon sign als was
again don e by th e com bin ation of 2D h om o- an d h eteron uclear correlation
NMR tech n iques; th e results are given in Table II. Th e proton couplin g con -
stan ts J(7aα,8) < 1, J(7aβ,8) ≈ 1.5, J(5,6α) ≈ 2.2 an d J(5,6β) ≈ 12.2 Hz in dicate
th at th e eigh t-m em bered rin g adopts a “ch air-boat” form in both com -
poun ds (Fig. 2) in accordan ce with th e lowest en ergy con form ation , calcu-
lated usin g m olecular dyn am ics an d en ergy m in im ization (m olecular
m ech an ics MM+ m eth od with HyperCh em version 6.01 from Hypercube
In c.). Th e six-m em bered rin g A assum es th e ch air form with 2-OH(eq),
3-OH(ax) in com poun d 15 an d 3-OH(ax), 4-OH(eq) in com poun d 16.
HO
HO
H
O
13
(i)
9
8
(i)
(i)
HO
HO
HO
H
H
O
O
HO
14
12
(ii)
(ii)
HO
HO
HO
O
O
H
H
HO
O
O
16
15
(i) OsO4; (ii) TFAA
SCHEME 2
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

On Steroids
2177
TABLE II
1H an d 13C NMR ch em ical sh ifts of dih ydroxylacton es 15 an d 16 in CDCl3
Proton
15
16
Carbon
15
16
1α
1β
2α
2β
3
≈1.58
≈1.58
–
1.33
1.59
1
41.39
68.27
68.66
38.00
41.27
35.61
176.19
69.56
41.30
46.64
40.22
24.92
40.17
41.90
51.54
24.96
27.81
56.15
11.72
13.08
35.84
18.44
35.89
23.70
39.47
27.98
22.79
22.55
32.00
27.79
68.79
70.97
49.66
28.24
178.40
69.87
41.74
47.14
40.69
25.12
40.34
41.84
51.70
24.98
27.57
56.18
11.70
13.04
35.85
18.44
35.89
23.70
39.46
27.98
22.78
22.55
2
1.80
3
3.78
1.77
4
3.96
4.03
5
4α
4β
5
≈1.73
≈1.73
2.24
–
6
3.34
7
2.04
7a
8
6α
6β
7aα
7aβ
8
2.17
3.21
2.69
2.50
9
4.53
4.67
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
4.15
4.19
1.56
1.60
9
1.15
1.12
11α
11β
12α
12β
14
15α
15β
16α
16β
17
18
19
20
21
22
23
24
25
26
27
1.81
1.83
1.35
1.34
1.13
1.12
1.94
1.93
1.58
1.58
≈1.55
≈1.55
1.27
≈1.55
≈1.55
1.25
1.81
1.75
1.12
1.14
0.673
1.008
1.35
0.716
0.999
1.35
0.885
1.33; 1.00
1.33; 1.13
≈1.14 (2 H)
1.52
0.874
1.33; 1.00
1.33; 1.13
≈1.13 (2 H)
1.52
0.866
0.860
0.866
0.860
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

2178
Šíša, Buděšínský, Kohout:
Th e syn th esis of an alogues with out an y oxygen fun ction in rin g B was
carried out from acetate 4, wh ich was tran sform ed in to (20R)-7a-h om o-
5α-ch olestan -3β-ol (17) by th e Kish n er–Wolff reduction 11. We attem pted to
obtain two diols, (20R)-7a-h om o-5α-ch olestan -2α,3α-diol (21) an d (20R)-
7a-h om o-5α-ch olestan -3α,4α-diol (22), in a sim ilar way, as described above
for th e syn th esis of diols 12 an d 14. However, we en coun tered a problem
with th e separation of olefin s 19 an d 20, as th eir polarities were practically
iden tical. Th erefore, we h ydroxylated th e m ixture, an d obtain ed a m ixture
of two diols, 21 an d 22 (Sch em e 3). Th eir structures were con firm ed by in -
depen den t syn th esis from keton es 12 an d 14. Th e Kish n er–Wolff reduction
con verted keton e 12 to diol 21 an d keton e 14 to diol 22. Th e last com -
poun d of th is series, (20R)-7a-h om o-7-oxa-5α-ch olestan -2α,3α-diol (24) was
obtain ed on reduction 13 of th e previously syn th esized14 lacton e 23 with so-
dium boroh ydride in th e presen ce of boron trifluoride eth erate in a good
yield (Sch em e 4). Th is m eth od seem s to be useful for th e preparation of
oxa-steroids.
Th e above syn th esis yielded products, wh ich were em ployed in our
screen in g project. Th eir brassin osteroid activities were determ in ed by th e
m odified bean secon d in tern ode bioassay15. Am on g all th e substan ces
15
16
FIG. 2
Th e con form ation s of rin gs A an d B in com poun ds 15 an d 16
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

On Steroids
2179
(i)
RO
AcO
H
H
O
17, R = H
18, R = Ts
4
(ii)
H
H
19
20
(iii)
HO
HO
HO
H
H
HO
21
22
(i)
(i)
12
14
(i) N2H2·H2O, KOH; (ii) collidine, ∆; (iii) OsO4
SCHEME 3
HO
HO
HO
HO
(i)
O
O
H
H
O
23
24
(i) BF3·Et2O
SCHEME 4
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

2180
Šíša, Buděšínský, Kohout:
tested, th e h igh est biological activity was displayed by com poun ds 14, 16
an d 22, i.e. by com poun ds, wh ich h ave a 3α,4α diol m oiety an d a carbon yl
in position 7a. However, th eir activities were lower in com parison with th at
of 24-epibrassin olide [(22R,23R,24R)-2α,3α,22,23-tetrah ydroxy-24-m eth yl-
7-oxa-7a-h om o-5α-ch olestan -6-on e] (Table III).
EXPERIMENTAL
Th e m eltin g poin ts were determ in ed on a Kofler block. Optical rotation s were m easured at
25 °C in ch loroform (un less oth erwise stated) an d [α]D values are given in 10–1 deg cm 2 g–1
.
In frared spectra were recorded on a Bruker IFS 88 spectrom eter in tetrach lorom eth an e (un -
less oth erwise stated), th e waven um bers are given in cm –1 1H NMR spectra were taken in
.
deuterioch loroform on a Varian UNITY-200 (at 200 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. Detailed
NMR structure an alyses of cyan oh ydrin s 3a, 3b an d dih ydroxylacton es 15, 16 were don e on
a Varian UNITY-500 (1H at 500 MHz; 13C at 125.7 MHz). Ch em ical sh ifts are given in ppm
(δ-scale), couplin g con stan ts (J) an d m ultiplet h alf-width s (W1/2) in Hz. All values were ob-
tain 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 prepared sam ples was ch ecked by m eltin g poin ts, th in -layer ch rom a-
tograph y (TLC) on silica gel G (ICN Bioch em icals, detection by sprayin g with sulfuric acid
an d h eatin g), IR an d 1H NMR spectra. Preparative TLC was carried out on 200 × 200 m m
TABLE III
Bean secon d in tern ode bioassay
Len gth en in g of th e secon d
in tern ode, m m a
Th e am oun t
Com poun d
applied, m olb
24-epiBRc
32.3
5.6
10–10
10–9
10–9
10–10
10–10
10–10
10–11
10–10
10–9
12
14
14
15
16
21
22
24
21.8
19.4
–5.4
17.2
8.3
18.0
8.8
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 as com pared to th at of th e
b
referen ce plan t for th e applied am oun t given .
Th e applied am oun t m ean s th e am oun t
causin g th e m axim um len gth en in g of th e secon d in tern ode (all com poun ds were applied in
c
th e am oun ts from 1 × 10–12 to 1 × 10–7 m ol per on e plan t). 24-epiBR m ean s 24-epibras-
sin olide [(22R,23R,24R)-2α,3α,22,23-tetrah ydroxy-24-m eth yl-7-oxa-7a-h om o-5α-ch olestan -
6-on e].
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

On Steroids
2181
plates coated with 0.7 m m layer of silica gel Woelm DC, detection by sprayin g th e plates
with 0.2% m eth an olic m orin e solution , by UV detection or by sprayin g with sulfuric 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).
“Worked up as usual” 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, saturated aqueous potassium h ydrogen carbon ate,
water, dryin g over an h ydrous sodium sulfate, filterin g an d evaporatin g th e solven t to dry-
n ess un der reduced pressure.
Ligh t petroleum refers to a fraction boilin g at 40–65 °C.
Bean Secon d In tern ode Bioassay
Bean seeds (Phaseolus vulgaris L., cv. Pin to) were germ in ated for two days. Selected germ i-
n ated 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
to 27 °C, ligh t: 48 W/m 2, ligh t/dark period: 16 h /8 h ). Groups of eigh t 7-day-old seedlin gs
with 1–2 m m lon g secon d in tern odes were treated with differen t am oun ts of th e tested com -
poun ds in lan olin e (2 m l). Th e con trol plan ts were treated with lan olin e. Th e m easurem en ts
were taken after 5 days. Th e differen ce in th e len gth of th e secon d in tern odes of th e treated
an d con trol plan ts was used as a m easure of th e activity.
(20R)-7-Oxo-5α-ch olestan -3β-yl Acetate (2)
(20R)-7-Oxo-ch olest-5-en -3β-yl acetate (1; 1 g, 2.26 m m ol) was dissolved in acetic acid (30 m l).
Th e Adam s catalyst (50 m g, 0.22 m m ol) was added an d th e m ixture was h ydrogen ated. Th e
reaction m ixture was poured in to water an d worked up as usual. Th e con sum ed am oun t of
h ydrogen (ca 110 m l, th eoretical am oun t about 60 m l) an d TLC in dicated th at th e 7-on e
group was also h ydrogen ated. Th e evaporated residue was th erefore dissolved in aceton e
(20 m l) an d treated with th e Jon es reagen t un til a perm an en t oran ge colour persisted. After
10 m in , th e excess reagen t was destroyed by th e addition of m eth an ol. After 5 m in an d fil-
tration , th e solution was diluted with eth er an d worked up as usual. Evaporation of th e
solvent in vacuo afforded 1 g of a solid residue, which was chrom atographed on silica gel (100 g);
elution with ligh t petroleum –eth er (7:3) gave 900 m g (2.02 m m ol; 89%) of pure keton e 2.
An alytical sam ple was crystallized from m eth an ol to afford: m .p. 146–149 °C, [α]D –46
(c 1.040), givin g iden tical ph ysical con stan ts, as described in th e literature11. IR: 1737, 1027
(OAc); 1712 (C7=O); 1380 (Me, i-Pr); 1367 (i-Pr). 1H NMR: 0.658 s, 3 H (3 × H-18); 0.858 d,
3 H, J = 6.5 an d 0.862 d, 3 H, J = 6.5 (3 × H-26 an d 3 × H-27); 0.905 d, 3 H, J = 6.7 (3 ×
H-21); 1.097 s, 3 H (3 × H-19); 2.025 s, 3 H (3α-OAc); 4.66 m , 1 H, W1/2 = 19.0 (H-3α). EI
MS, m/z: 444 (M+), 429 (M+ – CH3), 384 (M+ – AcOH). For C29H48O3 (444.7) calculated:
78.33% C, 10.88% H; foun d: 78.30% C, 10.79% H.
(20R)-7β-Cyan o-7α-h ydroxy-5α-ch olestan -3β-yl Acetate (3a)
Keton e 2 (550 m g, 1.24 m m ol) was dissolved in dioxan e (5 m l) an d eth an ol (5 m l) an d th e
solution was cooled to 0 °C. Th e stirred solution was treated with powdered potassium cya-
n ide (730 m g, 11.21 m m ol), an d acetic acid was th en added dropwise over 30 m in un der
con stan t stirrin g at th e sam e tem perature. Th e reaction m ixture was stirred at 0 °C for 1 h
an d at room tem perature for an oth er 2 h . It was th en poured in to ice–water m ixture an d,
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after stan din g for 1 h , th e precipitate was separated by suction . Th e product was dissolved in
eth er, th e eth ereal solution wash ed with a sodium h ydrogen carbon ate solution , water,
dried, an d evaporated. Th e solid residue (510 m g) was ch rom atograph ed on silica gel (50 g);
elution with ligh t petroleum –eth er (8:2) gave 290 m g (0.62 m m ol; 50%) of a product, wh ich
con tain ed two isom ers. Th is m ixture was crystallized from eth yl acetate to afford practically
on e product, th e m ore polar on e of th e two isom ers. An oth er crystallization of h is m aterial
from eth yl acetate afforded an an alytical sam ple of cyan oh ydrin 3a: m .p. 164–167 °C, in ac-
cordan ce with th e literature11. IR: 3602, 3447, 1052 (OH); 2231 (C≡N); 1723 (C=O); 1254
(C–O); 1027 (OAc); 1382 (Me, i-Pr); 1366 (i-Pr). 1H an d 13C NMR data, see Table I. EI MS,
m/z: 471 (M+), 444 (M+ – HCN), 426 (444 – H2O), 384 (444 – AcOH). For C30H49NO3 (471.7)
calculated: 76.39% C, 10.71% H, 2.97% N; foun d: 75.95% C, 10.71% H, 2.71% N.
(20R)-7α-Cyan o-7β-h ydroxy-5α-ch olestan -3β-yl Acetate (3b)
Th e m ixture of th e two isom ers (50 m g), obtain ed in th e previous experim en t, was separated
by preparative TLC on 4 plates in ligh t petroleum –eth er (8:2) to afford isom er 3a (40 m g)
an d isom er 3b (10 m g), wh ich was m ore lipoph ilic th en th e form er an d m uch m ore un sta-
ble (disappearan ce on TLC in several h ours). 1H an d 13C NMR data, see Table I.
(20R)-7-Oxo-7a-h om o-5α-ch olestan -3β-yl Acetate (4)
A m ixture of cyan oh ydrin s 3a an d 3b from th e above experim en ts (5 g, 10.60 m m ol) was
dissolved in acetic acid (200 m l), th e Adam s catalyst was added (750 m g) an d th e resultan t
m ixture was h ydrogen ated un til th e th eoretical am oun t of h ydrogen h ad been taken up
(477 m l). Th e catalyst was filtered off an d th e filtrate was treated with water (20 m l). A solu-
tion of sodium n itrite (10 g) in water (30 m l) was th en added dropwise with stirrin g at 0 °C.
Th e reaction m ixture was th en stirred at room tem perature for addition al 2 h , treated with
water (100 m l) an d, after stan din g overn igh t, th e precipitate was isolated by suction . Th e
product was dissolved in eth er, th e eth ereal solution wash ed with a sodium h ydrogen car-
bon ate solution , water, dried, an d evaporated. Th e sem icrystallin e residue (3.4 g) was
ch rom atograph ed on silica gel (250 g); elution with toluen e an d toluen e–eth er (99:1) gave
1.1 g (2.40 m m ol; 23%) of th e 7a-h om oketon e 4. An alytical sam ple was crystallized from
m eth an ol: m .p. 119–120 °C, [α]D –34 (c 0.684), givin g iden tical ph ysical con stan ts, as de-
scribed in th e literature11. IR: 1735, 1243, 1025 (OAc); 1705 (cycloh eptan on e); 1383, 1365
(i-Pr); 1373 (Me). 1H NMR: 0.662 s, 3 H (3 × H-18); 0.847 2d, 6 H, J = 8.2 (3 × H-26 an d 3 ×
H-27); 0.879 s, 3 H (3 × H-19); 0.908 d, 3 H, J = 5.8 (3 × H-21); 2.030 s, 3 H (3α-OAc);
4.63 m , 1 H, W1/2 = 23.1 (H-3α). EI MS, m/z: 458 (M+), 440 (M+ – H2O), 398 (M+ – AcOH),
380 (440 – AcOH an d 398 – H2O). For C30H50O3 (458.7) calculated: 78.55% C, 10.99% H;
foun d: 76.78% C, 10.89% H.
(20R)-3β-Hydroxy-7a-h om o-5α-ch olestan -7-on e (5)
A solution of acetate 4 (1 g, 2.18 m m ol) in m eth an ol (100 m l) was treated with a solution of
potassium carbon ate (1 g) in water (10 m l). After stan din g overn igh t at room tem perature,
m eth an ol was rem oved un der reduced pressure, th e residue was dissolved in eth er an d th e
eth ereal solution worked up as usual. Th e crude product (90 m g, 2.16 m m ol; 99%) was crys-
tallized from aceton e–water (10:1): m .p. 116–118 °C, [α]D –31 (c 0.518), givin g iden tical
ph ysical con stan ts, as described in th e literature11. IR: 3622, 3448, 1038 (OH); 1704 (C=O);
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1384, 1366 (i-Pr); 1378 (Me). 1H NMR: 0.661 s, 3 H (3 × H-18); 0.801 s, 3 H (3 × H-19);
0.858 d, 3H, J = 6.7 an d 0.861 d, 3 H, J = 6.7 (3 × H-26 an d 3 × H-27); 0.891 d, 3 H, J = 6.2
(3 × H-21); 3.56 m , 1 H, W1/2 = 29.4 (H-3α). FAB MS, m/z: 417 (M+ + H), 399 (417 – H2O).
For C27H48O2 (416.7) calculated: 80.71% C, 11.62% H; foun d: 80.18% C, 11.14% H.
(20R)-7-Oxo-7a-h om o-5α-ch olestan -3β-yl Toluen esulfon ate (6)
A solution of alcoh ol 5 (800 m g, 2.16 m m ol) in pyridin e (10 m l) was treated with
4-toluen esulfon yl ch loride (800 m g) an d th e m ixture was allowed to stan d at room tem pera-
ture overn igh t. Th e reaction m ixture was decom posed with ice an d water, th e product taken
up in to eth er an d th e solution worked up as usual. Th e evaporated residue was dissolved in
a very sm all am oun t of ch loroform an d eth er was added to precipitate product 6 (600 m g,
1.05 m m ol; 49%). An alytical sam ple was crystallized from aceton e: m .p. 124–126 °C, [α]D
–32 (c 1.140). IR: 1705 (C=O); 1371, 1178, 557, 575 (SO2); 1600, 1496 (arom atic). 1H NMR:
0.641 s, 3 H (3 × H-18); 0.769 s, 3 H (3 × H-19); 0.858 d, 6 H, J = 6.4 (3 × H-26 an d 3 ×
H-27); 0.885 d, 3 H, J = 4.2 (3 × H-21); 2.452 s, 3 H (CH3 (Tos)); 4.38 m , 1 H, W1/2 = 24.1
(H-3α); 7.33 m , 2 H an d 7.79 m , 2 H (C6H4 (Tos)). EI MS, m/z: 398 (M+ – tosylate). For
C
35H54O4S (570.9) calculated: 73.64% C, 9.33% H, 5.62% S; foun d: 73.84% C, 9.77% H,
5.31% S.
(20R)-7-Oxo-7a-h om o-5α-ch olestan -3β-yl Meth an esulfon ate (7)
A solution of alcoh ol 5 (1 g, 2.40 m m ol) in pyridin e (15 m l) was treated with m eth an e-
sulfon yl ch loride (1.5 m l) an d th e solution was allowed to stan d for 2 h . Th e reaction m ix-
ture was decom posed with ice an d water, th e product taken up in to eth er an d th e eth ereal
solution worked up as usual. Th e evaporated residue was dissolved in 2 m l of ch loroform
an d eth er was added to precipitate crystallin e product 7 (1.1 g, 2.22 m m ol; 93%). An alytical
sam ple was crystallized from m eth an ol: m .p. 143–144 °C, [α]D –30 (c 0.900), givin g iden tical
ph ysical con stan ts, as described in th e literature12. IR: 1705 (C=O); 1367, 1344, 1176, 938,
598, 528 (SO2). 1H NMR: 0.661 s, 3 H (3 × H-18); 0.816 s, 3 H (3 × H-19); 0.861 d, 6 H, J =
6.1 (3 × H-26 an d 3 × H-27); 0.908 d, 3 H, J = 6.5 (3 × H-21); 3.010 s, 3 H (CH3 (Mes));
4.68 m , 1 H, W1/2 = 24.5 (H-3α). EI MS, m/z: 494 (M+), 398 (M+ – m esylate), 383 (398 –
CH3). For C29H50O4S (494.8) calculated: 70.40% C, 10.19% H, 6.48% S; foun d: 69.20% C,
10.13% H, 6.70% S.
(20R)-7a-Hom o-5α-ch olest-2-en -7-on e (8)
a) From (20R)-7-oxo-7a-homo-5α-cholestan-3β-yl methanesulfonate (7): Mesylate 7 was h eated
to reflux (700 m g, 1.41 m m ol) in sym -collidin e (15 m l) un der n itrogen atm osph ere for 2 h .
Sym -collidin e was th en distilled off un der reduced pressure, th e residue treated with water,
an d th e product taken up in to eth er. Usual work-up an d evaporation left 650 m g of a prod-
uct, wh ich was ch rom atograph ed on silica gel (100 g) in h eptan e–eth er (19:1). Th e fraction s
with a m ore polar com poun d were collected, an d th e solven t evaporated to afford 142 m g
(0.36 m m ol; 25%) of com poun d 8, wh ich was crystallized from m eth an ol: m .p. 108–109 °C,
[α]D –40 (c 0.920), givin g iden tical ph ysical con stan ts, as described in th e literature12. IR:
3023 (=CH); 1704 (C=O); 1666 (C=C); 1384 (i-Pr, Me); 1367 (i-Pr). 1H NMR: 0.683 s, 3 H (3 ×
H-18); 0.861 d, 3 H an d 0.865 d, 3 H, J = 6.1 (3 × H-26 an d 3 × H-27); 0.907 s, 3 H (3 ×
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H-19); 0.910 d, 3 H, J = 6.4 (3 × H-21); 5.58 m , 2 H (H-2 an d H-3). EI MS, m/z: 398 (M+), 383
(M+ – CH3). For C28H46O (398.7) calculated: 84.36% C, 11.63% H; foun d: 83.34% C, 11.64% H.
b) From (20R)-7-oxo-7a-homo-5α-cholestan-3β-yl toluenesulfonate (6): Tosylate 6 (800 m g,
1.40 m m ol) in sym -collidin e (15 m l) was treated in th e sam e way, as given above un der a).
Usual work-up an d evaporation left 640 m g of a product, wh ich was ch rom atograph ed on a
silica gel (100 g) in h eptan e–eth er (19:1). Th e fraction s with a m ore polar com pon en t af-
forded 140 m g (0.35 m m ol; 25%) of olefin 8, iden tical in all respects with th e com poun d
obtain ed above un der a).
c) From (20R)-3β-chloro-7a-homo-5α-cholestan-7-one (10) and (20R)-3α-chloro-7a-homo-5α-
cholestan-7-one (11): Lith ium brom ide (65 m g, 0.75 m m ol) an d pyridin ium 4-toluen e-
sulfon ate (5.5 m g, 0.028 m m ol) were added to a m ixture of ch lorides 10 an d 11 (60 m g,
0.14 m m ol) in N,N-dim eth ylacetam ide (2 m l) an d th e m ixture was h eated at 160 °C for
2.5 h . After coolin g, th e reaction m ixture was poured in to water an d extracted with eth er.
Th e eth ereal extract was worked up in th e usual m an n er to give 52 m g (0.13 m m ol; 92%)
of a m ixture of olefin s 8 an d 9. Preparative TLC on 4 plates in ligh t petroleum –eth er (7:3)
afforded 15 m g (0.04 m m ol; 29%) of isom er 8, iden tical in all respects with th e com poun d
obtain ed above un der a).
(20R)-7a-Hom o-5α-ch olest-3-en -7-on e (9)
a) From (20R)-7-oxo-7a-homo-5α-cholestan-3β-yl methanesulfonate (7): Th e layers with
a
m ore lipoph ilic com poun d from th e preparation of 8 un der a) afforded 380 m g (0.80 m m ol;
57%) of olefin 9, wh ich was crystallized from m eth an ol: m .p. 70–72 °C, [α]D +27 (c 0.810),
givin g iden tical ph ysical con stan ts, as described in th e literature12. IR: 3028 (=CH); 1704
(C=O); 1384 (i-Pr, Me); 1367 (i-Pr). 1H NMR: 0.665 s, 3 H an d 0.676 s, 3 H (3 × H-18 an d 3 ×
H-19); 0.861 d, 3 H an d 0.865 d, 3 H, J = 6.4 (3 × H-26 an d 3 × H-27); 0.896 d, 3 H, J = 6.4
(3 × H-21); 5.20 dd, 1 H, J(4,3) = 8.7, J(4,5α) = 1.2 (H-4); 5.73 m , 1 H, W1/2 = 16.2 (H-3). EI
MS, m/z: 398 (M+), 383 (M – CH3). For C28H46O (398.7) calculated: 84.36% C, 11.63% H;
foun d: 83.99% C, 11.71% H.
b) From (20R)-7-oxo-7a-homo-5α-cholestan-3β-yl toluenesulfonate (6): Work-up of th e frac-
tion s with a m ore lipoph ilic com pon en t from th e preparation of 8 un der b) afforded 355 m g
(0.89 m m ol; 64%) of olefin 9, iden tical in all respects with th e com poun d obtain ed above
un der a).
c) From (20R)-3β-chloro-7a-homo-5α-cholestan-7-one (10) and (20R)-3α-chloro-7a-homo-5α-
cholestan-7-one (11): Work-up of th e preparative TLC zon es with a m ore lipoph ilic com -
poun d from th e syn th esis of 8 un der c) afforded 34 m g (0.08 m m ol; 57%) of isom er 9, iden -
tical in all respects with th e com poun d obtain ed above un der a).
(20R)-3β-Ch loro-7a-h om o-5α-ch olestan -7-on e (10)
Work-up of th e m oth er liquor (100 m g) from th e crystallization of th e crude residue from
th e syn th esis of tosylate 6 afforded, in addition to a furth er am oun t of tosylate 6, a m ixture
of two ch loro com poun ds 10 an d 11, wh ich were separated by preparative TLC on 4 plates
in ligh t petroleum –eth er (7:3). Work-up of th e layers with a m ore lipoph ilic com poun d af-
forded 39 m g (0.09 m m ol; 6%) of ch loride 10. 1H NMR: 0.656 s, 3 H (3 × H-18); 0.823 s, 3 H
(3 × H-19); 0.857 d, 6 H, J = 6.4 (3 × H-26 an d 3 × H-27); 0.877 d, 3 H, J = 6.4 (3 × H-21);
3.79 m , 1 H, W 1/ 2 = 24.4 (H-3α). EI MS, m/z: 434 an d 436 (M+), 398 (M+ – HCl). For
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2185
C28H47ClO (435.1) calculated: 77.29% C, 10.89% H, 8.15% Cl; foun d: 77.01% C, 10.71% H,
7.73% Cl.
(20R)-3α-Ch loro-7a-h om o-5α-ch olestan -7-on e (11)
Work-up of th e TLC plates from th e above experim en t with a m ore polar com poun d af-
forded 27 m g (0.06 m m ol; 4%) of ch loride 11. 1H NMR: 0.665 s, 3 H (3 × H-18); 0.778 s, 3 H
(3 × H-19); 0.863 d, 6 H, J = 6.4 (3 × H-26 an d 3 × H-27); 0.896 d, 3 H, J = 6.7 (3 × H-21);
4.46 m , 1 H, W 1/ 2 = 7.3 (H-3β). EI MS, m/z: 434 an d 436 (M+), 398 (M+ – HCl). For
C
28H47ClO (435.1) calculated: 77.29% C, 10.89% H, 8.15% Cl; foun d: 77.12% C, 10.76% H,
7.74% Cl.
(20R)-2α,3α-Dih ydroxy-7a-h om o-5α-ch olestan -7-on e (12)
A solution of osm ium tetroxide (12 m g, 0.05 m m ol) in 2-m eth ylpropan -2-ol (0.12 m l) was
added to a solution of alken e 8 (234 m g, 0.59 m m ol) in aceton e (12 m l). N-Meth yl-
m orph olin e N-oxide (234 m g, 2.00 m m ol) was added an d th e m ixture was stirred un der n i-
trogen for 5 h . A solution of sodium sulfite (5 m l; 10%) was th en added an d th e m ixture
was stirred for 30 m in , poured in to water, extracted with ch loroform an d th e extract worked
up as usual. Th e residue (212 m g) was purified by preparative TLC on 12 plates in CHCl3–
propan -2-ol–dieth yl eth er (14:1:5). Work-up of th e zon es with a m ore polar com poun d
afforded 106 m g (0.25 m m ol; 42%) of (20R)-2α,3α-dih ydroxy-7a-h om o-5α-ch olestan -7-on e
(12): m .p. 142–144 °C, [α]D –27 (c 0.300). IR: 3387, 1079, 1047, 1021 (OH); 1702 (C=O);
1383, 1367 (i-Pr); 1378 (Me). 1H NMR: 0.662 s, 3 H (3 × H-18); 0.806 s, 3 H (3 × H-19);
0.865 d, 6 H, J = 6.4 (3 × H-26 an d 3 × H-27); 0.894 d, 3 H, J = 6.4 (3 × H-21); 3.66 m , 1 H,
W1/2 = 21.5 (H-2β); 3.93 m , 1 H, W1/2 = 7.4 (H-3β). FAB MS, m/z: 433 (M+ + H), 415 (433 –
H2O). For C28H48O3 (432.7) calculated: 77.33% C, 11.18% H; foun d: 76.99% C, 10.81% H.
(20R)-2β,3β-Dih ydroxy-7a-h om o-5α-ch olestan -7-on e (13)
Work-up of th e zon es of th e preparative TLC plates from th e above experim en t with a m ore
lipoph ilic com poun d afforded 22 m g (0.05 m m ol; 9%) of 2β,3β-diol 13: m .p. 154–155 °C,
[α]D –19 (c 0.057). IR: 3617, 3573, 3457, 1038, 1053 (OH); 1694 (C=O); 1384, 1367 (Me,
i-Pr). 1H NMR: 0.663 s, 3 H (3 × H-18); 0.859 d, 3 H an d 0.863 d, 3 H, J = 6.6 (3 × H-26 an d
3 × H-27); 0.893 d, 3 H, J = 6.2 (3 × H-21); 0.907 s, 3 H (3 × H-19); 3.64 m , 1 H, W1/2 = 19.6
(H-2β); 4.00 m , 1 H, W1/2 = 8.5 (H-3β). EI MS, m/z: 432 (M+), 414 (433 – H2O). For C28H48O3
(432.7) calculated: 77.33% C, 11.18% H; foun d: 76.96% C, 11.25% H.
(20R)-3α,4α-Dih ydroxy-7a-h om o-5α-ch olestan -7-on e (14)
A solution of osm ium tetroxide (15 m g, 0.06 m m ol) in 2-m eth ylpropan -2-ol (0.15 m l) was
added to a solution of alken e 9 (305 m g, 0.71 m m ol) in aceton e (15 m l). N-Meth yl-
m orph olin e N-oxide (305 m g, 2.60 m m ol) was th en added an d th e m ixture was stirred un -
der n itrogen for 5 h . A solution of sodium sulfite (5 m l; 10%) was added an d th e m ixture
was stirred for 30 m in , poured in to water, extracted with ch loroform an d th e extract worked
up as usual. Th e crystallin e residue (290 m g, 0.67 m m ol; 94%) con tain ed on ly 3α,4α-isom er
14 (TLC). An alytical sam ple was crystallized from m eth an ol: m .p. 146–148 °C, [α]D –52
(c 0.283). IR: 3399, 1027, 1017 (OH); 1700 (C=O); 1383, 1367 (i-Pr); 1377 (Me). 1H NMR:
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0.659 s, 3 H (3 × H-18); 0.799 s, 3 H (3 × H-19); 0.860 d, 6 H, J = 6.5 (3 × H-26 an d 3 ×
H-27); 0.888 d, 3 H, J = 6.1 (3 × H-21); 3.00 dd, 1 H, J(5α,4β) = 3.0, J(5α,6α) = 17.9 (H-5α);
3.29 dd, 1 H, J(5α,4β) = 3.0, J(5α,6α) = 10.1 (H-4β); 3.95 m , 1 H, W1/2 = 8.0 (H-3β). EI MS,
m/z: 432 (M+), 414 (432 – H2O). For C28H48O3 (432.7) calculated: 77.33% C, 11.18% H;
foun d: 77.01% C, 10.96% H.
(20R)-2α,3α-Dih ydroxy-7a-oxa-7a,7b-dih om o-5α-ch olestan -7-on e (15)
A solution of trifluoroperoxyacetic acid, fresh ly prepared from trifluoroacetic an h ydride
(650 m g, 3.10 m m ol) an d h ydrogen peroxide (30%; 110 m g) in dich lorom eth an e (4 m l), was
added to a solution of diol 12 (200 m g, 0.46 m m ol) in dich lorom eth an e (10 m l). After
stan din g at room tem perature for 2 h , th e reaction m ixture was poured in to water an d taken
up in ch loroform . Th e ch loroform extract was wash ed with water, saturated solution of po-
tassium h ydrogen carbon ate, water, an d dried over an h ydrous sodium sulfate. Rem oval of
th e solven t gave 180 m g of a residue. Purification by preparative TLC on 12 plates in
CHCl3–propan -2-ol–dieth yl eth er (14:1:5) afforded 87 m g (0.19 m m ol; 42%) of product 15:
m .p. 230–203 °C, [α]D –11 (c 0.700). IR: 3556, 3515, 3462, 3438, 1042 (OH); 1740, 1711
(C=O); 1385, 1366 (i-Pr, Me). 1H an d 13C NMR data, see Table II. FAB MS, m/z: 471 (M+
+
Na), 449 (M+ + H), 431 (449 – H2O), 413 (431 – CO). For C28H48O4 (448.7) calculated:
74.95% C, 10.78% H; foun d: 73.75% C, 10.66% H.
(20R)-3α,4α-Dih ydroxy-7a-oxa-7a,7b-dih om o-5α-ch olestan -7-on e (16)
A solution of trifluoroperoxyacetic acid, fresh ly prepared from trifluoroacetic an h ydride
(650 m g, 3.10 m m ol) an d h ydrogen peroxide (30%; 110 m g) in dich lorom eth an e (4 m l), was
added to a solution of diol 14 (200 m g, 0.46 m m ol) in dich lorom eth an e (10 m l). After
stan din g at room tem perature for 2 h , th e reaction m ixture was poured in to water an d taken
up in ch loroform . Th e ch loroform extract was wash ed with water, saturated solution of po-
tassium h ydrogen carbon ate, water, an d dried over sodium sulfate. Rem oval of th e solven t
gave 185 m g of a residue. Preparative TLC on 12 plates in CHCl3–propan -2-ol–dieth yl eth er
(14:1:5) afforded 90 m g (0.19 m m ol; 44%) of product 16: m .p. 224–227 °C, [α]D –8 (c 1.120).
IR: 3461, 3354, 1054, 1036, 1011 (OH); 1719 (C=O); 1383, 1371, 1367 (i-Pr, Me). 1H an d
13C NMR data, see Table II. FAB MS, m/z: 471 (M+ + Na), 413 ((M+ + H) – H2O – CO). For
C
28H48O4 (448.7) calculated: 74.95% C, 10.78% H; foun d: 74.15% C, 10.84% H.
(20R)-7a-Hom o-5α-ch olestan -3β-ol (17)
Keton e 4 (100 m g, 0.22 m m ol) was treated with h ydrazin e h ydrate (5 m l; 90%) an d th e m ix-
ture h eated un der reflux for 2 h (un til th e disappearan ce of th e startin g m aterial on TLC).
Th e m ixture was th en cooled to room tem perature, an d eth ylen e glycol (5 m l) an d potas-
sium h ydroxide (250 m g) were added. Th e reaction m ixture was h eated carefully to 195 °C
(with out reflux in order to rem ove h ydrazin e h ydrate) an d th en at reflux for 2 h (un til th e
decom position of th e h ydrazon e was apparen t on TLC). Th e reaction m ixture was poured
in to a saturated sodium ch loride solution (40 m l) an d th e product was extracted with di-
eth yl eth er. Usual work-up gave a residue (88 m g), wh ich was purified by preparative TLC
on 4 plates in ligh t petroleum –eth er (1:1) to afford 75 m g (19 m m ol; 85%) of com poun d 17.
An alytical sam ple was crystallized from aceton e: m .p. 89–91 °C, [α]D +25 (c 0.200), givin g
iden tical ph ysical con stan ts, as described in th e literature11. IR: 3620, 3350, 1043 (OH);
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2187
1383, 1367 (i-Pr, Me). 1H NMR: 0.658 s, 3 H (3 × H-18); 0.826 s, 3 H (3 × H-19); 0.861 d, 3 H
an d 0.864 d, 3 H, J = 6.7 (3 × H-26 an d 3 × H-27); 0.894 d, 3 H, J = 6.7 (3 × H-21); 3.52 m ,
1 H, W 1/2 = 24.3 (H-3α). EI MS, m/z: 402 (M+), 384 (402 – H2O), 369 (384 – Me). For
C
28H50O (402.7) calculated: 83.51% C, 12.51% H; foun d: 82.34% C, 12.68% H.
(20R)-7a-Hom o-5α-ch olestan -3β-yl Toluen esulfon ate (18)
A solution of alcoh ol 17 (90 m g, 0.22 m m ol) in pyridin e (1 m l) was treated with 4-toluen e-
sulfon yl ch loride (90 m g, 0.52 m m ol) an d th en allowed to stan d at room tem perature over-
n igh t. Th e reaction m ixture was decom posed with ice an d water, th e product was taken up
in to eth er an d th e eth ereal solution was worked up as usual to afford 118 m g (0.21 m m ol;
96%) of tosylate 18: m .p. 88–90 °C, [α]D +22 (c 0.318). IR: 3068, 3032 (=CH); 1370, 1179,
558, 557 (SO2); 1600, 1496, 1306, 1290, 2111, 1189, 1119, 1100, 1021, 832, 815, 706, 669,
458 (arom atic); 1039 (Me). 1H NMR: 0.633 s, 3 H (3 × H-18); 0.790 s, 3 H (3 × H-19);
0.855 d, 3 H an d 0.859 d, 3 H, J = 6.6 (3 × H-26 an d 3 × H-27); 0.880 d, 3 H, J = 6.4 (3 ×
H-21); 2.44 s, 3 H (CH3 (Tos)); 4.35 m , 1 H, W1/2 = 25.1 (H-3α); 7.32 m , 2 H an d 7.79 m ,
2 H (C6H4 (Tos)). EI MS, m/z: 556 (M+), 384 (M+ – TosOH), 369 (384 – Me). For C35H54O4S
(556.9) calculated: 75.49% C, 10.14% H, 5.76% S; foun d: 75.60% C, 9.94% H, 5.14% S.
(20R)-7a-Hom o-5α-ch olest-2-en e (19) an d (20R)-7a-Hom o-5α-ch olest-3-en e (20)
Tosylate 18 (110 m g, 0.20 m m ol) was h eated in sym -collidin e (5 m l) un der n itrogen atm o-
sph ere for 2 h . Sym -collidin e was th en distilled off un der reduced pressure, th e residue
treated with water, an d th e product was taken up in to eth er. Usual work-up an d evaporation
left 85 m g of a product, con tain in g a m ixture of two isom eric olefin s 19 an d 20, wh ich were
in separable due to th eir practically iden tical polarity.
(20R)-7a-Hom o-5α-ch olestan -2α,3α-diol (21)
a) From (20R)-2α,3α-dihydroxy-7a-homo-5α-cholestan-7-one (12): A solution of keton e 12
(40 m g, 0.09 m m ol) was treated with h ydrazin e h ydrate (5 m l; 90%) an d th en h eated un der
reflux for 2 h (un til th e disappearan ce of th e startin g m aterial on TLC). Th e m ixture was
th en cooled to room tem perature, an d eth ylen e glycol (5 m l) an d potassium h ydroxide
(150 m g) were added. Th e reaction m ixture was h eated carefully to 195 °C (with out reflux in
order to rem ove h ydrazin e h ydrate) an d th en at reflux for 2 h (un til TLC m on itorin g
sh owed a com plete decom position of th e h ydrazon e). Th e reaction m ixture was poured in to
a saturated sodium ch loride solution (20 m l) an d th e product was isolated with dieth yl
eth er. Usual work-up an d evaporation gave a residue (37 m g), wh ich was purified by pre-
parative TLC on 4 plates in ligh t petroleum –eth er (1:1) to afford 30 m g (0.07 m m ol; 78%) of
diol 21. An alytical sam ple was crystallized from aceton e: m .p. 135–137 °C, [α]D +50.2
(c 0.215). IR: 3625, 3585, 3391, 1052, 1020 (OH); 1384, 1367 (i-Pr, Me). 1H NMR: 0.657 s,
3 H (3 × H-18); 0.844 s, 3 H (3 × H-19); 0.862 d, 6 H, J = 6.5 (3 × H-26 an d 3 × H-27);
0.898 d, 3 H, J = 6.6 (3 × H-21); 3.65 m , 1 H, W1/2 = 21.8 (H-2β); 3.89 m , 1 H, W1/2 = 7.3
(H-3β). FAB MS, m/z: 401 [α]D (M+ + H) – H2O], 383 (401 – H2O). For C28H50O2 (418.7) cal-
culated: 80.32% C, 12.04% H; foun d: 80.02% C, 12.06% H.
b) From a mixture of (20R)-7a-homo-5α-cholest-2-ene (19) and (20R)-7a-homo-5α-cholest-3-ene
(20): A solution of osm ium tetroxide (4 m g, 0.02 m m ol) in 2-m eth ylpropan -2-ol (0.04 m l)
was added to a solution of alken es 19 an d 20 (85 m g, 0.20 m m ol) in aceton e (4 m l).
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

2188
Šíša, Buděšínský, Kohout:
N-Meth ylm orph olin e N-oxide (85 m g, 0.73 m m ol) was added an d th e m ixture was stirred
un der n itrogen for 5 h . A solution of sodium sulfite (2 m l; 10%) was th en added an d th e
m ixture stirred for 30 m in , poured in to water, extracted with ch loroform an d th e extract
was worked up as usual. Th e sem icrystallin e residue (76 m g, 0.18 m m ol; 91%) con tain ed
m ain ly two diols, 21 an d 22. Th is residue was purified by preparative TLC on 4 plates in
CHCl3–propan -2-ol–dieth yl eth er (14:1:5). Work-up of th e zon es with a m ore polar com -
poun d afforded 27 m g (0.07 m m ol; 32%) of (20R)-7a-h om o-5α-ch olestan -2α,3α-diol (21).
(20R)-7a-Hom o-5α-ch olestan -3α,4α-diol (22)
a) From (20R)-3α,4α-dihydroxy-7a-homo-5α-cholestan-7-one (14): Th e solution of dih ydroxy
keton e 14 (40 m g, 0.09 m m ol) was treated with h ydrazin e h ydrate (5 m l; 90%) an d th en
h eated at reflux for 2 h (un til th e disappearan ce of th e startin g m aterial on TLC). Th e m ix-
ture was th en cooled to room tem perature an d eth ylen e glycol (5 m l) an d potassium h y-
droxide (150 m g) were added. Th e reaction m ixture was h eated carefully to 195 °C (with out
reflux in order to rem ove h ydrazin e h ydrate) an d th en at reflux for 2 h (un til th e decom po-
sition of th e h ydrazon e was in dicated by TLC). Th e reaction m ixture was poured in to a satu-
rated sodium ch loride solution (20 m l) an d th e product was extracted with dieth yl eth er.
Usual work-up an d evaporation gave a residue (38 m g), wh ich was purified by preparative
TLC on 2 plates in ligh t petroleum –eth er (1:1) to afford 30 m g (0.07 m m ol; 78%) of diol 22.
An alytical sam ple was crystallized from aceton e: m .p. 86–90 °C, [α]D +6 (c 1.001). IR (KBr):
3619, 3567 (OH). 1H NMR: 0.655 s, 3 H (3 × H-18); 0.835 s, 3 H (3 × H-19); 0.861 d, 6 H, J =
7.0 (3 × H-26 an d 3 × H-27); 0.895 d, 3 H, J = 6.4 (3 × H-21); 3.34 dd, 1 H, J(4β,5α) = 10.2,
J(4β,3β) = 3.5 (H-4β); 3.93 m , 1 H, W1/2 = 12.6 (H-3β). EI MS, m/z: 418 (M+), 400 (418 –
H2O). For C28H50O2 (418.7) calculated: 80.32% C, 12.04% H; foun d: 80.18% C, 12.35% H.
b) From a mixture of (20R)-7a-homo-5α-cholest-2-ene (19) and (20R)-7a-homo-5α-cholest-3-ene
(20): Work-up of th e correspon din g zon es with a m ore lipoph ilic com poun d from th e purifi-
cation of com poun d 21 un der b) afforded 40 m g (0.10 m m ol; 48%) of 3α,4α-diol 22, iden ti-
cal in all respects with com poun d 22 obtain ed un der a).
(20R)-7-Oxa-7a-h om o-5α-ch olestan -2α,3α-diol (24)
A solution of (20R)-2α,3α-dih ydroxy-7-oxa-7a-h om o-5α-ch olestan -6-on e14 (23) (100 m g,
0.23 m m ol) in boron trifluoride eth erate (2.5 m l) was dropped at 0 °C over 10 m in to a solu-
tion of sodium boroh ydride (150 m g) in diglym e (2.5 m l)13. Th e reaction m ixture was
allowed to stan d at 0 °C for 2 h , an d th en decom posed with potassium carbon ate. Th e reac-
tion m ixture was poured in to water, th e product extracted with eth er an d th e eth ereal solu-
tion was worked up as usual. Evaporation afforded 80 m g of a residue, wh ich was purified
by preparative TLC on 8 plates in toluen e–eth er (1:1) to afford 45 m g (0.11 m m ol; 48%) of
product 24 (m ore lipoph ilic) an d 20 m g (0.05 m m ol; 20%) of th e startin g com poun d 23:
m .p. 145–147 °C, [α]D +45 (c 0.259). IR: 3379, 1056, 1047, 1032, 1022 (OH); 1142, 1130
(COC); 1383, 1377, 1367 (i-Pr, Me). 1H NMR: 0.693 s, 3 H (3 × H-18); 0.862 d, 6 H, J = 6.7 (3 ×
H-26 an d 3 × H-27); 0.897 d, 3 H, J = 7.0 (3 × H-21); 0.976 s, 3 H (3 × H-19); 3.17–3.35 m ,
2 H (H-7aα an d H-7aβ); 3.62–3.75 m , 3 H (H-6α, H-6β an d H-2β); 3.94 m , 1 H, W1/2 = 8.8
(H-3β). EI MS, m/z: 420 (M+), 405 (420 – Me), 402 (420 – H2O). For C28H48O3 (420.7) calcu-
lated: 77.09% C, 11.50% H; foun d: 75.54% C, 11.62% H.
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2189
The authors are indebted to Dr P. Fiedler for recording and interpretation of IR spectra. Our thanks
are due to Ms M. Snopková and Dr R. Pohl for the measurements of 1H NMR spectra on the 200 MHz
instrument. Mass spectra were measured by Dr P. Loudová of the Laboratory of Mass Spectrometry.
Elemental analyses were done and optical rotations measured by Dr L. Šuranská and Dr V. Pechanec
under the direction of Dr Holasová of the Analytical laboratory. Bean second internod bioassays were
done by Ms J. Balonová and Ms M. Šubová under the direction of Prof. M. Strnad (Institute of Experi-
mental Botany, Academy of Sciences of the Czech Republic, Olomouc, Czech Republic). The technical
assistance of Ms J. Neumannová is gratefully acknowledged. This work was supported by grants No.
203/01/0083, No. A4055204 and No. Z4 055905.
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