7-OXO-7a-OXA-BRASSINOSTEROIDS WITH CHOLESTANE SIDE-CHAIN

Article abstract of DOI:10.1135/cccc19941219

New 7-oxa-7a-oxa analogs of brassinolide with cholestane side-chain were prepared.Analogs VIII and IX with 2,3-diol grouping exhibit a weak brassinolide activity.Of the synthesized compounds, the highest brassinolide activity was found for 3α,4α-dihydroxy-7a-oxa-B-homo-5α-cholestan-7-one (X).

Full text of DOI:10.1135/cccc19941219

On Steroids
1219
7-OXO-7a-OXA-BRASSINOSTEROIDS WITH CHOLESTANE SIDE-CHAIN*
Ladislav KOHOUT
Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic, 166 10 Prague 6, The Czech Republic
Received October 22, 1993
Accepted November 28, 1993
New 7-oxo-7a-oxa analogs of brassinolide with cholestane side-chain were prepared. Analogs VIII and
IX with 2,3-diol grouping exhibit a weak brassinolide activity. Of the synthesized compounds, the hig-
hest brassinolide activity was found for 3α,4α-dihydroxy-7a-oxa-B-homo-5α-cholestan-7-one (X).
In the present communication we describe the synthesis of the so far unknown 7-oxo-
7a-oxa analogs of the plant growth hormone brassinolide1,2.
The synthesis (Scheme 1) starts from 3β-hydroxy-5α-cholestan-7-one3 (I). Com-
pound I was converted into tosylate II which on boiling in 2,4,6-collidine afforded a
mixture of two isomeric olefins. The desired more polar olefin, which was the main
reaction product, was identical4,5 with the known 2(3)-olefin III; consequently, the
more lipophilic olefin was the 3(4)-isomer IV. Olefin III was hydroxylated with os-
mium tetroxide in the presence of N-methylmorpholine N-oxide to give a mixture of
two diols V and VI which were separated by chromatography. Their structure was deter-
1
mined by H NMR spectroscopy. Comparison of chemical shifts of H-19 with the cal-
culated values (Table I) has shown that the lipophilic diol is 2β,3β-dihydroxy-
5α-cholestan-7-one (VI) whereas the polar diol is 2α,3α-dihydroxy-5α-cholestan-7-one
(V). When treated with trifluoroperacetic acid in dichloromethane, compound V affor-
ded only one product, although the reaction can give rise to two lactones: the 7-oxo-
1
7a-oxa and 7-oxa-7a-oxo compounds. For the former structure, the H NMR spectrum
would exhibit one CH−O−C proton signal whereas in the spectrum the 7-oxa-7a-oxo
lactone one would find two protons of the CH2−O−C type. In our case the spectrum
corresponded to the first alternative, showing one proton at 4.12 ppm as a doublet of
doublets, and the product thus was 2α,3α-dihydroxy-7a-oxa-B-homo-5α-cholestan-7-
one (VIII).
* Part CCCLXXV in the series On Steroids; Part CCCLXXIV: Collect. Czech. Chem. Commun. 59,
649 (1994).
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

1220
Kohout:
SCHEME 1
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

On Steroids
1221
Also compound VI reacted with trifluoroperacetic acid under formation of a single
lactone which was assigned the structure 2β,3β-dihydroxy-7a-oxa-B-homo-5α-choles-
tan-7-one (IX) on the basis of reasoning analogous to that above.
The 3(4)-olefin IV was also hydroxylated with osmium tetroxide in the presence of
N-methylmorpholine N-oxide to give diol VII as the sole product. In principle, both the
3α,4α- and 3β,4β-diols are possible, however, the former should be preferred for
stereochemical reasons. Indeed, an analysis of 1H NMR spectra confirmed this expecta-
tion (the calculated6 position of the H-19 signal is 1.33 ppm for the 3β,4β-diol and 1.08
ppm for the 3α,4α-diol; the value experimentally found is 1.07 ppm). Similarly to diols
V and VI, also the diol VII was subjected to Baeyer–Villiger reaction, affording 3α,4α-
dihydroxy-7a-oxa-B-homo-5α-cholestan-7-one (X).
An inspection of Dreiding models shows that the ring B in lactone X can exist in two
1
interconvertible conformations (Fig. 1). In the H NMR spectra, one of the H-6 protons
has (in addition to the geminal interaction) a zero coupling constant with the H-5α
proton, whereas for the second H-6 proton the coupling constant is high (J = 10.7 Hz).
As seen from Newman projection (Fig. 2), this observation is compatible with both
conformations: for the C(8)-chair form the coupling constants J(5α,6α) and J(5α,6β)
should amount to 10.7 Hz and 0, respectively; for the C(7)-twist chair form the zero
coupling constant should correspond to J(5α,6α) and the value of 10.7 Hz to J(5α,6β).
Decisive in this respect was a NOE experiment which proved steric interaction of H-19
with the proton resonating at 2.55 ppm. This proton is 6β and axial and therefore the
B-ring in compound X exists in conformation TC7.
Thus, by the described procedures we obtained three analogs of brassinolide (com-
pounds VIII, IX and X) and three analogs of castasterone (compounds V, VI and VII).
According to preliminary bean second internode bioassays7, all the compounds de-
scribed exhibit a weak brassinolide activity. Surprisingly, compound X was the most
potent.
TABLE I
1
Chemical shifts (ppm, δ-scale) of H-19 in H NMR spectra of diols V and VI
H-19
Compound
Calculateda
Found
2α, 3α-Dihydroxy-5α-cholestan-7-one (V)
2β, 3β-Dihydroxy-5α-cholestan-7-one (VI)
1.08
1.35
1.07
1.28
a
According to ref.6.
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

1222
Kohout:
EXPERIMENTAL
The melting points were determined on a Kofler block and are uncorrected. Infrared spectra were
recorded on a Perkin–Elmer PE 580 spectrometer in tetrachloromethane (unless stated otherwise),
wavenumbers are given in cm−1. Proton NMR spectra were taken in deuteriochloroform on a Varian
XL-200 (FT mode, 200 MHz) instrument with tetramethylsilane as internal reference. Chemical shifts
are given in ppm (δ-scale), coupling constants (J) and multiplet half-widths (W1/2) in Hz. The data
were interpreted as the first-order spectra. Mass spectra were obtained with a ZAB-EG spectrometer
at 70 eV. The identity of the samples prepared 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. The “usual work-up” of a solu-
tion denotes washing with water, 5% aqueous potassium hydrogen carbonate, water, drying over so-
dium sulfate, filtering and evaporating the solvent to dryness in vacuo. The light petroleum used was
a fraction boiling at 40 – 62 °C.
FIG. 1
Possible conformations of 3α,4α-dihydroxy-7a-oxa-B-homo-5α -cholestan-7-one (X): a C(8)-chair,
b C(7)-twist chair
FIG. 2
Newman projection along the C(6)−C(5) bond in 3α,4α-dihydroxy-7a-oxa-B-homo-5α-cholestan-7-
one (X): a C(8)-chair, b C(7)-twist chair
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

On Steroids
1223
3β-Tosyloxy-5α-cholestan-7-one (II)
p-Toluenesulfonyl chloride (5.0 g, 26.3 mmol) was added to a solution of 3β-hydroxy-5α-cholestan-
7-one4 (I; 5.0 g, 12.42 mmol) in pyridine (25 ml). After standing at room temperature overnight, the
mixture was poured on ice, extracted with chloroform and worked up in the usual manner. Crystal-
lization of the crude product from chloroform–ether afforded 2.1 g (56%) of tosylate II, m.p.
131 – 134 °C (decomposition). For C34H52O3S (540.9) calculated: 75.51% C, 9.69% H, 5.93% S;
found: 75.12% C, 9.45% H, 5.50% S.
5α-Cholest-2-en-7-one (III)
The chromatography described in the preparation of olefin IV (vide infra) was continued by elution
with benzene–light petroleum (1 : 4) and the eluted crude more polar olefin (3.7 g) was crystallized
from methanol–acetone to give 1.6 g of product. Mother liquors on evaporation and crystallization
from the same solvent mixture afforded further amount (0.7 g) of the product, raising the total yield
to 2.3 g (48%). The product melted at 96 – 99 °C, in accord with the reported5 value of 98 – 100 °C
1
(an earlier reference4 reported m.p. 156 – 158 °C). H NMR spectrum: 0.67 s, 3 H (3 × H-18); 1.02 s, 3 H
(3 × H-19); 0.86 d, 6 H (3 × H-26 and 3 × H-27, J = 6.5); 0.91 d, 3 H (3 × H-21, J = 6.5); 5.60 m, 2 H
(H-2 and H-3, W1/2 = 6). IR spectrum: 3 066, 3 026, 1 655 (C=C); 1 711 and 1 438 (CH2C=O). For
C27H44O (384.6) calculated: 84.31% C, 11.53% H; found: 84.44% C, 11.49% H.
5α-Cholest-3-en-7-one (IV)
A solution of tosylate II (5.0 g, 8.98 mmol) in 2,4,6-collidine (20 ml) was heated at 150 °C for 8 h.
After pouring into water, the reaction mixture was extracted with ether and worked up as usual. The
obtained mixture of two olefins (shown by TLC to contain majority of the more polar III) was sub-
jected to chromatography on silica gel (420 g) in light petroleum–benzene (2 : 1). Fractions contain-
ing the lipophilic olefin afforded 520 mg of residue which was crystallized from methanol to give
315 mg (9%) of crystalline olefin IV, m.p. 86 – 88 °C. Mass spectrum, m/z: 384 (M), 366 (M − H2O).
1H NMR spectrum: 0.67 s, 3 H (3 × H-18); 1.05 s, 3 H (3 × H-19); 0.86 d, 6 H (3 × H-26 and
3 × H-27, J = 6.5); 0.92 d, 3 H (3 × H-21, J = 6.5); 5.24 d, 1 H (H-4, J = 5); 5.64 dm, 1 H
(H-3, J = 5). IR spectrum: 3 055, 3 022, 1 640, 1 652 sh (C=C); 1 711 and 1 432 (CH2C=O). For
C27H44O (384.6) calculated: 84.31% C, 11.53% H; found: 84.37% C, 11.52% H.
2α,3α-Dihydroxy-5α-cholestan-7-one (V)
Osmium tetroxide (48 mg, 0.19 mmol) in 2-methyl-2-propanol (0.48 ml) was added to a solution of
olefin III (960 mg, 2.5 mmol) in acetone (48 ml). After addition of N-methylmorpholine N-oxide
(960 mg, 8.2 mmol), the reaction mixture was stirred in a nitrogen atmosphere at room temperature
for 5 h and then left overnight. A solution of sodium sulfite (10%) was added, the mixture was
stirred for 30 min and poured into water. The product was extracted with chloroform and worked up
in the usual manner. The residue (910 mg) was crystallized from ethanol to give pure 2α,3α-diol V
(330 mg, 32%), m.p. 217 – 219 °C. 1H NMR spectrum: 0.65 s, 3 H (3 × H-18); 1.07 s, 3 H
(3 × H-19); 0.86 d, 6 H (3 × H-26 and 3 × H-27, J = 6); 0.91 d, 3 H (3 × H-21, J = 6.5);
3.80 m, 1 H (H-2β, W1/2 = 22); 4.00 m, 1 H (H-3β, W1/2 = 5). IR spectrum: 3 633, 3 577, 3 332, 1 037
(O−H); 1 710 (C=O). For C27H46O3 (418.7) calculated: 77.46% C, 11.07% H; found: 77.37% C,
11.07% H.
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

1224
Kohout:
2β,3β-Dihydroxy-5α-cholestan-7-one (VI)
Mother liquors from crystallization in the preceding experiment were evaporated to dryness and the
residue was column chromatographed on silica gel in chloroform–2-propanol (199 : 1 to 33 : 1).
Fractions containing the lipophilic compound afforded 86 mg (9%) of diol VI, m.p. 207 – 208 °C
1
(ethanol). H NMR spectrum: 0.64 s, 3 H (3 × H-18); 1.28 s, 3 H (3 × H-19); 0.86 d, 6 H (3 × H-26
and 3 × H-27, J = 6); 0.91 d, 3 H (3 × H-21, J = 6.5); 3.64 m, 1 H (H-3α, W1/2 = 21); 4.04 d, 1 H
(H-2α, J = 3, W1/2 = 7.5). IR spectrum: 3 629, 3 577 (O−H); 1 711 (C=O). For C27H46O3 (418.7)
calculated: 77.46% C, 11.07% H; found: 77.37% C, 11.02% H.
3α,4α-Dihydroxy-5α-cholestan-7-one (VII)
Osmium tetroxide (158.4 mg, 0.83 mmol) and N-methylmorpholine N-oxide (3.15 g, 27.0 mmol)
were added to a solution of olefin IV (3.15 g; 8.2 mmol) in acetone (157 ml) and the mixture was
stirred at room temperature for 28 h. A solution of sodium sulfite (10%, 5 ml) was added, the mix-
ture was stirred for 30 min and then poured into water. The product was taken up in chloroform,
isolated as usual and the residue (3.25 g) was chromatographed on a silica gel column, using success-
ively light petroleum–ether (1 : 1), light petroleum–ether–chloroform (1 : 2 : 1) and finally chloro-
form–ether (1 : 1) as eluents. Crystallization of the product (2.76 g) from ethanol afforded 1.1 g (40%)
1
of diol VII, m.p. 228 – 230 °C. Mass spectrum, m/z: 418 (M), 400 (M − H2O). H NMR spectrum:
0.65 s, 3 H (3 × H-18); 1.07 s, 3 H (3 × H-19); 0.86 d, 6 H (3 × H-26 and 3 × H-27, J = 6);
0.91 d, 3 H (3 × H-21, J = 6.5); 2.1 – 2.7 m, 3 H (H-8 and 2 × H-6); 3.51 dd, 1 H (H-4β, J = 2.2;
J′ = 10.8); 3.98 dm, 1 H (H-3β, W1/2 = 6.5). IR spectrum: 3 629, 3 621 sh, 3 564, 3 437, 1 070,
1 049, 1 030 (O−H); 1 702 (C=O). For C27H46O3 (418.7) calculated: 77.46% C, 11.07% H; found:
77.37% C, 11.02% H.
2α,3α-Dihydroxy-7a-oxa-B-homo-5α-cholestan-7-one (VIII)
A solution of trifluoroperacetic acid (freshly prepared from trifluoroperacetic anhydride (904 mg) and
50% hydrogen peroxide (0.147 ml) in dichloromethane (5 ml)) was added to a solution of diol V
(209.3 mg, 0.50 mmol) in dichloromethane (3 ml). After standing at room temperature for 4 h, the
reaction mixture was poured into water and the product was extracted with chloroform. The chloro-
form extract was washed with 10% sodium hydrogen carbonate and water, dried over sodium sulfate
and the solvent was distilled off in vacuo. The residue was purified by chromatography on a column
of silica gel (95 g) in chloroform–2-propanol (49 : 1). Crystallization of the obtained material
(165 mg) from aqueous ethanol afforded 49 mg (26%) of lactone VIII, m.p. 212 – 214 °C. Mass
1
spectrum, m/z: 434 (M), 416 (M − H2O), 398 (416 − H2O), 304 (M − H2O − C8H16). H NMR spec-
trum: 0.67 s, 3 H (3 × H-18); 0.99 s, 3 H (3 × H-19); 0.86 d, 6 H (3 × H-26 and 3 × H-27, J = 6);
0.91 d, 3 H (3 × H-21, J = 6); 3.64 m, 1 H (H-2β, W1/2 = 21); 3.95 m, 1 H (H-3β, W1/2 = 7.5);
4.12 dd, 1 H (H-8, J = 8; J′ = 10). IR spectrum: 3 608, 3 567, 1 034, 1 019 (O−H); 1 720, 1 156,
1 034, 1 019 (lactone). For C27H46O4 (434.7) calculated: 74.61% C, 10.67% H; found: 79.61% C,
10.85% H.
2β,3β-Dihydroxy-7a-oxa-B-homo-5α-cholestan-7-one (IX)
A solution of trifluoroperacetic acid (freshly prepared from trifluoroperacetic anhydride (3.72 g) and
50% hydrogen peroxide (0.6 ml) in dichloromethane (20.6 ml)) was added to a solution of diol VI
(0.86 g, 2.05 mmol) in dichloromethane (12.3 ml). After standing at room temperature for 20 h, the
mixture was worked up as described in the preceding experiment. The obtained residue (1.2 g) was
twice crystallized from aqueous ethanol; yield 0.45 g (51%) of lactone IX, m.p. 233 – 235 °C (ethanol).
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

On Steroids
1225
1
Mass spectrum, m/z: 434 (M), 416 (M − H2O), 398 (416 − H2O). H NMR spectrum: 0.67 s, 3 H
(3 × H-18); 1.18 s, 3 H (3 × H-19); 0.86 d, 6 H (3 × H-26 and 3 × H-27, J = 6); 0.90 d, 3 H (3 × H-21,
J = 6.5); 2.89 dd, 1 H (H-6, J = 10; J′ = 14.6); 3.65 m, 1 H (H-3α, W1/2 = 21); 3.99 d, 1 H (H-2α,
J = 2); 4.18 dd, 1 H (H-8, J = 8.5; J′ = 10). IR spectrum (chloroform): 3 620, 3 613 sh, 3 567, 3 435
(O−H); 1 719, 1 304, 1 153 (lactone). For C27H46O4 (434.7) calculated: 74.61% C, 10.67% H; found:
74.30% C, 10.15% H.
3α,4α-Dihydroxy-7a-oxa-B-homo-5α-cholestan-7-one (X)
A solution of trifluoroperacetic acid (freshly prepared from trifluoroperacetic anhydride (2.16 g) and
50% hydrogen peroxide (0.35 ml) in dichloromethane (12 ml)) was added to a solution of diol VII
(0.50 g, 1.19 mmol) in dichloromethane (7.15 ml). After standing at room temperature for 6 h, the
mixture was worked up as described in the preceding experiment. The residue (520 mg) was twice
crystallized from anhydrous ethanol; yield 169 mg (33%) of lactone X, m.p. 250 – 252 °C. Mass
1
spectrum, m/z: 434 (M), 416 (M − H2O), 398 (416 − H2O). H NMR spectrum: 0.67 s, 3 H (3 × H-18);
0.99 s, 3 H (3 × H-19); 0.861 d, 3 H and 0.864 d, 3 H (3 × H-26 and 3 × H-27, J = 6.5); 2.08 – 3.00 m,
2 H (2 × H-6); 3.37 dd, 1 H (H-4β, J = 11; J′ = 2.5), 4.01 m, 1 H (H-3β, W1/2 = 7.5); 4.24 dd, 1 H
(H-8, J = 9; J′ = 10.5). IR spectrum (chloroform): 3 611, 3 436, 1 052 (O−H); 1 719, 1 156, 1 306
(lactone). For C27H46O4 (434.7) calculated: 74.61% C, 10.67% H; found: 74.15% C, 10.35% H.
The author is indebted to Dr M. Strnad of the Institute of Experimental Botany, Olomouc, for prelimi-
nary bean second internode bioassays and to Dr J. Smolikova for interpretation of IR spectra measured
by Mrs K. Matouskova. Proton NMR spectra were recorded by Mrs J. Jelinkova and Mrs M. Snopkova.
Mass spectra were measured by Dr J. Kohoutova. The technical assistance of Mrs J. Neumannova is
gratefully acknowledged.
REFERENCES
1. Grove M. D., Spencer G. F., Rohwedder W. K., Mandava N., Worley J. F., Warthen J. D., jr.,
Steffens G. L., Flippen-Anderson J. L., Cook J. C., jr.: Nature 281, 216 (1979).
2. Kohout L.: Collect. Czech. Chem. Commun. 59, 457 (1994).
3. Wintersteiner O., Moore M.: J. Am. Chem. Soc. 66, 1507 (1943).
4. Davies A. R., Summers G. H. R.: J. Chem. Soc., C 1967, 1227.
5. Hodge P., Khan M. N.: J. Chem. Soc., Perkin Trans. 1 1975, 809.
6. Bridgeman J. E., Cherry P. C., Clegg A. S., Evans J. M., Jones E. R. H., Kasal A., Kumar V.,
Meakins G. D., Morisawa Y., Richards E. E., Woodgate P. D.: J. Chem. Soc., C 1970, 250.
7. Mitchell J. W., Livingstone G. A.: Methods of Studying Plant Hormones and Growth Regulating
Substances, p. 26, Agriculture Handbook No. 336. U.S. Government Printing Office,
Washington, D.C. 1968.
Translated by M. Tichy.
Collect. Czech. Chem. Commun. (Vol. 59) (1994)