SYNTHESIS OF A SCAFFOLD FOR THE CREATION OF NON-PEPTIDE LIBRARIES

Article abstract of DOI:10.1135/cccc19952147

5β-Cholanic acid derivative XVII was prepared as a skeleton for the creation of non-peptide libraries.In positions 3, 7 and 12, different functional groups were designed in order to provide for the attachment of various building blocks linked to them.

Full text of DOI:10.1135/cccc19952147

On Steroids
2147
SYNTHESIS OF A SCAFFOLD FOR THE CREATION OF NON-PEPTIDE
LIBRARIES*
Alexander KASAL, Ladislav KOHOUT and Michal LEBL
Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic, 166 10 Prague 6, Czech Republic
Received September 13, 1995
Accepted October 12, 1995
5β-Cholanic acid derivative XVII was prepared as a skeleton for the creation of non-peptide libraries.
In positions 3, 7 and 12, different functional groups were designed in order to provide for the attach-
ment of various building blocks linked to them.
Recently, Ernest et al.1 used a naphthalene derivative as a scaffold for the construction
of protein β-turn mimics, later for the same purpose Hirschmann2,3 employed a disub-
stituted steroid derivative as a scaffold. Disubstituted steroid molecule was employed
successfully for the construction of libraries of synthetic receptors4. We have conceived
the use of a scaffold for the generation of non-peptide libraries (for discussion of
various library approaches see e.g.5–7) which would contain a rigid nucleus with three
amino or pro-amino groups and a spacer for binding the molecule to a support. We
have chosen cholic acid (I) as the most convenient starting material. Its synthetic poten-
tial was recognized by Hirschmann2 who pointed to two major reasons for the choice:
the well known rigidity of the steroid molecule, securing distinct reactivity of indivi-
dual functional groups, and the wealth of accumulated knowledge of partial transforma-
tions of individual hydroxy groups in the classical steroid literature.
For practical reasons (polarity, solubility), it was considered convenient to carry out
the whole sequence with an esterified carboxylic group (compound II, ref.8) which
would only be regenerated at the end (Scheme 1). The trihydroxy ester II has been
reported to be selectively acylated or oxidized first in the position 3, then 7 and eventu-
ally 12. The former reaction was most conveniently carried out by means of ethyl chlo-
roformate in pyridine under the formation of a dihydroxy ester III (ref.9; partial
acetylation by acetanhydride was less selective). For partial oxidation of diol III in
* Part CCCLXXIX in the series On Steroids; Part CCCLXXVIII: Collect. Czech. Chem. Commun. 60,
137 (1995).
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Kasal, Kohout, Lebl:
SCHEME 1
Collect. Czech. Chem. Commun. (Vol. 60) (1995)

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2149
position 7, N-bromoacetamide in acetone10 was most effective, the yield of 7-ketone IV
was over 80%.
For the conversion of the 7-oxo group into the 7-amino group, sodium borohydride
reduction of oxime V was considered first. The reduction, however, was so slow that
the ester groupings present were partly affected. Eventually 7-amino derivative VI was
prepared by catalytic hydrogenation. As expected10,11, the amino group occupied the
1
axial position (see the narrow signal of 7β-proton in the H NMR spectra of VI and of
its derivatives).
In some runs the hydrogenation stopped12 at the stage of intermediary hydroxylamino
derivative VII, the further reduction of which being the slowest step. Eventually, ex-
perimental conditions were worked out for the reductive amination13 of ketone IV with
ammonium acetate in the presence of sodium cyanoborohydride; stereospecificity of
the amine formation was lower in this case (65.5% of VI, 3.1% of another amine; the
desired 7α-isomer VI could be best purified after conversion to a Boc derivative X).
Because of solubility problems, acylation of amine VI was first attempted in a non-
aqueous solution (pyridine). The desired carbamate X was only isolated by chromato-
graphy in 46% yield. A side product (yield 49%) was formed as well, its IR spectrum
(νas 2 267 cm–1) and elemental analysis prompted the structure of an isocyanate VIII. Its
structure was confirmed by methanolysis leading to methyl carbamate IX. The same
reaction with tert-butyl alcohol was very slow (10 days at reflux temperature) and
afforded compound X besides products of other reactions (e.g., of alcoholysis of the
3-carbonate group).
Later Boc derivative X was cleanly produced by the Schotten–Baumann14 modifica-
tion using a two-phase system: aqueous potassium hydroxide and a tetrahydrofuran
solution of amine VI and di-tert-butyl dicarbonate.
The same sequence of reactions (oxidation to XI, oximation to XII, reduction to XIII)
was used in the conversion of the 12α-hydroxy group into an amino group. The equa-
1
torial configuration of amino group at carbon 12 was again confirmed by the H NMR
spectrum of XIII (doublet of doublets of the 12α-proton at δ 3.24, J = 9.7 and 1.2 Hz),
in this case the approach of hydrogen from the β-side was hindered by the angular
methyl group and an equatorial amino group was created. The above described reductive
amination of ketone VI was applicable in this case (ketone XI) as well though an even
higher temperature had to be used.
The 12β-amino derivative XIII was converted into corresponding allyloxy carbamate
XIV by treatment with allyl chloroformate in pyridine. The 3α-acyloxy derivative XIV
was hydrolyzed by potassium carbonate which recovered the 3α-hydroxy group. Com-
pound XV was oxidized to 3-ketone XVI and only then the carboxyl in the side chain
1
was regenerated by lithium hydroxide hydrolysis. H NMR and IR spectra of product
XVII verify that both amine protecting groups survived the reaction conditions. The
compound XVII will be bound to beads through the carboxyl and then the third amino
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Kasal, Kohout, Lebl:
group will analogously be created in the position 3. The use of the scaffold for the
generation of non-peptide libraries will be published elsewhere.
EXPERIMENTAL
Melting points were determined on a micro melting point apparatus Boetius and are uncorrected. Op-
tical rotations and infrared spectra (wavenumbers in cm–1) were measured in chloroform unless stated
otherwise. 1H NMR spectra were recorded on a Varian UNITY-200 (200 MHz, FT mode) spec-
TABLE I
Characteristics parameters of 200 MHz 1H NMR spectra of compounds I–XVII in deuteriochloro-
form; for other conditions see Experimental
Compound H-18a H-19a H-3b H-7c H-12d
Other signals
e
I
0.65 0.85 3.59 4.0134.10.96
0.67 0.88 3.44 3.85 3.96 0.98e, 3.66f
0.69 0.914.44 3.98 3.85 0.98 e, 1.29g, 4.16h, 3.67f
II
III
IV
V
0.68 1.19 4.54
–
4.00 0.97e, 1.29g, 3.67f, 4.16h
4.00 0.98e, 1.29g, 3.11i, 3.66f, 4.16h, 7.31j
0.69 1.06 4.55
–
VI
VII
VIII
IX
X
0.69 0.92 4.413.08 3.97 0.98 e, 1.29g, 3.67f, 4.16h
0.67 0.94 4.44 3.08 3.97 0.96e, 1.30g, 3.67f, 4.17h
0.69 0.914.45 3.90 3.98 0.97 e, 1.31g, 3.67f, 4.18h
0.710.97 4.45 3.70 4.02 0.99 e, 1.33g, 3.68f, 3.69f, 4.20h, 4.91k
0.69 0.94 4.43 3.67k 3.910.97 e, 1.30g, 1.44l, 3.67f, 4.18h
XI
XII
XIII
XIV
1.03 1.06 4.43 3.67k
0.92 1.03 4.45 3.67k
0.73 0.94 4.33 3.67k 3.24o 0.97e, 1.31g, 1.44l, 3.33m, 3.67f, 4.18h, 4.86m
–
–
0.84e, 1.31g, 1.43l, 3.66f, 3.83m, 4.18h
0.93e, 1.30g, 1.43l, 3.33n, 3.66f, 4.18h
0.810.95 4.44
3.69k 4.06o 0.88e, 1.30g, 1.42l, 3.69f, 4.17h, 4.45p, 5.28q, 5.29r,
5.90s, 5.10m, 5.54m
XV
0.810.93 3.50
0.85 1.04
3.68k 4.05o 0.88e, 1.45l, 3.68f, 4.59p, 5.24q, 5.32r, 5.92s, 4.95m
3.72 4.13o 0.91e, 1.42l, 3.63f, 4.55p, 5.22q, 5.28r, 5.89s, 5.69m
3.45 4.13o 0.90e, 1.47l, 4.33t, 4.77u, 5.15q, 5.23r, 5.86v, 6.78m
XVI
XVII
–
–
0.82 1.01
a
b
c
d
e
f
g
s, 3 H; m, W = 39; m, W = 16; t, J = 6 and 6; d, 3 H, J = 6.1 (H-21); s, 3 H (OCH3); t,
h
i
j
3 H, J = 7.0 (CH3CH2O); q, 1 H, J = 7.0 (CH3CH2O); dd, 1 H, J = 13.4 and 1.5 (H-6α); m, 1 H,
(C=NOH); overlapping signals; s, 9 H (COOC(CH3)3); m m, 1 H, W = 32 (NH); n dd, 1 H, J =
k
l
o
p
q
8.8 and 2.4 (H-11α); dd, 1 H, J = 9.7 and 1.2 (H-12); d, 2 H, J = 5.6 (COOCH2CH=CH2); d,
1 H, J = 10.3 ( cis-COOCH2CH=CH2); r d, 1 H, J = 16.9 ( trans-COOCH2CH=CH2); s tdd, 1 H, J =
t
u
5.6, 10.3 and 16.9 (COOCH2CH=CH2); dd, 1 H, J = 13.6 and 4.9 (CHCH=CH2); dd, 1 H, J =
13.6 and 5.8 (CHCH=CH2); m, W = 45, 1 H (CH 2CH=CH2).
v
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trometer in deuteriochloroform with tetramethylsilane as internal standard. Chemical shifts are given
in ppm (δ-scale), coupling constants and width of multiplets in Hz. Selected parameters are given in
Table I. Thin-layer chromatography was performed on silica gel (ICN Biochemicals). For column
chromatography silica gel 60–120 µ was used. Methyl 3α,7α,12α-trihydroxy-5β-cholanate (II) was
prepared according to ref.15.
Methyl 3α-Ethoxycarbonyloxy-7α,12α-dihydroxy-5β-cholanate (III)
Ethyl chloroformate (30 ml, 107 mmol) was slowly added to a solution of compound II (45.2 g, 107 mmol)
in pyridine (130 ml) under stirring at 0 °C. The mixture was kept at 0 °C for an additional 45 min
and then poured onto ice (800 ml), the precipitate was filtered off, dissolved in chloroform and
washed with water. Evaporation of the solvent and crystallization from methanol yielded compound
III (44.8 g, 85%). M.p. 151–153 °C, [α]D +41° (c 1.1) (ref.10 records 176–177 °C).
Methyl 3α-Ethoxycarbonyloxy-12α-hydroxy-7-oxo-5β-cholanate (IV)
N-Bromoacetamide (2.5 g, 18.5 mmol) and then acetic acid (5.0 ml) were added to a solution of
compound III (5.0 g, 10.1 mmol) in aqueous acetone (190 ml, 20% of water). After 1 h the mixture
was poured into water (500 ml), the precipitate was filtered off and washed with water. TLC (25%
ether in benzene): RF 0.40, traces of lipophilic impurities were removed by crystallization. M.p. 183–185 °C
(methanol, 4.05 g, 81.3%), ref.10 records 157–159 °C.
Methyl 3α-Ethoxycarbonyloxy-12α-hydroxy-7-oximino-5β-cholanate (V)
Hydroxylamine hydrochloride (7.0 g, 100.7 mmol) was added to a solution of ketone IV (20.0 g, 40.6
mmol) in pyridine (50 ml) and the mixture was kept at 90 °C under stirring. After 8 h the solution
was poured onto ice (300 ml) and oily product was separated, dissolved in chloroform and washed
with water. TLC (chloroform treated with ammonium hydroxide, 5% of acetone): RF 0.25, no starting
material (RF 0.50) was present (when neutral solvent was used, e.g., 50% ether in benzene, com-
pounds IV and V exert identical RF 0.51). IR spectrum: 3 600, 3 400 (OH); 3 592, 3 288 (N–OH); 1 732
(C=O); 1 653 (C=N); 1 271, 1 259 (C–O). Product (16.2 g, 79%) crystallized from toluene, m.p.
156–157 °C, [α]D +21° (c 1.0). For C28H45NO7 (507.7) calculated: 66.25% C, 8.93% H, 2.76% N;
found: 66.28% C, 9.05% H, 2.85% N.
Methyl 7α-Amino-3α-ethoxycarbonyloxy-12α-hydroxy-5β-cholanate (VI)
A) Mother liquors from the preparation of compound VII (2.89 g) were shaken with chloroform
(20 ml) and ammonium hydroxide (4 ml), the organic phase was washed with water and dried over
anhydrous sodium sulfate. Chloroform was evaporated in vacuo and the residue (2.35 g, 67%, based
on oxime used) crystallized from toluene. M.p. 189–192 °C, [α]D +32° (c 0.9). IR spectrum: 3 618,
3 444 (OH); 1 733, 1 438 (COOR); 1 620 (NH2); 1 272, 1 261 (C–O). For C28H47NO6 (493.7) cal-
culated: 68.12% C, 9.60% H, 2.84% N; found: 68.17% C, 9.69% H, 2.76% N.
B) A mixture of ketone IV (128 mg, 0.26 mmol), ammonium acetate (160 mg, 2.07 mmol) and
sodium cyanoborohydride (40 mg, 0.64 mmol) in methanol (3 ml) was stirred at 50 °C. The reaction
was complete within 18 h (TLC), the reagent was decomposed by brine (1 ml). The mixture was
diluted with toluene (6 ml) and concentrated in a vacuum to halve the volume. The mixture was
separated between brine (10 ml) and toluene (10 ml), the organic layer was washed with water, dried
and evaporated. Thin layer chromatography (2 plates, 200 × 200 × 1 mm, 96% of chloroform treated
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Kasal, Kohout, Lebl:
with ammonia and 4% of methanol). The major component (85 mg, 66%, m.p. 189–192 °C) was
identical in all respects with amine VI, a less polar admixture (4 mg) was not studied further.
Methyl 7α-(N-Hydroxyl)amino-3α-ethoxycarbonyloxy-12α-hydroxy-5β-cholanate (VII)
A suspension of platinum dioxide, hydrate (200 mg, 0.88 mmol) and oxime V (3.6 g, 7.1 mmol) in
acetic acid (30 ml) was stirred in hydrogen atmosphere for 60 h. The solvent was evaporated and the
product (710 mg, 20%) crystallized from toluene. M.p. 203–206 °C (methanol); [α]D +32° (c 1.1). IR
spectrum: 3 619 (OH); 3 588 (NH); 1 733, 1 271 (COOR). For C28H47NO7 (509.7) calculated:
65.98% C, 9.29% H, 2.75% N; found: 66.08% C, 9.47% H, 2.67% N.
Methyl 3α-Ethoxycarbonyloxy-12α-hydroxy-7α-isocyanato-5β-cholanate (VIII)
Collected lipophilic fractions from the preparation of compound X (8.23 g, 49% based on amine VI)
were crystallized from ether–heptane, m.p. 135–137 °C, [α]D +23° (c 1.3). IR spectrum: 3 620 (OH);
2 267 (N=C=O); 1 733, 1 270; 1 260 (COOR). For C29H45NO7 (519.7) calculated: 67.03% C, 8.73% H,
2.70% N; found: 67.02% C, 8.76% H, 2.79% N.
Methyl 7α-(N-Methoxycarbonyl)amino-3α-ethoxycarbonyloxy-12α-hydroxy-5β-cholanate (IX)
A solution of isocyanate VIII (135 mg, 0.23 mmol) in absolute methanol (13.5 ml) was heated to
reflux for 6 h; TLC (25% ether in benzene): RF 0.20, no starting material (RF 0.65). The solution was
concentrated in vacuo and the residue was chromatographed on three silica gel plates (200 × 200 × 1 mm)
in benzene–ether (1 : 1). Product (123.5 mg, 86%) crystallized from ether, m.p. 167–169 °C, [α]D
+21° (c 1.4). IR spectrum: 3 621, 3 509 (OH); 3 452 (NH); 1 731 (COOR); 1 689, 1 506 (CONH);
1 270, 1 261 (C–O). For C30H49NO8 (551.7) calculated: 65.31% C, 8.95% H, 2.54% N; found:
65.26% C, 8.95% H, 2.39% N.
In analogous treatment of isocyanate VIII with tert-butyl alcohol (120 h), most of the starting
compound (ca 60%) remained unchanged, however, the desired compound X was accompanied by a
more polar product with a free 3α-hydroxy group (1H NMR spectrum).
Methyl 7α-(N-tert-Butoxycarbonyl)amino-3α-ethoxycarbonyloxy-12α-hydroxy-5β-cholanate (X)
A) Di-tert-butyl dicarbonate (19.0 g, 87.1 mmol) was added to a solution of amine VI (16.1 g, 32.6 mmol)
in pyridine (55 ml) and toluene (55 ml) under stirring at 20 °C. After 20 min, the solution was
diluted with toluene (100 ml) and washed with aqueous potassium hydrogen carbonate and water.
The solution was dried with anhydrous sodium sulfate, concentrated in vacuo to one tenth of its volume
and applied on a column of silica gel (600 g). Mixture toluene–ether (9 : 1) eluted the lipophilic compo-
nent (8.23 g, see the preparation of compound VIII) and 25% ether in toluene eluted compound X (8.91 g,
46%), m.p. 181–182 °C (heptane); [α]D +25° (c 1.6). IR spectrum: 3 619, 3 529 (OH); 3 455, 3 404
(NH); 1 734 (COOCH3); 1 705 (COO); 1 495 (amide II); 1 270, 1 059, 1 024 (C–O). For C33H55NO8
(593.8) calculated: 66.76% C, 9.34% H, 2.36% N; found: 66.58% C, 9.48% H, 2.29% N.
B) Aqueous potassium hydroxide (10%, 1.4 ml, 2.5 mmol) was added to a solution of amine VI
(1.2 g, 2.4 mmol) in tetrahydrofuran (15 ml). The mixture was shaken with di-tert-butyl dicarbonate
(880 mg, 4.0 mmol) for 30 min and then it was acidified with aqueous citric acid (10%, 10 ml, 5.2 mmol).
The mixture was diluted with ether (50 ml), washed with brine and dried over anhydrous magnesium
sulfate. TLC of the product revealed the presence of amide X only. Yield: 1.1 g (76%), m.p. 181–182 °C
(heptane).
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Methyl 7α-(N-tert-Butoxycarbonyl)amino-3α-ethoxycarbonyloxy-12-oxo-5β-cholanate (XI)
Compound X (6.79 g, 13.8 mmol) was oxidized in acetone (30 ml) at 0 °C with Jones reagent. After
5 min the excess of the reagent was reduced by a few drops of methanol and the mixture was poured
into aqueous potassium hydrogen carbonate (5%, 100 ml). The product was extracted with chloro-
form, washed with water and dried over anhydrous sodium sulfate. Product XI (6.56 g, 97%) crystal-
lized from acetone and heptane, m.p. 104–106 °C; [α]D +111° (c 1.1). IR spectrum: 1 734 (COOR);
1 706 (NCOOR); 1 495 (amide II); 1 393, 1 367 (C(CH3)3); 1 269, 1 255 (C–O). For C33H53NO8
(591.8) calculated: 66.98% C, 9.03% H, 2.37% N; found: 67.14% C, 9.23% H, 2.26% N.
Methyl 7α-(N-tert-Butoxycarbonyl)amino-3α-ethoxycarbonyloxy-12-oximino-5β-cholanate (XII)
Compound XI (6.4 g, 10.8 mmol) was dissolved in pyridine (15 ml) and hydroxylamine hydro-
chloride (3.0 g, 43.2 mmol) was added. The mixture was stirred at 80 °C for 2 h, the cool reaction
mixture was poured onto ice (200 ml) and left in a refrigerator overnight. The solid product (6.4 g,
98%) was collected by filtration, dissolved in chloroform and washed with water. TLC (chloroform
treated with ammonia and 5% of acetone): RF 0.50, no starting ketone (RF 0.67). M.p. 116–117 °C
(ether); [α]D +126° (c 1.1). IR spectrum: 3 589, 3 271 (N–OH); 3 455 (NH); 1 732 (COOR); 1 706
(NCOOR); 1 494 (amide II); 1 392, 1 367 (C(CH3)3); 1 270, 1 257 (C–O); 937 (N–O). For
C33H54N2O8 (606.8) calculated: 65.32% C, 8.97% H, 4.62% N; found: 65.27% C, 9.18% H, 4.53% N.
Methyl 12β-Amino-7α-(N-tert-butoxycarbonyl)amino-3α-ethoxycarbonyloxy-5β-cholanate (XIII)
A) Oxime XII (3.3 g, 5.4 mmol) was hydrogenated according to the procedure used for the preparation
of amine VI. The crude product was purified by column chromatography (250 ml of silica, chloroform
with 2.5% of methanol and 1% of acetic acid). The major component (2.8 g, 79%, acetic acid salt of
amine XIII, melting at 103–109 °C) was shaken in chloroform (20 ml) with ammonium hydroxide (5 ml)
and then with water. Free base XIII melts at 180–182 °C (benzene–heptane); [α]D +20° (c 1.1). IR
spectrum: 3 455 (NH); 1 734 (COOR); 1 705 (NHCOOR); 1 492 (amide II); 1 270, 1 170 (C–O). For
C33H56N2O7 (592.8) calculated: 66.86% C, 9.52% H, 4.73% N; found: 66.89% C, 9.42% H, 4.50% N.
B) A mixture of ketone XI (1.0 g, 1.69 mmol), ammonium acetate (1.2 g, 15.6 mmol) and sodium
cyanoborohydride (245 mg, 3.9 mmol) in tert-butyl alcohol (6 ml) was stirred at 75 °C in nitrogen
atmosphere. The reaction was complete within 5 h (TLC), the reagent was decomposed by aqueous
citric acid (10%, 10 ml). The mixture was separated between brine (40 ml) and ethyl acetate (40 ml),
the organic layer was washed with water, dried and evaporated. The product was chromatographed
on a column of silica gel (50 g). A mixture of toluene, ether and methanol (10 : 10 : 1) eluted 380 mg
1
(38%) of an oily product, H NMR spectrum of which was identical with that of compound XIII.
Methyl 12β-(N-Allyloxycarbonyl)amino-7α-(N-tert-butoxycarbonyl)amino-3α-ethoxycarbonyloxy-
5β-cholanate (XIV)
A solution of allyl chloroformate (0.8 ml, 7.5 mmol) in toluene (2 ml) was added to a solution of
amine XIII (1.44 g, 2.4 mmol) in pyridine (3 ml) within 10 min under stirring at 0 °C. After 1 h the
mixture was poured onto ice, the product separated was dissolved in chloroform and washed with
water and dried over sodium sulfate. On evaporation, the residue (1.5 g, 91%) crystallized from
methanol at –18 °C. M.p. 130–133 °C, [α]D +85° (c 1.0). IR spectrum: 3 452, 3 399 (NH); 1 730, 1 392,
1 367 (COOR); 1 706, 1 268, 1 250 (NCOOR); 1 528, 1 511 (amide II). For C37H60N2O9 (676.9)
calculated: 65.65% C, 8.93% H, 4.14% N; found: 65.37% C, 8.85% H, 4.01% N.
Collect. Czech. Chem. Commun. (Vol. 60) (1995)

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Kasal, Kohout, Lebl:
Methyl 12β-(N-Allyloxycarbonyl)amino-7α-(N-tert-butoxycarbonyl)amino-3α-hydroxy-
5β-cholanate (XV)
A) Potassium carbonate (130 mg, 0.94 mmol) in water (1 ml) was added to a solution of com-
pound XIV (640 mg, 0.94 mmol) in methanol (50 ml) and the mixture was kept at 20 °C for 18 h and
then at 50 °C for 30 min. The solution was concentrated in vacuo to volume 5 ml, brine was added
and the precipitate filtered and washed with water. The product XV (464 mg, 81%) crystallized from
acetone, m.p. 214–216 °C; [α]D +65° (c 0.9). IR spectrum: 3 609 (OH); 3 452, 3 347 (NH); 3 088,
1 654 (H–C=C); 1 705, 1 392, 1 367, 1 243, 1 067 (COOR); 1 528, 1 513 (amide II). For
C34H56N2O7 (604.8) calculated: 67.52% C, 9.33% H, 4.63% N; found: 67.47% C, 9.28% H, 4.49% N.
B) Compound XII (1.0 g, 1.69 mmol) was treated with ammonium acetate (1.27 g, 16.5 mmol) and
sodium cyanoborohydride (245 mg, 3.9 mmol) as described above (preparation of XIII, procedure B).
The crude product was acylated with allyl chloroformate (0.8 ml, 7.5 mmol) in pyridine (1 ml) and
toluene (1 ml) at 0 °C. The crude product was hydrolyzed in a solution of potassium carbonate (250 mg,
1.8 mmol) in aqueous methanol (98%, 110 ml) as described above. The product was chromato-
graphed on a column of silica gel (100 g) using 50% ether in toluene. The major component (449 mg)
crystallized from acetone (4.5 ml) yielding 349 mg of compound XV (34% from ketone XII), m.p.
206–211 °C, [α]D +68° (c 1.6). Further crystallization yielded a product identical with the product
described under A (identity of IR spectra).
Methyl 12β-(N-Allyloxycarbonyl)amino-7α-(N-tert-butoxycarbonyl)amino-3-oxo-5β-cholanate (XVI)
Hydroxy derivative XV (1.52 g, 2.5 mmol) was oxidized according to the procedure used for the
preparation of compound XI. The product (1.4 g, 92%) crystallized from acetone–heptane, m.p. 217–218 °C;
[α]D +82° (c 1.1). IR spectrum: 3 369 (NH); 3 100, 1 650 (H–C=C); 1 704, 1 247, 1 164 (COOR);
1 524 (amide II). For C34H54N2O7 (602.8) calculated: 67.74% C, 9.03% H, 4.65% N; found: 67.70% C,
9.11% H, 4.39% N.
12β-(N-Allyloxycarbonyl)amino-7α-(N-tert-butoxycarbonyl)amino-3-oxo-5β-cholanic Acid (XVII)
Ester XVI (2.09 g, 3.5 mmol) was dissolved in methanol (80 ml) and lithium hydroxide monohydrate
(1.5 g, 40.5 mmol) in water (7 ml) was added. The mixture was kept at 50 °C for 1 h. Alkalinity of
the mixture was suppressed by the addition of citric acid (2.7 g, 14.0 mmol) and the volume was
reduced one half on a vacuo evaporator. Brine (100 ml) was added and the precipitate was filtered
off and washed with water and dissolved in chloroform. The extract was washed with water, dried
over anhydrous sodium sulfate and evaporated in vacuo. The residue crystallized from acetone–heptane,
m.p. 217–218 °C (1.4 g, 69%). [α]D +177° (c 0.9). IR spectrum: 3 628 (OH); 3 450 (NH); 1 708
(NHCOOR); 1 648, 1 009 (H2C=CH–CH2O); 1 502 (amide II); 1 270, 1 198 (C–O). For C33H52N2O7
(588.8) calculated: 67.32% C, 8.90% H, 4.76% N; found: 67.14% C, 8.95% H, 4.55% N. Mother
liquors were purified by chromatography on silica gel column in chloroform–acetone (9 : 1), yielding
an additional 269 mg of the product (total yield 82%).
The authors are indebted to Mrs M. Sedlackova for technical assistance. The IR spectra were
measured by Mrs K. Matouskova and interpreted by Dr L. Bednarova, the NMR spectra were taken by
Mrs M. Snopkova. Our thanks are due to members of the Analytical Department (Dr V. Pechanec, Head)
for carrying out elemental analyses.
Collect. Czech. Chem. Commun. (Vol. 60) (1995)

On Steroids
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