Addition of azoimide to unsaturated ketones in the steroid series. Synthesis of N-(17β-hydroxy-3-oxo-5α-androstan-15β-yl)-succinamoic acid and its evaluation as hapten for dihydrotestosterone immunoanalysis

Article abstract of DOI:10.1135/cccc19971931

Addition of azoimide to 17-oxoandrosta-5,15-dien-3β-yl acetate or 17-oxo-5α-androst-15-en-3β-yl acetate gave in both cases corresponding 15β-azido derivatives. 15β-Azido-17-oxo-5α-androstan-3β-yl acetate was selectively reduced to 17β-hydroxy derivative and protected as methoxymethyl ether. Subsequent reduction of azide group and condensation with methyl hydrogen succinate gave a protected succinylamino derivative. Deacetylation and oxidation then led to dihydrotestosterone (DHT) series. Successive removal of protecting groups gave N-(17β-hydroxy-3-oxo-5α-androstan-15β-yl)succinamoic acid, an experimental hapten for DHT. Its conjugate with bovine serum albumin was used for immunization of rabbits and corresponding antisera were evaluated in RIA using [³H]-DHT as a tracer. The results are comparable with standard kits based on antisera generated using DHT derivatives with connecting bridge in position 7.

Full text of DOI:10.1135/cccc19971931

On Steroids
1931
ADDITION OF AZOIMIDE TO UNSATURATED KETONES IN THE STEROID
SERIES. SYNTHESIS OF N-(17β-HYDROXY-3-OXO-5α-ANDROSTAN-15β-YL)-
SUCCINAMOIC ACID AND ITS EVALUATION AS HAPTEN FOR
DIHYDROTESTOSTERONE IMMUNOANALYSIS*
a1
a2
b
b
Ivan CERNY , Vladimir POUZAR , Oldrich LAPCIK and Richard HAMPL
a Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
166 10 Prague 6, Czech Republic; e-mail: 1cerny@uochb.cas.cz, 2pouzar@uochb.cas.cz
b Institute of Endocrinology, 116 94 Prague 1, Czech Republic; e-mail: rhampl@endo.lib.cas.cz
Received September 24, 1997
Accepted November 12, 1997
Addition of azoimide to 17-oxoandrosta-5,15-dien-3β-yl acetate or 17-oxo-5α-androst-15-en-3β-yl
acetate gave in both cases corresponding 15β-azido derivatives. 15β-Azido-17-oxo-5α-androstan-3β-yl
acetate was selectively reduced to 17β-hydroxy derivative and protected as methoxymethyl ether.
Subsequent reduction of azide group and condensation with methyl hydrogen succinate gave a pro-
tected succinylamino derivative. Deacetylation and oxidation then led to dihydrotestosterone (DHT)
series. Successive removal of protecting groups gave N-(17β-hydroxy-3-oxo-5α-androstan-15β-
yl)succinamoic acid, an experimental hapten for DHT. Its conjugate with bovine serum albumin was
used for immunization of rabbits and corresponding antisera were evaluated in RIA using [3H]-DHT
as a tracer. The results are comparable with standard kits based on antisera generated using DHT
derivatives with connecting bridge in position 7.
Key words: Steroids; Azoimide; DHT; Haptens.
Continuing our work on 15-substituted derivatives of the androstane series, we have
focused on 15-amino derivatives. For the introduction of the nitrogen substituent into
steroid molecule, we adopted the reaction of azoimide with conjugated enones2. One of
the potential uses of such derivatives is their transformation into haptens with the
amidic connecting bridge3.
The preliminary experiments were done on 3β-hydroxyandrosta-5,15-dien-17-one4
(1) with generation of azoimide in situ from sodium azide and acetic acid. The starting
compound was dissolved in acetic acid, saturated aqueous sodium azide was added, and
the reaction mixture was stirred at room temperature for four hours. After the work-up,
we obtained a mixture, from which after acetylation and column chromatography 15β-
* Part CCCXCII in the series On Steroids; Part CCCXCI see ref.1.
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

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Cerny, Pouzar, Lapcik, Hampl:
azido-17-oxoandrost-5-en-3β-yl acetate (3) was separated as the main product. Under
these conditions the reaction was not complete, probably due to heterogeneity of the
reaction mixture. Moreover, the TLC monitoring was difficult because the product and
starting compound had similar mobility. After several additional experiments, we found
that both these drawbacks can be eliminated by using acetate 2 as a starting compound
and by addition of tetrahydrofuran into the reaction mixture (see Experimental). Such a
modified reaction was used for further experiments. The subsequent reduction step was
also modelled in the 5-ene series: sodium borohydride in a methanol–ethyl acetate mix-
ture smoothly reduced ketone 3 to 17β-hydroxy derivative 4 (Scheme 1).
O
O
N
3
RO
AcO
3
R = H
1,
2,
R = Ac
OH
N
3
AcO
4
SCHEME 1
In the 5α-androstane series, we reacted 3β-hydroxy-5α-androstan-15-en-17-one5 (5)
with acetic anhydride in pyridine to give acetate 6. This compound was, without purifi-
cation, subjected to the azoimide addition and after column chromatography and crys-
tallization from ethanol, we obtained 15β-azido-17-oxo-5α-androstan-3β-yl acetate (7)
in 65% yield. The borohydride reduction of ketone 7 gave 17β-hydroxy derivative 8
which was subsequently protected by reaction with chloromethyl methyl ether yielding
methoxymethyl ether 9.
For the preparation of amine 10, we tried to use hydrogenation of 9 over palladium
on activated carbon in acetic acid, but deactivation of the catalyst during reaction made
this method unusable. Of the other methods we succeeded in reducing 9 with sodium
borohydride in the presence of nickel(II) chloride6: we obtained 15β-amino-17β-
(methoxymethoxy)-5α-androstan-3β-yl acetate (10) as a semisolid mass (Scheme 2).
This product was condensed with methyl hydrogen succinate in the presence of N,N′-
dicyclohexylcarbodiimide and afforded amide 11 which was, without further purifica-
tion, deacetylated with sodium methoxide in methanol to methyl N-[3β-hydroxy-
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

On Steroids
1933
O
O
N
3
RO
AcO
H
H
7
R = H
5,
6,
R = Ac
OH
O
O
N
N
3
3
AcO
AcO
H
H
8
9
O
O
O
O
NH
NHCO(CH ) COOCH
2 2
2
3
AcO
AcO
H
H
10
11
SCHEME 2
17β-(methoxymethoxy)-5α-androstan-15β-yl]succinamate (12). Subsequent oxidation
with the Jones reagent completed transformation into the dihydrotestosterone (DHT)
series giving methyl N-[17β-(methoxymethoxy)-3-oxo-5α-androstan-15β-yl]succina-
mate (13) (Scheme 3).
The removal of protecting groups was performed in two steps. The 4-toluenesulfonic
acid monohydrate in a benzene–methanol mixture split off the methoxymethyl group.
As a side product, the corresponding dimethyl ketal 14 was observed in 1H NMR spec-
trum of the crude product from this reaction (3.19 s and 3.14 s: CH3O–C(3)–OCH3;
0.95 s and 0.85 s: H-18 and H-19). This step was therefore completed by treatment with
4-toluenesulfonic acid monohydrate in acetone to give 15. In the second step aqueous-
methanolic potassium hydroxide saponified the ester. The N-(17β-hydroxy-3-oxo-5α-
androstan-15β-yl)succinamoic acid (16) obtained was preliminary tested as a hapten for
DHT immunoassays.
The conjugate with bovine serum albumin (BSA) was prepared by the standard
method7 and the amount of hapten 16 bound was assayed by the titration of free amino
groups with 2,4,6-trinitrobenzenesulfonic acid8 to yield 14 units per molecule of BSA.
Rabbits were immunized by 250 µg of the conjugate four times in 4–6 weeks periods.
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Cerny, Pouzar, Lapcik, Hampl:
The antisera obtained (Serum No. 21 and 22) were found to bind [3H]-DHT and
displayed a good sensitivity in DHT radioimmunoassay using [3H]-DHT as indicator
(Table I). However, the cross-reactivity with testosterone is too high for direct use in
DHT assays. Nevertheless, it is comparable with the routinely used systems based on
DHT haptens with bridge in position 7, where the analysis must be combined with
oxidative treatment removing interfering testosterone before the assay.
O
O
O
O
NHCO(CH ) COOCH
NHCO(CH ) COOCH
2 2 3
2 2
3
AcO
HO
H
H
H
H
H
H
11
12
15
16
O
O
OH
NHCO(CH ) COOCH
2 2
NHCO(CH ) COOCH
2 2
3
3
O
O
13
OH
OH
CH O
3
NHCO(CH ) COOCH
NHCO(CH ) COOH
2 2
2 2
3
O
CH O
3
14
SCHEME 3
Additionally, we tested the antisera in assays using 125I labelled indicators. We used
compound 17, corresponding to condensation of amine 10 with activated 3-(4-hydroxy-
phenyl)propionate (Bolton–Hunter reagent9) for homologous assay and L-tyrosine
methyl ester conjugate of (15E)-17β-hydroxy-5α-androstane-3,15-dione 15-(O-carboxy-
methyl)oxime1 (18) for heterologous assay. Both compounds were radioiodinated by
the same method10 and radioligands were purified by TLC, giving ligand A and ligand
B, respectively. For the hapten 17, the radioligand (ligand A) could not be displaced by
nonradioactive DHT, probably due to a strong bridge effect, so that this system could
not be used in the DHT assay (Table I). The ligand B, derived from 15-CMO DHT 18,
was useful as a tracer in DHT RIA although the results were not better than those in
assays using [3H]-DHT (Table I).
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

On Steroids
1935
OH
OH
OH
O
H
N
N
HN
OCH
OH
3
O
O
O
H
H
H
O
O
18
17
In summary, we developed a method for preparation of 15β-amino derivatives in the
androstane series and made use of it in preparation of a model hapten for DHT with
amidic bridge. Previous immunoassays provided active antisera generated with this
hapten, but, compared with classical methods, a mere change of connecting position
could not avoid the interference of 3-oxo-4-ene steroids.
EXPERIMENTAL
Melting points were determined on a Boetius micromelting point apparatus (Jena, Germany). Optical
rotations were measured at 25 °C on a Perkin–Elmer 141 MC polarimeter; [α]D values are given in °
[10–1 deg cm2 g–1]. Infrared spectra (wavenumbers in cm–1) were recorded on a Bruker IFS 88 spec-
1
trometer in chloroform. H NMR spectra were taken on a Varian UNITY-200 (200 MHz) or a Varian
UNITY-500 (500 MHz) spectrometer at 23 °C in deuteriochloroform with tetramethylsilane as an in-
ternal standard. Chemical shifts are given in ppm (δ-scale), coupling constants (J) and multiplet
widths (W) in Hz. The purity of the products and reaction course were checked by thin-layer chro-
matography (TLC) performed on silica gel G (ICN Biochemicals) followed by spraying with concen-
trated sulfuric acid and heating. Column chromatography was performed on silica gel (60–120 µm,
Service laboratory of the Institute). Prior to evaporation on a rotary evaporator in vacuo (bath tem-
perature 50 °C), solutions in organic solvents were dried over magnesium sulfate. Analytical samples
were dried over phosphorus pentoxide at 40 °C/26 Pa for 12 h.
15β-Azido-17-oxoandrost-5-en-3β-yl Acetate (3)
Ketone 1 (1.0 g, 3.49 mmol) was acetylated with acetic anhydride (2 ml, 21.2 mmol) in pyridine (20 ml)
overnight. The mixture was poured on ice and extracted with ethyl acetate. The organic layer was
washed with 10% HCl, water, saturated aqueous KHCO3 and water, dried, and the solvents were
evaporated. Resulting acetate 2, without purification, was dissolved in tetrahydrofuran (10 ml) and
acetic acid (10 ml). To the stirred solution was added sodium azide (2 g, 30.8 mmol) in water (5 ml).
After 6 h, the mixture was poured on cold saturated aqueous KHCO3 and extracted with ethyl acetate
(100 ml). The organic phase was washed with saturated aqueous KHCO3, dried, and solvents were
evaporated. The residue was crystallized from hot ethanol to give 930 mg (72%) of azide 3, m.p.
162–164 °C, [α]D –62° (c 1.2, chloroform). IR spectrum (chloroform): 2 116, 2 088 (azide); 1 734
(C=O, acetate and ketone); 1 670 (C=C); 1 367 (CH3, acetate); 1 264, 1 025 (C–O, acetate); 1 076
1
(C–O). H NMR spectrum: 5.42 m, 1 H, W = 10 (H-6); 4.61 m, 1 H, W = 32 (H-3α); 4.33 ddd, 1 H,
J ≈ 7.0, 6.0, 2.0 (H-15α); 2.72 dd, 1 H, J ≈ 20.0, 2.0 (H-16β); 2.55 dd, 1 H, J ≈ 20.0, 6.5 (H-16α);
2.04 s, 3 H (CH3COO); 1.10 s, 3 H (3 × H-18); 1.09 s, 3 H (3 × H-19). For C21H29N3O3 (371.5)
calculated: 67.90% C, 7.87% H, 11.31% N; found: 68.09% C, 8.00% H, 11.57% N.
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

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Cerny, Pouzar, Lapcik, Hampl:
15β-Azido-17-hydroxyandrost-5-en-3β-yl Acetate (4)
To a stirred solution of 3 (1.5 g, 4.0 mmol) in a mixture of ethyl acetate (15 ml) and methanol (30 ml),
sodium borohydride (180 mg, 4.8 mmol) was gradually added at 10 °C during 5 min. After additional
5 min of stirring at the same temperature, the excess hydride was destroyed by acetic acid (0.3 ml)
and water (0.3 ml). The solution was concentrated under reduced pressure, diluted with ethyl acetate,
and washed successively with saturated aqueous NaCl, 10% HCl, water, saturated aqueous KHCO3,
and water. After drying and evaporation, the residue was crystallized from ethanol–petroleum ether
giving 1.08 g (72%) of azide 4, m.p. 121–122 °C, [α]D –112° (c 0.6, chloroform). IR spectrum (chloroform):
3 610, 3 484 (O–H); 2 110, 2 085 (azide); 1 723 (C=O, acetate); 1 671 (C=C); 1 366 (CH3, acetate);
1
1 255, 1 047, 1 029 (C–O). H NMR spectrum: 5.40 m, W = 12 (H-6); 4.61 m, 1 H, W = 32 (H-3α);
3.96 ddd, 1 H, J ≈ 8.5, 6.5, 3.0 (H-15α); 3.63 t, 1 H, J ≈ 8.5 (H-17α); 2.62 dt, 1 H, J ≈ 15.0, 2 × 8.5
(H-16α); 2.04 s, 3 H (CH3COO); 1.71 ddd, 1 H, J ≈ 15.0, 8.5, 3.0 (H-16β); 1.07 s, 3 H (3 × H-19);
0.92 s, 3 H (3 × H-18). For C21H31N3O3 (373.5) calculated: 67.53% C, 8.37% H, 11.25% N; found:
67.24% C, 8.48% H, 11.13% N.
15β-Azido-17-oxo-5α-androstan-3β-yl Acetate (7)
Ketone 5 (1.0 g, 3.47 mmol) was processed as in the preparation of 3 to give 1.1 g (86%) of crude
azide 7. The chromatography on a silica gel column (150 ml) in a benzene–petroleum ether–acetone
(50 : 50 : 1) mixture and crystallization from ethanol gave 730 mg (57%) of 7, m.p. 95–98 °C, [α]D –18°
(c 1.1, chloroform). IR spectrum (chloroform): 2 115, 2 088 (azide); 1 736 (C=O, ketone); 1 728
1
(C=O, acetate); 1 367 (CH3, acetate); 1 255, 1 025 (C–O, acetate). H NMR spectrum: 4.69 m, 1 H,
TABLE I
Properties of the antisera induced by haptene 16 – bovine serum albumin conjugate in radioimmuno-
assays
Serum No.
Technique
21
22
RIA with [3H]-DHT:
dilution
1 : 6 4000
1 : 3 200
50% intercept
sensitivitya
30.7 pg/tube
19.4 pg/tube
<1 pg/tube
cross-reactivityb
RIA with ligand A:
dilution
36.1%
27.8%
1 : 320 000
808 pg/tube
1 : 160 000
377 pg/tube
50% intercept
RIA with ligand B:
dilution
1 : 2 000
156.8 pg/tube
39.9%
1 : 3 000
59.4 pg/tube
27.8%
50% intercept
cross-reactivityb
a
b
Expressed as an amount of the analyte distinguishable with 95% probability from a zero sample.
Cross-reactivity against testosterone.
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

On Steroids
1937
W = 32 (H-3α); 4.33 ddd, 1 H, J = 5.2, 1.7, 6.5 (H-15α); 2.68 dd, 1 H, J = 19.8, 1.7 (H-16β); 2.54 dd,
1 H, J = 19.8, 6.5 (H-16α), 2.03 s, 3 H (CH3COO); 1.08 s, 3 H (3 × H-18); 0.89 s, 3 H (3 × H-19).
For C21H31N3O3 (373.5) calculated: 67.53% C, 8.37% H, 11.25% N; found: 67.81% C, 8.48% H,
11.39% N.
15β-Azido-17β-hydroxy-5α-androstan-3β-yl Acetate (8)
Ketone 7 (900 mg, 2.41 mmol) in a mixture of ethyl acetate (5 ml) and methanol (10 ml) was pro-
cessed as in the preparation of 4 using sodium borohydride (120 mg, 3.2 mmol) to give 890 mg
(99%) of 8 as foam, [α]D –70° (c 0.3, chloroform). IR spectrum (chloroform): 3 610 (O–H); 2 110,
1
2 087 (azide); 1 723 (C=O, acetate); 1 367 (CH3, acetate); 1 256, 1 026 (C–O). H NMR spectrum:
4.67 m, 1 H, W = 32 (H-3α); 3.96 ddd, 1 H, J = 8.5, 6.4, 3.1 (H-15α); 3.59 t, 1 H, J = 8.5 (H-17α);
2.59 dt, 1 H, J = 14.5, 2 × 8.5 (H-16α); 2.01 s, 3 H (CH3COO); 0.89 s, 3 H (3 × H-18); 0.86 s, 3 H
(3 × H-19). For C21H33N3O3 (375.5) calculated: 67.17% C, 8.86% H, 11.19% N; found: 67.31% C,
8.93% H, 11.21% N.
15β-Azido-17β-(methoxymethoxy)-5α-androstan-3β-yl Acetate (9)
Hydroxy derivative 8 (850 mg, 2.26 mmol) was dissolved in dichloromethane (10 ml) and N,N-diiso-
propylethylamine (1.5 ml, 10.7 mmol). To a stirred solution chloromethyl methyl ether (0.4 ml, 5.3 mmol)
was added dropwise and the stirring continued at room temperature for 4 h. Then the reaction mix-
ture was poured on ice cold saturated aqueous NaCl and extracted with ether (50 ml). The ethereal
layer was washed twice with 10% HCl, saturated aqueous KHCO3 and saturated aqueous NaCl. After
drying, the solution was filtered through a short column of aluminum oxide and concentrated giving
1 g (105%) of oily 9, which successively solidified. Recrystallization from ethanol yielded 800 mg
(84%) of 9, m.p. 84–85 °C, [α]D –74° (c 1.0, chloroform). IR spectrum (chloroform): 2 110, 2 089 sh
(azide); 1 724 (C=O, acetate); 1 255, 1 027 (C–O, acetate); 1 150, 1 103, 1 045 (C–O, CH3OCH2O);
913 (CH2, CH3OCH2O). 1H NMR spectrum: 4.68 m, 1 H, W = 35 (H-3α); 4.63 s, 2 H (OCH2O);
3.97 ddd, 1 H, J = 8.5, 6.4, 2.8 (H-15α); 3.46 t, 1 H, J = 8.5 (H-17α); 3.36 s, 3 H (CH3OCH2);
2.58 dt, 1 H, J = 14.7, 2 × 8.5 (H-16α); 2.02 s, 3 H (CH3COO); 0.93 s, 3 H (3 × H-18); 0.86 s, 3 H
(3 × H-19). For C23H37N3O4 (419.6) calculated: 65.84% C, 8.89% H, 10.02% N; found: 66.06% C,
8.91% H, 10.12% N.
Methyl N-[3β-Hydroxy-17β-(methoxymethoxy)-5α-androstan-15β-yl]succinamate (12)
To a stirred solution of azide 9 (600 mg, 1.43 mmol) in a mixture of ethanol (20 ml) and dioxane
(20 ml), sodium borohydride (240 mg, 6.34 mmol) and then dropwise 0.1 M ethanolic solution of
nickel chloride hexahydrate (1.4 ml, 0.14 mmol) were added. During the addition of the nickel solu-
tion, a black precipitate was formed. After 1 h stirring, acetic acid (ca 2 ml) and water (ca 1 ml)
were added to dissolve most of the catalyst. The mixture was concentrated to a small volume, poured
into saturated aqueous KHCO3 (100 ml), and extracted with ethyl acetate (ca 70 ml). After drying,
the solvents were evaporated and the residue coevaporated with toluene (three times) giving 600 mg
(106%) of crude 15β-amino-17β-(methoxymethoxy)-5α-androstan-3β-yl acetate (10) as a semisolid
paste homogeneous on TLC in a chloroform–propan-2-ol–25% aqueous ammonia (100 : 10 : 0.5)
mixture. Amine 10 was dissolved in benzene (10 ml), methyl hydrogen succinate (450 mg, 3.41 mmol)
was added and then, after dissolution, 1 M N,N′-dicyclohexylcarbodiimide in benzene (2 ml, 2 mmol),
all under stirring. The stirring continued 1 h and the completion of reaction was checked by TLC in
the above mentioned mixture. Two drops of water were added and solvents were evaporated. The
solid residue was extracted with ethyl acetate (3 × 15 ml) and the solution was washed with saturated
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

1938
Cerny, Pouzar, Lapcik, Hampl:
aqueous KHCO3, saturated aqueous NaCl, dried and evaporated to give the foamy residue containing
1
methyl N-[3β-acetoxy-17β-(methoxymethoxy)-5α-androstan-15β-yl]succinamate (11). H NMR spec-
trum: 6.03 bd, 1 H, J = 8.6 (NH); 4.69 m, 1 H, W = 35 (H-3α); 4.60 s, 2 H (OCH2O); 4.26 ddt, 1 H,
J = 7.6, 3.7, 2 × 8.6 (H-15α); 3.69 s, 3 H (COOCH3); 3.48 t, 1 H, J = 8.6 (H-17α); 3.33 s, 3 H
(CH3OCH2); 2.40–2.80 bm, 5 H (CH2CH2 succinate + H-16α); 2.02 s, 3 H (CH3COO); 0.97 s, 3 H
(3 × H-18); 0.88 s, 3 H (3 × H-19).
Acetate 11 was dissolved in absolute methanol (50 ml), methanolic sodium methoxide (1 ml, from
ca 100 mg Na in 5 ml of methanol) was added, and the mixture was left aside at room temperature
for 24 h. Solid CO2 (about 0.7 g) was added and most of the methanol was evaporated. The mixture
was partitioned between chloroform (30 ml) and saturated aqueous NaCl, the organic layer was
washed with saturated aqueous NaCl, dried, and solvents were evaporated. Chromatography on the
column of silica gel (50 g) in a chloroform–methanol (100 : 1) mixture gave 460 mg (69%) of oily
succinamate 12. IR spectrum (chloroform): 3 610 (O–H); 3 454 (N–H); 1 731 (C=O, ester); 1 663, 1 513
1
(CONH); 1 440 (CH3, ester); 1 154, 1 103, 1 044 (C–O, CH3OCH2O). H NMR spectrum: 6.05 bd,
1 H, J = 8.6 (NH); 4.60 s, 2 H (OCH2O); 4.25 ddt, 1 H, J = 7.6, 3.7, 2 × 8.6 (H-15α); 3.70 s, 3 H
(CH3OCO); 3.60 m, 1 H, W = 35 (H-3α); 3.48 t, 1 H, J = 8.6 (H-17α); 3.34 s, 3 H (CH3OCH2);
2.40–2.80 bm, 5 H (CH2CH2 succinate + H-16α); 0.98 s, 3 H (3 × H-18); 0.87 s, 3 H (3 × H-19).
For C26H43NO6 (465.5) calculated: 67.07% C, 9.31% H, 3.01% N; found: 67.26% C, 9.03% H,
3.09% N.
Methyl N-[17β-(Methoxymethoxy)-3-oxo-5α-androstan-15β-yl]succinamate (13)
A solution of hydroxy derivative 12 (210 mg, 0.45 mmol) in acetone (5 ml) was treated with excess
Jones reagent. After 5 min at room temperature, the excess reagent was destroyed with methanol (0.2 ml)
and the mixture was poured into saturated aqueous KHCO3 and extracted with ethyl acetate. The or-
ganic layer was washed twice with saturated aqueous KHCO3 and water, dried, and evaporated. Col-
umn chromatography on silica gel (25 g) in a chloroform–methanol (100 : 1) mixture gave 185 mg
(88%) of foamy 13. IR spectrum (chloroform): 3 454, 3 409 (N–H); 1 731 (C=O, ester); 1 707
(C=O, ketone); 1 665, 1 513 (CONH); 1 440 (CH3, ester); 1 153, 1 103, 1 045 (C–O, CH3OCH2O).
1H NMR spectrum: 6.04 bd, 1 H, J = 8.6 (NH); 4.61 s, 2 H (OCH2O); 4.28 ddt, 1 H, J = 7.6, 3.7, 2 × 8.6
(H-15α); 3.70 s, 3 H (COOCH3); 3.49 t, 1 H, J = 8.6 (H-17α); 3.34 s, 3 H (CH3OCH2); 2.40–2.80 bm,
5 H (CH2CH2 succinate + H-16α); 1.07 s, 3 H (3 × H-19); 1.00 s, 3 H (3 × H-18). For C26H41NO6
(463.6) calculated: 67.36% C, 8.91% H, 3.02% N; found: 67.48% C, 9.03% H, 3.10% N.
Methyl N-(17β-Hydroxy-3-oxo-5α-androstan-15β-yl)succinamate (15)
To a solution of amide 13 (165 mg, 0.36 mmol) in a mixture of benzene (5 ml) and methanol (5 ml),
4-toluenesulfonic acid monohydrate (80 mg) was added. The mixture was stirred at 40 °C for 12 h
then evaporated to about half of the volume and poured into saturated aqueous KHCO3. The product
was extracted with ethyl acetate (30 ml) in three portions. The organic layer was washed with satu-
rated aqueous NaCl and water, dried and solvents were evaporated. The foamy product (160 mg) was
dissolved in acetone (5 ml) and a solution of 4-toluenesulfonic acid monohydrate (5 mg) in water
(0.5 ml) was added. After 2 h stirring at room temperature, the mixture was poured into saturated
aqueous KHCO3 and extracted with ethyl acetate. The organic layer was washed with saturated
aqueous NaCl, dried and solvents were evaporated. Crystallization from acetone gave 95 mg (64%)
of 15, m.p. 188–189 °C, [α]D –3° (c 0.9, chloroform). IR spectrum (chloroform): 3 615 (O–H); 3 453,
3 412 (N–H); 1 729 (C=O, ester); 1 707 (C=O, ketone); 1 665, 1 513 (CONH); 1 468, 1 439 (CH3,
1
ester); 1 052 (C–O). H NMR spectrum: 6.04 bd, 1 H, J = 8.7 (NH); 4.28 ddt, 1 H, J = 7.6, 3.7, 2 × 8.7
(H-15α); 3.70 s, 3 H (COOCH3); 3.63 t, 1 H, J = 8.7 (H-17α); 2.40–2.80 bm, 5 H (CH2CH2 succi-
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

On Steroids
1939
nate + H-16α); 1.07 s, 3 H (3 × H-19); 0.98 s, 3 H (3 × H-18). For C24H37NO5 (419.6) calculated:
68.71% C, 8.89% H, 3.34% N; found: 68.89% C, 9.01% H, 3.42% N.
N-(17β-Hydroxy-3-oxo-5α-androstan-15β-yl)succinamoic Acid (16)
Ester 15 (89 mg, 0.21 mmol) was dissolved in methanol (5 ml), 2 M aqueous sodium hydroxide (2 ml)
was added, and the mixture was stirred at room temperature for 1 h. Reaction mixture was acidified
with ice-cold 10% H2SO4, most of the methanol was evaporated, and the product was extracted with
ethyl acetate (5 ml) in three portions. The extract was washed with water (twice) and the solvent was
evaporated. Chromatography of the residue on silica gel column (10 g) in a mixture of chloroform–
methanol (20 : 1) with 0.5% of acetic acid gave 60 mg (70%) of glassy acid 16. IR spectrum (KBr):
3 360 (N–H); 3 290 sh (O–H); 2 641, 2 585, 2 544 (COOH, dimer); 1 729 (C=O, COOH); 1 705
(C=O, ketone); 1 653, 1 525 (CONH); 1 052 (C–O). 1H NMR spectrum (500 MHz): 6.66 bd, 1 H, J = 8.4
(NH); 4.19 dddd, 1 H, J = 8.8, 5.8, 3.7, 8.4 (H-15α); 3.63 t, 1 H, J = 8.8 (H-17α); 2.40–2.80 bm, 5 H
(CH2CH2 succinate + H-16α); 1.07 s, 3 H (3 × H-19); 0.95 s, 3 H (3 × H-18). For C23H35NO5
(405.5) calculated: 68.12% C, 8.70% H, 3.45% N; found: 68.29% C, 8.90% H, 3.42% N.
The authors are indebted Dr L. Bednarova and Dr P. Fiedler for taking and interpretation of IR
1
spectra. Our thanks are due to Mrs M. Snopkova for measurements of 200 MHz H NMR and to Dr M.
1
Budesinsky for measurements and interpretation of 500 MHz H NMR spectra. The elemental analyses
were carried out in the Analytical Laboratory of this Institute (Dr V. Pechanec, Head).
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Collect. Czech. Chem. Commun. (Vol. 62) (1997)