Azido analogs of neuroactive steroids

Article abstract of DOI:10.1016/j.steroids.2011.04.008

Analogs of pregnanolone (3α-hydroxy-5β-pregnan-20-one), modified in position 17 were prepared. Compounds with 20-keto pregnane side chain replaced completely by azide (17α- and 17β-azido-5β-androstan- 3α-ol), compounds with its part replaced (20-azido-21-

Full text of DOI:10.1016/j.steroids.2011.04.008

Steroids 76 (2011) 1043–1050
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Steroids
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Azido analogs of neuroactive steroids
a
Lukásˇ Vidrna , Ivan Cerny´ a,∗, Vladimír Pouzara, Jirˇina Borovskáb,
ˇ
Vojteˇch Vyklicky´ b, Ladislav Vyklicky´ Jr.b, Hana Chodounskáa
a Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., 166 10 Prague 6, Czech Republic
b Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., 142 20 Prague 4, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Analogs of pregnanolone (3␣-hydroxy-5␤-pregnan-20-one), modified in position 17 were prepared.
Compounds with 20-keto pregnane side chain replaced completely by azide (17␣- and 17␤-azido-
5␤-androstan-3␣-ol), compounds with its part replaced (20-azido-21-nor-5␤-pregnan-3␣-ol), and
compounds with keto group only replaced ((20R)- and (20S)-20-azido-5␤-pregnan-3␣-ol) were syn-
thesized using tosylate displacements with sodium azide or Mitsunobu reaction with azoimide. All five
azido steroids were converted into corresponding sulfates. Subsequent tests for inhibition of glutamate
induced response on NMDA receptors revealed that modification of pregnanolone sulfate side chain with
azide did not disturb the activity and some of sulfates tested were more active than parent compound.
© 2011 Elsevier Inc. All rights reserved.
Received 27 January 2011
Received in revised form 4 April 2011
Accepted 8 April 2011
Available online 25 April 2011
Keywords:
Neuroactive steroids
Synthesis
Azides
NMDA receptor
1. Introduction
derivatives and on 3-keto derivative with 5␣-androstane skeleton
and 17␤-hydroxy group [18]. Nucleophilic substitution by sodium
Steroidal azides are very often described in connection with
preparation of corresponding amines [1–5]. Optical [6], thermal
and photochemical properties [7–9] of steroidal azides were also
studied. Recently, they were used in preparation of nitrogen hete-
rocycles [10] or as intermediates for click chemistry [11].
azide has been used also for preparation of 20-azidopregnane
derivatives [19–21]. In this case, concurrent elimination reac-
tion was noticed and the mechanism and solvent influence were
studied [21].
In connection with broader project on neuroactive steroids we
started with preparation of analogs of pregnanolone, modified in
position 17. We designed analogs with the 20-keto pregnane side
chain replaced with azido group only or with one or two carbon
side chain bearing azido group. The rest of neurosteroid structure
has to be conserved, so derivatives of 3␣-hydroxy-5␤-androstane,
21-nor-5␤-pregnane or 5␤-pregnane are to be synthesized.
In position 17 of androstane skeleton, 17␣-azides are acces-
sible by sulfonate substitution by sodium azide or Mitsunobu
reaction with azoimide. The first approach was applied to 5␣-
androstane derivatives with 3-keto group [12], 3␤-acetoxy group
[13], or 3␤-acetoxy-5-ene moiety [14]. On 3-keto-4-ene deriva-
tives, effects of various solvents [15] or sulfonate leaving groups
[16] were studied. So far mentioned examples covered substi-
tution of pseudo-equatorial 17␤-sulfonate with sodium azide to
give 17␣-azide. Also substitution in 14␤-androstane series was
described [17], where leaving 17␣-sulfonate gave 17␤-azide. The
second approach, Mitsunobu reaction, was applied on 3-keto-4-ene
2. Experimental
2.1. General
Melting points were determined on a Boetius micro melting
point apparatus (Germany) and are uncorrected. Optical rota-
tions were measured at 25 ◦C on a AUTOPOL IV polarimeter
(Rudolph Research Analytical, USA), and [␣]D values are given in
10−1 deg cm2 g−1. Infrared spectra (wavenumbers in cm−1) were
recorded on a Bruker IFS 88 spectrometer. 1H and 13C NMR spec-
tra were taken on Bruker AVANCE-400 instrument at 23 ◦C in
deuterochloroform and referenced to tetramethylsilane as the
internal standard. 13C NMR spectra for pregnane derivatives 15a
and 15b were measured on Bruker AVANCE-500 instrument under
the above conditions, secondary referencing was performed using
the solvent signal at position ı(CDCl3) = 77.0. In addition to 1D
proton and carbon NMR spectra, homonuclear 2D-spectra (H,H-
PFG-COSY, ROESY and J-resolved) together with heteronuclear
2D-spectra (H,C-PFG-HSQC) were used for complete structural
assignment of signals (Table 1). Chemical shifts are given in ppm
(ı-scale); coupling constants (J) and width of multiplets (W) are
given in Hz.
∗
Corresponding author. Tel.: +420 220 183 385; fax: +420 220 183 385.
E-mail address: cerny@uochb.cas.cz (I. Cerny´).
ˇ
0039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.04.008

1044
L. Vidrna et al. / Steroids 76 (2011) 1043–1050
Table 1
Carbon-13 chemical shifts of selected azides in CDCl3.
Carbon
3a
37.12
3b
37.00
4a
4b
9
10
15a
15b
1
2
3
4
5
6
7
8
35.47
30.60
71.80
36.47
42.08
27.09
26.73
36.16
40.29
34.66
20.36
32.79
45.99
49.64
24.64
28.65
71.62
17.67
23.32
–
35.40
30.55
71.74
36.43
42.05
27.01
26.13
35.98
40.59
34.68
20.37
37.44
44.57
52.23
23.59
27.00
71.38
12.27
23.33
–
37.06
37.18
35.43
30.59
71.81
36.49
42.12
27.14
26.77
35.72
40.77
34.69
20.51
38.22
42.22
49.86
24.59
26.51
55.74
12.50
23.36
52.91
–
35.29
30.48
71.78
36.38
41.98
27.09
26.34
35.56
40.38
34.56
20.53
39.24
42.21
56.22
24.09
26.86
56.07
12.31
23.32
61.12
19.54
35.32
30.54
71.84
36.38
42.03
27.11
26.44
35.74
40.47
34.58
20.62
38.74
42.70
55.80
24.34
26.52
55.22
12.40
23.35
59.09
18.84
37.18
212.91
42.31
44.30
26.53
26.08
35.84
40.55
34.99
20.74
32.71
45.95
49.50
24.57
28.66
71.51
17.65
22.63
–
37.14
212.84
42.29
44.20
26.46
25.50
35.69
40.92
34.99
20.77
37.34
44.58
52.18
23.55
26.99
71.27
12.31
22.64
–
213.02
42.35
44.29
26.69
25.86
35.42
41.06
34.99
20.91
38.14
42.25
55.68
24.54
26.58
49.80
12.51
22.66
52.80
–
9
10
11
12
13
14
15
16
17
18
19
20
21
–
–
–
–
Thin-layer chromatography (TLC) was performed on silica gel
G (ICN Biochemicals), detection by spraying with concentrated
sulfuric acid followed by heating. For column chromatography, Sil-
ica gel 60 (Merck, 63–100 ␮m) was used. Prior to evaporation on
a rotary evaporator in vacuo (0.25 kPa, bath temperature 40 ◦C),
solutions in organic solvents were dried over anhydrous sodium
sulfate.
17␤-Hydroxy-5␤-androstan-3-one (1a), 17␣-hydroxy-5␤-
androstan-3-one (1b), 3-oxo-21-nor-5␤-pregnan-20-yl pivalate
(6), and (20R)- and (20S)-20-hydroxy-5␤-pregnan-3␣-yl acetates
(12a and 12b) were prepared according to Refs. [22–25].
2.2.3. 17˛-Azido-5ˇ-androstan-3-one (3a)
A. Tosylate 2a (4.0 g, 9.0 mmol) and sodium azide (4.0 g,
61.5 mmol) in hexamethylphosphoramide (25 mL) in argon atmo-
sphere were stirred and heated to 90 ◦C for 60 h. The reaction
mixture was diluted with ethyl acetate (200 mL) and washed
successively with water, 10% sulfuric acid, saturated aqueous potas-
sium hydrogen carbonate, and water again. After drying, the solvent
was evaporated leaving crude oily product (2.9 g). Chromatography
on a column of silica gel (70 mL) in benzene yielded 2.47 g (74%)
of oily azide 3a. After crystallization from ethanol, 1.55 g (55%) of
product 3a was obtained, m.p. 85–86 ◦C, [␣]D −23 (c 0.38, chloro-
form). IR: 2101 (N3); 1708 (C O); 1382 (CH3). 1H NMR: 3.54 dd, 1H,
J = 0.6, Jꢀ = 6.6 (H-17␤); 2.70 dd, 1H, J = 13.6, Jꢀ = 14.8 (H-4␣); 1.02 s,
3H (3× H-19); 0.77 s, 3H (3× H-18). For 13C NMR data see Table 1.
Calcd. for C19H29N3O (315.5): C, 72.34; H, 9.27; N, 13.32. Found: C,
72.46; H, 9.14; N, 13.28.
2.2. Chemical synthesis
2.2.1. 3-Oxo-5ˇ-androstan-17ˇ-yl tosylate (2a)
To a stirred solution of hydroxy derivative 1a (3.0 g, 10.3 mmol)
in pyridine (50 mL) cooled with ice-water bath, tosyl chloride (6.0 g,
31.5 mmol) was added in portions. After all reagent dissolved, the
cooling bath was removed and the reaction mixture was left aside
at room temperature overnight. Then the mixture was poured into
water with ice (500 mL) and the product was filtered off and dried
on air. After trituration with ethanol (30 mL), the crude product
(4.2 g, 91%) was used further without purification. Analytical sam-
ple was crystallized from a mixture of chloroform/diethyl ether,
m.p. 201–203 ◦C. 1H NMR: 7.78 m, 2H (H-2ꢀ, H-6ꢀ); 7.32 m, 2H (H-
3ꢀ, H-5ꢀ); 4.27 dd, 1H, J = 9.2, Jꢀ = 7.8 (H-17␣); 2.62 dd, 1H, J = 15.0,
Jꢀ = 13.6 (H-4␣); 2.45 s, 3H (CH3); 1.00 s, 3H (3× H-19); 0.82 s, 3H
(3× H-18). Calcd. for C26H36O4S (444.6): C, 70.23; H, 8.16. Found:
C, 70.50; H, 8.21.
B. The stirred solution of hydroxy derivative 1a (1.0 g,
3.44 mmol) and triphenylphosphine (1.08 g, 4.12 mmol) in benzene
(10 mL) under argon was cooled with ice bath and azoimide in ben-
zene (3 mL of solution prepared from 650 mg sodium azide, 0.8 mL
water, 4 mL benzene, and 0.28 mL conc. sulfuric acid, Ref. [26]) was
added. Then, diethyl azodicarboxylate (40% solution in toluene,
1.81 mL, 3.98 mmol) was added dropwise and the reaction mix-
ture was heated to 70 ◦C for 4 h. After cooling, the solvents were
evaporated and chromatography on silica gel column (50 mL) in
petroleum ether/diethyl ether (10:1) yielded 670 mg (62%) of oily
azide 3a, identical with the product from procedure A.
2.2.4. 17ˇ-Azido-5ˇ-androstan-3-one (3b)
A. Tosylate 2b (2.3 g, 5.2 mmol), sodium azide (2.5 g, 38.5 mmol),
and hexamethylphosphoramide (15 mL) were processed accord-
ing to procedure in Section 2.2.3A Crude oily product (1.5 g) was
chromatographed on a silica gel column (150 mL) in a mixture of
petroleum ether/acetone (50:1). Firstly, a mixture of eliminated
products was eluted (700 mg). Further elution with petroleum
ether/acetone (20:1) yielded 450 mg (27%) of oily azide 3b, which
spontaneously crystallize. M.p. 67–68 ◦C, [␣]D +35 (c 0.35, chloro-
form). IR: 2101 (N3); 1708 (C O); 1388, 1381 (CH3). 1H NMR: 3.34
t, 1H, J = 9.0 (H-17␣); 2.67 dd, 1H, J = 15.1, Jꢀ = 13.4 (H-4␣); 1.03 s,
3H (3× H-19); 0.78 s, 3H (3× H-18). For 13C NMR data see Table 1.
Calcd. for C19H29N3O (315.5): C, 72.34; H, 9.27; N, 13.32. Found: C,
72.43; H, 9.15; N, 13.21.
2.2.2. 3-Oxo-5ˇ-androstan-17˛-yl tosylate (2b)
Hydroxy derivative 1b (2.0 g, 6.9 mmol), pyridine (13 mL), and
tosyl chloride (4.0 g, 20.1 mmol) were processed according to pro-
cedure in Section 2.2.1 only at 5 ◦C for 24 h. The mixture was then
poured into water with ice (400 mL) and the product was extracted
with ethyl acetate (100 mL). The ethyl acetate extract was washed
with 10% aqueous citric acid, saturated aqueous potassium hydro-
gen carbonate (2×), water and dried. Solvents were evaporated and
the crude foamy tosylate 2b (3.0 g, 98%) was immediately processed
further. 1H NMR: 7.78 m, 2H (H-2ꢀ, H-6ꢀ); 7.34 m, 2H (H-3ꢀ, H-5ꢀ);
4.49 d, 1H, J = 5.6 (H-17␤); 2.68 bt, 1H, J = 14.1 (H-4␣); 2.45 s, 3H
(CH3); 1.00 s, 3H (3× H-19); 0.70 s, 3H (3× H-18).

L. Vidrna et al. / Steroids 76 (2011) 1043–1050
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B. Hydroxy derivative 1b (100 g, 0.34 mmol), triphenylphos-
phine (108 mg, 0.41 mmol) in benzene (1 mL), azoimide in benzene
(from 65 mg sodium azide), and diethyl azodicarboxylate (40% solu-
tion in toluene, 180 ␮L, 0.40 mmol) were processed according to
procedure in Section 2.2.3B at 70 ◦C for 2 h. Chromatography on
silica gel column (10 mL) in petroleum ether/diethyl ether (10:1)
yielded 70 mg of a mixture of eliminated products and 20 mg (18%)
of oily azide 3b, identical with the product from procedure A.
175 mg (59%), m.p. 148–154 ◦C, [␣]D +35 (c 0.43, chloroform). IR:
2543 (N+–H); 2100 (N3); 1638, 1548, 1490 (pyridine ring); 1388,
1375 (CH3); 1261, 1174, 1049 (SO3−). 1H NMR: 8.98 m, 2H (H-2ꢀ,
H-6ꢀ); 8.49 tt, 1H, J = 1.6, Jꢀ = 7.8 (H-4ꢀ); 8.01 m, 2H (H-3ꢀ, H-5ꢀ); 4.47
tt, 1H, J = 4.9, Jꢀ = 11.3 (H-3␤); 3.31 t, 1H, J = 9.0 (H-17␣); 0.92 s, 3H
(3× H-19); 0.73 s, 3H (3× H-18). Calcd. for C24H36N4O4S (476.6): C,
60.48; H, 7.61; N, 11.75. Found: C, 60.27; H, 7.62; N, 11.61.
2.2.9. 20-Hydroxy-21-nor-5ˇ-pregnan-3-one (7)
2.2.5. 17˛-Azido-5ˇ-androstan-3˛-ol (4a)
3-Oxo-21-nor-5␤-pregnan-20-yl pivalate (6, 1.36 g, 3.50 mmol)
in benzene (17 mL) was refluxed in argon atmosphere with a solu-
tion of potassium hydroxide (3.5 g, 62.38 mmol) in 96% ethanol
(70 mL) for 6 h and was left aside at room temperature overnight.
The reaction mixture was poured into 5% hydrochloric acid
(120 mL) with ice and extracted with ethyl acetate (120 mL). The
extract was washed with aqueous saturated potassium hydrogen
carbonate and water, dried, and the solvents were evaporated.
Crude product (1 g) was chromatographed on a silica gel col-
umn (100 mL) in a mixture of petroleum ether/benzene/acetone
(20:20:1) to give 720 mg (68%) of hydroxy derivative 7. Analytical
sample was crystallized from acetone, m.p. 166–168 ◦C, [␣]D +25
(c 0.34, chloroform). IR: 3623, 3474 (OH); 1707 (C O); 1384 (CH3).
1H NMR: 3.73 ddd, 1H, J = 5.5, Jꢀ = 6.7, Jꢀꢀ = 10.8 (H-20); 3.56 ddd, 1H,
J = 4.3, Jꢀ = 7.5, Jꢀꢀ = 10.8 (H-20ꢀ); 2.70 dd, 1H, J = 13.7, Jꢀ = 14.7 (H-4␣);
1.03 s, 3H (3× H-19); 0.68 s, 3H (3× H-18). Calcd. for C20H32O2
(304.5): C, 78.90; H, 10.59. Found: C, 78.98; H, 10.80.
Azide 3a (1.1 g, 3.49 mmol) was dissolved in a mixture of ethyl
acetate (4 mL) and methanol (12 mL) and cerium(III) chloride hep-
tahydrate (1.43 g, 3.84 mmol) was added to a stirred solution. After
all the cerium(III) chloride was completely dissolved, sodium boro-
hydride (132 mg, 3.49 mmol) was added in portions over 5 min at
room temperature. The reaction mixture was stirred for 15 min and
then poured into 5% hydrochloric acid and extracted with ethyl
acetate (80 mL). The ethyl acetate extract was washed with sat-
urated aqueous potassium hydrogen carbonate (2×), water and
dried. Solvents were evaporated and the crude product (1.14 g) was
crystallized from acetone. The yield of azide 4a was 900 mg (81%),
m.p. 135–137 ◦C, [␣]D −25 (c 0.34, chloroform). IR: 3609, 3448 (OH);
2100 (N3); 1381 (CH3); 1033 (C–OH). 1H NMR: 3.62 tt, 1H, J = 4.8,
Jꢀ = 11.1 (H-3␤); 3.50 dd, 1H, J = 0.7, Jꢀ = 6.7 (H-17␤); 0.92 s, 3H (3×
H-19); 0.73 s, 3H (3× H-18). For 13C NMR data see Table 1. Calcd.
for C19H31N3O (317.5): C, 71.88; H, 9.84; N, 13.24. Found: C, 71.99;
H, 10.02; N, 13.01.
2.2.10. 3-Oxo-21-nor-5ˇ-pregnan-20-yl tosylate (8)
2.2.6. 17ˇ-Azido-5ˇ-androstan-3˛-ol (4b)
Hydroxy derivative 7 (485 mg, 1.59 mmol) in pyridine (10 mL)
with tosyl chloride (1.0 g, 5.0 mmol) was processed as described
in Section 2.2.1. Then the mixture was poured into water with ice
(100 mL) and the product was extracted with ethyl acetate (60 mL).
The ethyl acetate extract was washed with 5% hydrochloric acid,
saturated aqueous potassium hydrogen carbonate (2×), water and
dried. Solvents were evaporated and the crude foamy tosylate 8
(700 g, 96%) was used without further purification. 1H NMR: 7.79
m, 2H (H-2ꢀ, H-6ꢀ); 7.35 m, 2H (H-3ꢀ, H-5ꢀ); 4.06 dd, 1H, J = 7.6,
Jꢀ = 9.6(H-20);3.95dd, 1H, J = 6.7, Jꢀ = 9.6(H-20ꢀ);2.68dd, 1H, J = 13.5,
Jꢀ = 14.8 (H-4␣); 2.46 m, 3H (CH3); 1.01 s, 3H (3× H-19); 0.59 s, 3H
(3× H-18).
Azide 3b (400 mg, 1.27 mmol), ethyl acetate (2 mL), methanol
(6 mL), cerium(III) chloride heptahydrate (650 mg, 1.74 mmol), and
sodium borohydride (60 mg, 1.59 mmol) were processed accord-
ing to procedure in Section 2.2.5. Crude product (380 mg) was
crystallized from acetone giving 245 mg (61%) of azide 4b, m.p.
135–136 ◦C, [␣]D +27 (c 0.47, chloroform). IR: 3609, 3452 (OH);
2100 (N3); 1387, 1378 (CH3); 1033 (C–OH). 1H NMR: 3.64 tt, 1H,
J = 4.7, Jꢀ = 11.0 (H-3␤); 3.32 t, 1H, J = 9.0 (H-17␣); 0.93 s, 3H (3× H-
19); 0.73 s, 3H (3× H-18). For 13C NMR data see Table 1. Calcd. for
C19H31N3O (317.5): C, 71.88; H, 9.84; N, 13.24. Found: C, 72.09; H,
9.98; N, 13.26.
2.2.7. 17˛-Azido-5ˇ-androstan-3˛-yl sulfate pyridinium salt
(5a)
2.2.11. 20-Azido-21-nor-5ˇ-pregnan-3-one (9)
Tosylate 8 (690 mg, 1.5 mmol), sodium azide (0.8 g, 12.3 mmol),
and hexamethylphosphoramide (10 mL) were processed according
to procedure in Section 2.2.3A, only reaction time was 4 h. Crude
oily product (480 mg) was chromatographed on a silica gel col-
umn (100 mL) in a mixture of petroleum ether/benzene/acetone
(50:50:1) to afford 440 mg (89%) of azide 9. Crystallization from
ethanol yielded 320 mg (65%), m.p. 85–87 ◦C, [␣]D +7 (c 0.26, chlo-
roform). IR: 2098 (N3); 1707 (C O); 1390, 1381 (CH3). 1H NMR: 3.24
AB(ABX), 2H, JAB = 12.2 (H-20, H-20ꢀ); 2.69 dd, 1H, J = 14.9, Jꢀ = 13.6
(H-4␣); 1.03 s, 3H (3× H-19); 0.66 s, 3H (3× H-18). For 13C NMR
data see Table 1. Calcd. for C20H31N3O (329.5): C, 72.91; H, 9.48; N,
12.75. Found: C, 73.00; H, 9.59; N, 12.54.
To a solution of azide 4a (200 mg, 0.63 mmol) in pyridine (5 mL)
complex pyridine–sulfur trioxide (350 mg, 2.20 mmol) was added
and the mixture was stirred in argon atmosphere and heated to
70 ◦C for 1 h. After cooling, the solvent was evaporated and the
semisolid residue was shortly boiled with hot water (4 mL). The
mixture was cooled in ice-bath and injected on to semi-preparative
column of Lichrosorb RP-18 (75 mL). Polar impurities were washed
out with water and the product was eluted with 95% aqueous
methanol. The crude sulfate (280 mg) was crystallized from a mix-
ture of methanol/diethyl ether. The yield of 5a was 155 mg (52%),
m.p. 165–169 ◦C, [␣]D −7 (c 0.42, chloroform). IR: 2535 (N+–H);
2100 (N3); 1637, 1549, 1490 (pyridine ring); 1381 (CH3); 1262,
1172, 1049 (SO3−). 1H NMR: 8.99 m, 2H (H-2ꢀ, H-6ꢀ); 8.50 tt, 1H,
J = 1.5, Jꢀ = 7.9 (H-4ꢀ); 8.03 m, 2H (H-3ꢀ, H-5ꢀ); 4.47 tt, 1H, J = 4.6,
Jꢀ = 11.3 (H-3␤); 3.50 dd, 1H, J = 0.7, Jꢀ = 6.7 (H-17␤); 0.91 s, 3H (3× H-
19); 0.72 s, 3H (3× H-18). Calcd. for C24H36N4O4S (476.6): C, 60.48;
H, 7.61; N, 11.75. Found: C, 60.65; H, 7.58; N, 11.57.
2.2.12. 20-Azido-21-nor-5ˇ-pregnan-3˛-ol (10)
Azide 9 (400 mg, 1.21 mmol), ethyl acetate (2 mL), methanol
(6 mL), cerium(III) chloride heptahydrate (480 mg, 1.29 mmol), and
sodium borohydride (46 mg, 1.21 mmol) were processed accord-
ing to procedure in Section 2.2.5. Crude product (390 mg) was
crystallized from acetone giving 240 mg (60%) of azide 10, m.p.
118–119 ◦C, [␣]D +3 (c 0.24, chloroform). IR: 3609, 3448 (OH); 2097
(N3); 1390, 1379 (CH3); 1033 (C–OH). 1H NMR: 3.63 ddt, 1H, J = 11.4,
Jꢀ = 15.8, Jꢀꢀ = 4.6 (H-3␤); 3.22 AB(ABX), 2H, JAB = 12.2 (H-20, H-20ꢀ);
0.93 s, 3H (3× H-19); 0.62 s, 3H (3× H-18). For 13C NMR data see
2.2.8. 17ˇ-Azido-5ˇ-androstan-3˛-yl sulfate pyridinium salt
(5b)
Azide 4b (200 mg, 0.63 mmol) was processed according to pro-
cedure in Section 2.2.7. The crude sulfate (287 mg) was crystallized
from a mixture of methanol/diethyl ether. The yield of 5b was

1046
L. Vidrna et al. / Steroids 76 (2011) 1043–1050
Table 1. Calcd. for C20H33N3O (331.5): C, 72.46; H, 10.03; N, 12.68.
Found: C, 72.59; H, 10.09; N, 12.56.
(31%) of azide 14b. Analytical sample was crystallized from ace-
tone/heptane, m.p. 107–108 ◦C, [␣]D −11 (c 0.14, chloroform). IR:
2105 (N3); 1721 (C O); 1379, 1364 (CH3); 1257, 1028 (C–O). 1H
NMR: 4.72 tt, 1H, J = 4.8, Jꢀ = 11.3 (H-3); 3.13 dq, 1H, J = 10.8, Jꢀ = 6.4
(H-20); 2.03 s, 3H (CH3CO); 1.26 d, 3H, J = 6.4 (3× H-21); 0.94 s, 3H
(3× H-19); 0.68 s, 3H (3× H-18). Calcd. for C23H37N3O2 (387.6): C,
71.28; H, 9.62; N, 10.84. Found: C, 71.59; H, 9.85; N, 10.50.
2.2.13. 20-Azido-21-nor-5ˇ-pregnan-3˛-yl sulfate pyridinium
salt (11)
Azide 10 (100 mg, 0.30 mmol), pyridine (2.5 mL), and com-
plex pyridine–sulfur trioxide (175 mg, 1.10 mmol) were processed
according to procedure in Section 2.2.7. The crude sulfate (130 mg)
was crystallized from a mixture of methanol/diethyl ether. The
yield of 11 was 85 mg (57%), m.p. 166–170 ◦C, [␣]D +35 (c 0.43,
chloroform). IR: 2543, 2022 (N+–H); 2097 (N3); 1628, 1549, 1490
(pyridine ring); 1379 (CH3); 1262, 1173, 1048 (SO3−). 1H NMR: 8.97
m, 2H (H-2ꢀ, H-6ꢀ); 8.49 tt, 1H, J = 1.5, Jꢀ = 7.9 (H-4ꢀ); 8.00 m, 2H (H-3ꢀ,
H-5ꢀ); 4.47 tt, 1H, J = 4.8, Jꢀ = 11.3 (H-3␤); 3.21 AB(ABX), 2H, JAB = 12.2
(H-20, H-20ꢀ); 0.92 s, 3H (3× H-19); 0.61 s, 3H (3× H-18). Calcd. for
2.2.18. (20S)-20-Azido-5ˇ-pregnan-3˛-ol (15a)
Azide 14a (135 mg, 0.35 mmol) in ethanol (27 mL) was stirred
with
a solution of sodium hydroxide (21 mg, 0.53 mmol) in
methanol (1 mL) at 40 ◦C for 2 h. Then, the reaction mixture was
poured into water (100 mL) and extracted with ethyl acetate (3×
50 mL). Collected organic phase was washed with water (100 mL),
5% hydrochloric acid (2× 50 mL), saturated aqueous sodium hydro-
gen carbonate (2× 50 mL), water (100 mL), and dried. Solvent was
evaporated to give alcohol 15a (114 mg, 95%): m.p. 120–123 ◦C, [␣]D
+51 (c 0.10, chloroform). IR: 3609 (OH); 2103 (N3); 1378 (CH3);
1031 (C–O). 1H NMR: 3.63 tt, 1H, J = 4.6, Jꢀ = 11.0 (H-3); 3.26 dq, 1H,
J = 8.9, Jꢀ = 6.4 (H-20); 1.32 d, 3H, J = 6.5 (3× H-21); 0.92 s, 3H (3× H-
C25H38N4O4S (490.7): C, 61.20; H, 7.81; N, 11.21. Found: C, 61.35;
H, 7.86; N, 11.21.
2.2.14. (20R)-5ˇ-Pregnan-3˛,20-diyl 3-acetate 20-tosylate (13a)
(20R)-Alcohol 12a (2.4 g, 6.62 mmol), pyridine (40 mL), and tosyl
chloride (4.0 g, 21.0 mmol) were processed according to procedure
in Section 2.2.1. The reaction mixture was then poured into water
with ice (400 mL) and the product was extracted with chloroform
(3× 50 mL). The extract was washed with 5% hydrochloric acid, sat-
urated aqueous sodium hydrogen carbonate (2×), water and dried.
Solvents were evaporated to afford (20R)-tosylate 13a (3.29 g, 96%):
m.p. 148–150 ◦C. IR: 1721 (C O); 1382, 1362 (CH3); 1258, 1251
(C–O); 1175 (SO2). 1H NMR: 7.79 m, 2H (H-2ꢀ, H-6ꢀ); 7.32 m, 2H
(H-3ꢀ, H-5ꢀ); 4.74 dq, 1H, J = 9.8, Jꢀ = 6.3 (H-20); 4.72 tt, 1H, J = 4.9,
Jꢀ = 11.3 (H-3); 2.44 s, 3H (CH3); 2.03 s, 3H, (CH3CO), 1.18 d, 3H,
J = 6.1 (3× H-21); 0.92 s, 3H (3× H-19); 0.66 s, 3H (3× H-18). Calcd.
for C30H44O5S (516.7): C, 69.73; H, 8.58. Found: C, 70.03; H, 8.76.
19); 0.66 s, 3H (3× H-18). Calcd. for C21
H35N3O (345.5): C, 73.00;
H, 10.21; N, 12.16. Found: C, 73.10; H, 10.36; N, 11.49.
2.2.19. (20R)-20-Azido-5ˇ-pregnan-3˛-ol (15b)
Azide 14b (154 mg, 0.40 mmol) in ethanol (31 mL) and sodium
hydroxide (24 mg, 0.60 mmol) in methanol (1 mL) were processed
according to procedure in Section 2.2.18 to give alcohol 15b
(128 mg, 93%): m.p. 147–149 ◦C, [␣]D −29 (c 0.16, chloroform). IR:
3609 (OH); 2105 (N3); 1378 (CH3); 1031 (C–O). 1H NMR: 3.63 tt,
1H, J = 4.6, Jꢀ = 10.9 (H-3); 3.13 dq, 1H, J = 10.7, Jꢀ = 6.4 (H-20); 1.26 d,
3H, J = 6.5 (3× H-21); 0.93 s, 3H (3× H-19); 0.68 s, 3H (3× H-18).
Calcd. for C21H35N3O (345.5): C, 73.00; H, 10.21; N, 12.16. Found:
C, 73.01; H, 10.36; N, 11.87.
2.2.15. (20S)-5ˇ-Pregnan-3˛,20-diyl 3-acetate 20-tosylate (13b)
(20S)-Alcohol 12b (1.2 g, 3.31 mmol), tosyl chloride (1.28 g,
6.71 mmol), and pyridine (20 mL) were processed according to pro-
cedure in Section 2.2.1 to afford (20S)-tosylate 13b (1.58 g, 92%),
m.p. 134–136 ◦C (Ref. [27] 124–126 ◦C). IR: 1722 (C O); 1381, 1362
(CH3); 1256, 1028 (C–O); 1175 (SO2). 1H NMR: 7.78 m, 2H (H-2ꢀ, H-
6ꢀ); 7.32 m, 2H (H-3ꢀ, H-5ꢀ); 4.71 tt, 1H, J = 4.9, Jꢀ = 11.6 (H-3); 4.67
dq, 1H, J = 9.6, Jꢀ = 6.2 (H-20); 2.44 s, 3H (CH3); 2.02 s, 3H, (CH3CO),
1.31 d, 3H, J = 6.2 (3× H-21); 0.91 s, 3H (3× H-19); 0.62 s, 3H (3× H-
18). Calcd. for C30H44O5S (516.7): C, 69.73; H, 8.58. Found: C, 70.02;
H, 8.87.
2.2.20. (20S)-20-Azido-5ˇ-pregnan-3˛-yl sulfate pyridinium salt
(16a)
A
mixture of azide 15a (100 mg, 0.29 mmol) and sulfur
trioxide–pyridine complex (200 mg, 1.26 mmol) was stirred in
freshly dried (phosphorus pentoxide) chloroform (5 mL) at room
temperature for 6 h. After overnight-standing at −20 ◦C, the undis-
solved sulfur trioxide–pyridine complex was quickly filtered off
and the filtrate was evaporated to afford sulfate 16a (87 mg, 60%):
m.p. 194–196 ◦C, [␣]D +36 (c 0.27, chloroform). IR: 2800–2200
(N+–H); 2103 (N3); 1637, 1548, 1489 (pyridine ring); 1378 (CH3);
1257, 1174, 1048 (SO3). 1H NMR: 8.98 m, 2H (H-2ꢀ, H-6ꢀ); 8.48 tt,
1H, J = 1.5, Jꢀ = 7.8 (H-4ꢀ); 8.00 m, 2H (H-3ꢀ, H-5ꢀ); 4.47 tt, 1H, J = 4.8,
Jꢀ = 11.2 (H-3); 3.26 dq, 1H, J = 9.5, Jꢀ = 6.5 (H-20); 1.31 d, 3H, J = 6.5
(3× H-21); 0.91 s, 3H (3× H-19); 0.65 s, 3H (3× H-18). Calcd. for
C26H40N4O4S (504.7): C, 61.88; H, 7.99; N, 11.10. Found: C, 61.58;
H, 8.10; N, 10.86.
2.2.16. (20S)-20-Azido-5ˇ-pregnan-3˛-yl acetate (14a)
Tosylate 13a (1.02 g, 1.97 mmol), sodium azide (1.29 g,
19.84 mmol), and hexamethylphosphoramide (10 mL) were pro-
cessed according to procedure in Section 2.2.3A at 50 ◦C for 3 h.
Crude oily product (750 mg) was chromatographed on a silica gel
column (100 mL) in a mixture of petroleum ether/diethyl ether
(100:1). Less polar fractions contained products of elimination
(300 mg) and further elution afforded 420 mg (55%) of azide 14a.
Analytical sample was crystallized from acetone, m.p. 109–110 ◦C,
[␣]D +62 (c 0.43, chloroform). IR: 2103 (N3); 1722 (C O); 1380,
1364 (CH3); 1257, 1027 (C–O). 1H NMR: 4.72 tt, 1H, J = 4.8, Jꢀ = 11.4
(H-3); 3.27 dq, 1H, J = 9.2, Jꢀ = 6.4 (H-20); 2.03 s, 3H (CH3CO); 1.32
d, 3H, J = 6.5 (3× H-21); 0.93 s, 3H (3× H-19); 0.66 s, 3H (3× H-18).
Calcd. for C23H37N3O2 (387.6): C, 71.28; H, 9.62; N, 10.84. Found:
C, 71.56; H, 9.75; N, 10.54.
2.2.21. (20R)-20-Azido-5ˇ-pregnan-3˛-yl sulfate pyridinium salt
(16b)
Azide 15b (50 mg, 0.14 mmol), sulfur trioxide–pyridine com-
plex (200 mg, 1.26 mmol) and chloroform (5 mL) were processed
according to procedure in Section 2.2.20 to give sulfate 16b (40 mg,
55%): m.p. 172–174 ◦C, [␣]D +16 (c 0.20, chloroform). IR: 2800–2200
(N+–H); 2105 (N3); 1628, 1549, 1490 (pyridine ring); 1379 (CH3);
1255, 1175, 1050 (SO3). 1H NMR: 8.96 m, 2H (H-2ꢀ, H-6ꢀ); 8.45 tt,
1H, J = 1.5, Jꢀ = 7.8 (H-4ꢀ); 7.96 m, 2H (H-3ꢀ, H-5ꢀ); 4.47 tt, 1H, J = 4.8,
Jꢀ = 11.2 (H-3); 3.13 dq, 1H, J = 10.8, Jꢀ = 6.3 (H-20); 1.25 d, 3H, J = 6.4
(3× H-21); 0.92 s, 3H (3× H-19); 0.67 s, 3H (3× H-18). Calcd. for
2.2.17. (20R)-20-Azido-5ˇ-pregnan-3˛-yl acetate (14b)
Tosylate 13b (500 mg, 0.97 mmol), sodium azide (0.65 g,
10.0 mmol), and hexamethylphosphoramide (5 mL) were pro-
cessed according to procedure in Section 2.2.3A to afford 115 mg
C26H40N4O4S (504.7): C, 61.88; H, 7.99; N, 11.10. Found: C, 61.60;
H, 8.11; N, 10.97.

L. Vidrna et al. / Steroids 76 (2011) 1043–1050
1047
OH
OTs
i)
O
O
H
H
H
ii)
1a, 17β-isomer;
1b, 17α-isomer
2a, 17β-isomer;
2b, 17α-isomer
iii)
N3
N3
iv)
HO
O
H
v)
3a, 17α-isomer;
3b, 17β-isomer
4a, 17α-isomer;
4b, 17β-isomer
N3
O
O
S
N+
H
O
-O
H
5a, 17α-isomer;
5b, 17β-isomer
Scheme 1. Syntheses of 17-azido 5␤-androstanes. (i) TsCl/Py, r.t. (5 ◦C), o.n. (24 h); (ii) NaN3/HMPA, 90 ◦C, 60 h (16 h); (iii) Ph3P, HN3, DEAD/benzene, 70 ◦C, 4 h (2 h); (iv)
NaBH4, CeCl3·7H2O/EtOAc, MeOH, r.t., 15 min; and (v) SO3-Py/Py, 70 ◦C, 1 h. Temperatures or times for 17␤-series if different are given in parentheses.
2.3. Biological assays
3. Results and discussion
For the electrophysiological experiments on NMDA receptors,
human embryonic kidney cells (HEK293) were transfected with
NR1-1a/NR2B/GFP plasmids as described previously [28]. Exper-
iments were performed 24–48 h after the end of transfection.
Receptor cells were voltage-clamped at a holding potential of
−60 mV and currents elicited were recorded. Whole-cell voltage-
3.1. 17-Azido 5ˇ-androstanes
For the preparation of steroidal azides in sterically hindered
position 17, corresponding tosylates were treated with sodium
azide in hexamethylphosphoramide (HMPA). From both isomers,
17␣-azide is more easily available (Scheme 1). Starting from 17␤-
hydroxy-5␤-androstan-3-one (1a), available from testosterone
[22], tosylate 2a was prepared. The nucleophilic substitution with
sodium azide took 60 h at 90 ◦C for completion. Resulting 17␣-
azido-5␤-androstan-3-one (3a) was obtained in 74% yield. In this
case, the orientation of 17␤-hydroxy group in 1a is pseudo-
equatorial, so Mitsunobu reaction with azoimide was used for
comparison. After 4 h at 70 ◦C, azide 3a, identical with that orig-
inated from tosylate 2a, was obtained in 62% yield.
The preparation of 17␤-azido derivative is more complicated.
Firstly, starting 17␣-hydroxy derivative 1b (Scheme 1) is to be
prepared from 17␤-isomer 1a by the reaction of corresponding
tosylate with sodium nitrite [23] or by the Mitsunobu reaction with
4-nitrobenzoic acid [30,31], and both approaches has by-products
clamp recordings were made with
a patch-clamp amplifier
(Axopatch 1D; Axon Instruments, Inc., Foster City, CA). Patch
pipettes (3–5 Mꢀ) pulled from borosilicate glass were filled with
Cs+-based intracellular solution (Cs-ICS) containing the following
(in mM): 125 gluconic acid, 15 CsCl, 5 EGTA, 10 HEPES, 3 MgCl2, 0.5
CaCl2, and 2 ATP-Mg salt (pH-adjusted to 7.2 with CsOH). Extra-
cellular solution (ECS) contained the following: (in mM): 160 NaCl,
2.5 KCl, 10 HEPES, 10 glucose, 0.2 EDTA, and 0.7 CaCl2 (pH-adjusted
to 7.3 with NaOH). Glycine (10 ␮M), a NMDA receptor co-agonist,
was present in the control and test solutions. All steroids solutions
were made from freshly prepared 20 mM stock in dimethyl sul-
foxide (DMSO). The same concentration of DMSO was added in all
extracellular solutions (see [29] for details).

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L. Vidrna et al. / Steroids 76 (2011) 1043–1050
OPiv
OH
i)
O
O
H
H
ii)
6
7
N3
OTs
iii)
v)
O
O
H
H
iv)
9
8
N3
N3
O
O
-O
S
HO
O
H
H
10
11
N+
H
Scheme 2. Syntheses of 20-azido 21-nor-5␤-pregnanes. (i) KOH/benzene, EtOH, rfl., 6 h; (ii) TsCl/Py, r.t., o.n.; (iii) NaN3/HMPA, 90 ◦C, 4 h; (iv) NaBH4,CeCl3·7H2O/EtOAc,
MeOH, r.t., 15 min; and (v) SO3-Py/Py, 70 ◦C, 1 h.
and gave only moderate yields. Tosyl derivative 2b was susceptible
to elimination reactions and it is better to keep it at lower tem-
perature and use gentle and quick work-up. Also treatment of 2b
with sodium azide is accompanied by elimination and rearrange-
ment reactions. Less polar fraction is formed by a mixture which
was not separated and contained eliminated products (for similar
side products on testosterone series see Refs. [16,32]). Desired 17␤-
azide 3b was obtained in 27% yield. For comparison, Mitsunobu
reaction with azoimide was performed, and regardless of pseudo-
axial orientation of hydroxy group in starting 1b, 18% of azide 3b
was obtained beside excess of eliminated products.
Ketone in azides 3a and 3b was reduced with sodium boro-
hydride in presence of cerium(III) chloride heptahydrate [25] to
suppress formation of 3␤-hydroxy derivative and 17␣-azido- and
17␤-azido-5␤-androstan-3␣-ols (4a and 4b) were prepared in 81%
and 61% yields, respectively. For the preparation of sulfates 5a and
5b, reaction with sulfur trioxide–pyridine complex in pyridine was
used and resulting products were purified on preparative C-18
reverse phase column.
(6). So obtained 20-hydroxyderivative 7 was transformed into tosy-
late 8 and subjected to azide displacement with sodium azide in
hexamethylphosphoramide. In this case, the reaction proceeded
smoothly and after 4 h at 90 ◦C was complete and gave 89% of azide
9. Reduction of 3-keto group was performed similarly to androstane
series with sodium borohydride and cerium(III) chloride heptahy-
drate and 3␣-hydroxy derivative 10 was prepared. Subsequent
reaction with sulfur trioxide–pyridine complex in pyridine gave
sulfate 11.
In pregnane series, a little different approach was adopted.
Acetates 12a and 12b, available by cerium(III) chloride mediated
borohydride reductions [25], were used as starting compounds
(Scheme 3). Tosylates 13a and 13b were prepared by standard
method used previously, gentle work-up is necessary also in
this case. Both tosylates are more reactive than 17-tosylates in
androstane series and treating with sodium azide in hexam-
ethylphosphoramide at 50 ◦C for 3 h gave azides 14a and 14b in
55% and 31% yields, respectively. In both cases, products from
elimination side-reactions were observed. In agreement with liter-
ature [21], hexamethylphosphoramide gave better yields of desired
azide: in preliminary experiments on preparation of 14a dimethyl-
sulfoxide and N,N-dimethylformamide were checked and lower
yields were obtained (DMSO/90 ◦C/6 h: 36%; DMF/90 ◦C/3 h: 43%).
Protecting acetyl group was removed from 14a and 14b with
methanolic sodium hydroxide and hydroxy derivatives 15a and 15b
3.2. 20-Azido 21-nor-5ˇ-pregnanes and 5ˇ-pregnanes
Starting compound for a synthesis of 20-azido 21-nor-pregnane
series (Scheme 2) was prepared by hydrolysis of protecting pivalate
group from known [24] 3-oxo-21-nor-5␤-pregnan-20-yl pivalate

L. Vidrna et al. / Steroids 76 (2011) 1043–1050
1049
OH
OTs
H
H
i)
AcO
AcO
H
H
12a, (20R)-isomer;
12b, (20S)-isomer;
ii)
13a, (20R)-isomer;
13b, (20S)-isomer;
N3
N3
H
H
iv)
O
O
-O
S
RO
O
H
H
14a, (20S)-isomer, R = Ac;
15a, (20S)-isomer, R = OH
iii)
iii)
N+
H
16a, (20S)-isomer;
16b, (20R)-isomer;
14b, (20R)-isomer; R = Ac;
15b, (20R)-isomer; R = OH
Scheme 3. Syntheses of 20-azido 5␤-pregnanes. (i) TsCl/Py, r.t., o.n.; (ii) NaN3/HMPA, 50 ◦C, 3 h; (iii) NaOH/MeOH, EtOH, 40 ◦C, 2 h; (iv) SO3-Py/CHCl3, r.t., 6 h.
Table 2
Azide 11
Glu
Inhibition of Glu-induced response in recombinant NMDA receptors cells by preg-
nanolone sulfate azido analogs.
Compounda
Conc. [␮M]
% Inhib. S.D.b
IC50 (␮M) S.D.
nc
17␣N3A 5a
17␤N3A 5b
20N3nP 11
20SN3P 16a
20RN3P 16b
10
5
15
100
100
85.0 2.9
68.8 9.7
83.9 6.4
85.0 7.6
37.5 5.2
2.4 0.5
2.7 1.0
3.0 1.4
24.0 11.7
154.8 21.9
6
6
5
5
6
1nA
2 s
a
Skeleton and position/configuration of azido group were indicated (A – 5␤-
androstane, nP – 21-nor-5␤-pregnane, P – 5␤-pregnane).
b
Relative degree of steroid inhibition of current responses induced in NR1-
1a/NR2B receptors cells by fast application of 100 ␮M glutamate.
c
Fig. 1. Example of traces obtained from HEK293 cells transfected with NR1-1a/NR2B
receptor. Azide 11 (15 ␮M) was applied simultaneously with 1 mM glutamate (dura-
tion of steroid and glutamate application is indicated by filled and open bars,
respectively).
Number of cells studied. Results are expressed as mean standard deviation
(S.D.).
hippocampal neurons (71.3 5.0% inhibition, IC50 = 47.2 9.9, Ref.
[33]). Surprisingly the potency of compounds 5a, 5b, and 11 was
approximately 10-fold higher.
were obtained. Corresponding sulfates 16a and 16b were prepared
with sulfur trioxide–pyridine complex in dry chloroform.
3.3. Biological activity
4. Conclusion
Synthetic analogs of pregnanolone sulfate were tested on
responses mediated by NMDA receptors. Fig. 1 shows NR1-1a/NR2B
receptor responses induced in HEK293 cells by 1 mM glutamate and
that induced by glutamate co-application with azide sulfate 11. At
a concentration of 15 ␮M this steroid inhibited the responses by
83.9 6.4% (n = 5) corresponding to IC50 = 3.0 1.4 ␮M. The values
of relative inhibition and estimated IC50 values of steroids 5a, 5b,
11, 16a, and 16b are listed in Table 2. The data indicate that the
value of IC50 varied for pregnanolone sulfate azido analogs 65-fold.
The activity of pregnane derivatives 16a and 16b was compara-
ble with previously published data for pregnanolone sulfate on the
same receptors (IC50 = 44.4 9.8 ␮M, Ref. [29]) and also on cultured
Syntheses of pregnanolone analogs with side chain in position
17 modified or replaced with azido group were developed. All five
pregnanolone analogs were prepared by nucleophilic substitution
of corresponding tosylates with sodium azide in hexamethylphos-
phoramide. In some cases, concurrent elimination reactions were
observed so yields of this key reaction varied between 27% and 89%.
For 17-azides 3a and 3b also alternative Mitsunobu reaction with
azoimide was checked with comparable yields. Sulfates, intended
for tests on NMDA receptor, were prepared by reactions with sul-
fur trioxide–pyridine complex in yields from 52% to 60%. All azido
steroid sulfates tested showed inhibitory activity on responses
induced by glutamate on NMDA receptor; in some cases higher

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L. Vidrna et al. / Steroids 76 (2011) 1043–1050
than that reported for pregnanolone sulfate [29]. Our results have
shown that in case of pregnanolone sulfate, azido group can fully
or partially replace side chain with oxygen function in position
17 without disturbing of inhibitory activity. These findings may
contribute to the further study of NMDA receptor.
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nature des solvants sur la mecanisme et la stereochimie de l’azidolyse des
tosyloxy-20 ␤ steroides. Application a la synthese d’amino-20 ␣ steroides. Bull
Soc Chim France 1967;6:2100–4.
Acknowledgments
We are indebted to Dr. M. Budeˇsˇínsky´ for help with interpreting
of NMR spectra and especially for full assignment of 13C NMR spec-
tra of pregnane azides 15a and 15b. We thank Mrs. L. Kloucˇková and
Mrs. D. Hybsˇová for skillful technical assistance. Dr. S. Vasˇícˇková
was taking the IR spectra and Dr. P. Fiedler was interpreting them.
Mass spectra were measured by the staff of the Laboratory of Mass
Spectrometry (Dr. J. Cvacˇka, Head). Elemental analyses were carried
out in the Analytical Laboratory (Dr. S. Mateˇjková, Head). This work
was carried out as a part of the CAS research projects Z4 055 0506
and Z5 011 0509, and supported by the projects LC06077, LC554,
1M0002375201 (MSMT) and 203/08/1498, 309/07/0271 (GACR).
[20] Goutarel R, Conreur C, Djakouré L, Leboeuf M, Cavé A. Alcaloides stéroidiques
– LXXVII: syntheses de mono- et di-amines stéroidiques. Alcaloides A (sara-
codine) et
B (saracocine) du Sarcococca pruniformis lindl. Tetrahedron
1968;24:7013–26.
[21] Leboeuf M, Cave A, Goutarel R. Alcaloides steroidiques LXXXVI. Substitutions,
eliminations et transpositions des tosyloxy-20 steroides. 1er partie: role de
l’hexamethylphosphotriamide (HMPT) dans la substitution et l’elimination.
Bull Soc Chim France 1969;5:1619–24.
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