Synthesis of C3, C5, and C7 pregnane derivatives and their effect on NMDA receptor responses in cultured rat hippocampal neurons

Article abstract of DOI:10.1016/j.steroids.2008.11.011

The synthesis of several novel 5α- and 5β-20-oxo-pregnane derivatives substituted in the position 3 and 7 of the steroid skeleton is described. Activity of synthesized compounds was studied in voltage-clamped cultured rat hippocampal neurons. Substituted

Full text of DOI:10.1016/j.steroids.2008.11.011

Steroids 74 (2009) 256–263
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of C3, C5, and C7 pregnane derivatives and their effect on NMDA
receptor responses in cultured rat hippocampal neurons
Eva Stastnaa,b, Hana Chodounskaa,∗, Vladimir Pouzara, Vojtech Kaprasa, Jirina Borovskaa,c
,
Ondrej Caisc, Ladislav Vyklicky Jr.c
a Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i.,166 10 Prague 6, Czech Republic
b Department of Organic and Nuclear Chemistry, Faculty of Science, Charles University, 128 43 Prague 2, Czech Republic
c Institute of Physiology, Academy of Sciences of the Czech Republic, 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:
The synthesis of several novel 5␣- and 5␤-20-oxo-pregnane derivatives substituted in the position 3 and
7 of the steroid skeleton is described. Activity of synthesized compounds was studied in voltage-clamped
cultured rat hippocampal neurons. Substituted derivatives inhibited NMDA-elicited neuronal activity.
The relationship between biological activity and structure is discussed.
Received 23 September 2008
Received in revised form 31 October 2008
Accepted 15 November 2008
Available online 24 November 2008
© 2008 Published by Elsevier Inc.
Keywords:
Pregnane derivatives
NMDA receptor
Structure–activity relationship
Patch-clamp recording
1. Introduction
devoted to recognition of the corresponding binding sites and some
of them were identified for the GABAA receptor [11], but not for
Neurosteroids are synthesized in the nervous tissue from choles-
terol and/or modified to neuroactive compounds from circulating
precursors [1]. They do not act through classic steroid hormone
nuclear receptors, but through neuronal membrane receptors for
essential transmitters (e.g. ␥-amino-butyric acid (GABA) and the
N-methyl-d-aspartate (NMDA) receptors), connected with ion-
channels. Such neurosteroids have been proposed to control
neuronal excitability by modulating ligand and/or voltage-gated
ion channels [2,3] and this action has been shown to affect a lot
of physiological processes (learning [4], aging [5], stress [6], etc.) as
well as certain neurological and psychiatric disorders (Alzheimer’s
disease [7], epilepsy [8], etc.).
Defining a binding site of neurosteroids and the knowledge
of the molecular mechanism present a molecular template for
design and development of novel therapeutic entities. The molec-
ular mechanism by which neurosteroids affect ligand-gated ion
channels is still not clearly understood. In general, neurosteroids
influence the frequency of single channel openings and the average
channel open duration [9,10]. Similarly, much attention has been
NMDA receptor. The results of current studies indicate that neuros-
teroids have their specific binding sites, independent of particular
agonists or other allosteric modulators [12,13].
The knowledge of NMDA receptor’s binding site is not as all-
embracing as for the GABA receptor; nevertheless, the effect of
neurosteroids on NMDA receptor seems to be mediated by inde-
pendent binding sites located at the extracellular domain of the
NMDA receptor [14,15]. The experiments with chimeric recep-
tors have shown crucial role of an extracellular loop between
the third and fourth transmembrane domains of the NR2 subunit
in the mechanism of potentiating and inhibitory effects of 20-
oxo-pregn-5-en-3␤-yl sulfate (PS) and 20-oxo-5␤-pregnan-3␣-yl
sulfate (3␣5␤S) [15,16].
Without doubt, a lot of other aspects of the receptor struc-
ture will influence the nature and extent of the neurosteroid
modulation: the stereochemistry of the neurosteroids should be
mentioned. Previous structure–activity studies have reported sev-
eral features which are important for activity of neurosteroids at
NMDA receptors: the structure should include a sulfonyloxy group
at the position 3 and a 20-oxo group as well [17]. The poten-
tiating effect is maintained if the sulfate group is replaced by
another negatively charged group, e.g. hemioxalate, hemisuccinate
or hemiglutarate [12,18].
∗
Corresponding author at: Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic, Flemingovo 2, 166 10 Prague 6, Czech
Republic.
Our research is focused on the synthesis of pregnane deriva-
tives as possible inhibitors of the NMDA receptor. This synthesis
E-mail address: hchod@uochb.cas.cz (H. Chodounska).
0039-128X/$ – see front matter © 2008 Published by Elsevier Inc.
doi:10.1016/j.steroids.2008.11.011

E. Stastna et al. / Steroids 74 (2009) 256–263
257
ketone); 1263, 1220 (C O). 1H NMR (200 MHz): 0.60 s, 3H (3 × H-
18); 0.79 (s, 3H, 3 × H-19); 2.05 (s, 3H, OAc); 2.12 (s, 3H, 3 × H-21);
2.56 (t, 1H, J = 8.8, H-17); 3.87 (m, 1H, H-7); 5.03 (quintet, 1H, J = 2.9,
H-3). FAB MS: 399 (12%, M+Na), 359 (8%, M+1−H2O), 299 (80%,
M+1−H2O, AcOH). C23H36O4: calcd. C, 73.37; H, 9.64. Found. C,
73.17; H, 9.76.
is a part of a research project searching for the binding site
of the given receptor via medicinal chemistry—exploring the
structure–activity-relationship. In the present report, we describe
the synthesis of some 3␣,7␣-derivatives of 5␣- and 5␤-pregnan-20-
one [(3␣5␣7AcHS (5), 3␣5␣7AcS (6), 3␣5␣7NicS (11), 3␣5␤7AcHS
(17), 3␣5␤7AcS (18), and 3␣5␤7NicS (23)] that were tested as
inhibitors of the NMDA receptor by the patch-clamp technique.
2.1.2.3. 3˛-Hydroxy-20-oxo-5˛-pregnan-7˛-yl acetate (4). A solu-
2. Experimental
tion of diacetate 2 (90 mg, 0.22 mmol) in benzene (10 mL)
was treated with a solution of potassium hydroxide (15.4 mg,
0.27 mmol) in methanol (1 mL). After standing overnight at room
temperature, the mixture was poured into water and extracted
with ethyl acetate (50 mL). The extract was washed with water,
dried and the solvent was evaporated. The residue was purified
by plate thin layer chromatography (2 plates) in a mixture of ace-
tone/petroleum ether (1:1) to give compound 4 (69 mg, 85%): m.p.
148–150 ◦C (ether/petroleum ether), [˛]D + 64.5 (c 0.346, CHCl3). IR
spectrum (CHCl3): 3616 (O H); 1717 (C O, acetate); 1701 (C O,
ketone); 1256 (C O). 1H NMR (200 MHz): 0.59 (s, 3H, 3 × H-18);
0.78 (s, 3H, 3 × H-19); 2.07 (s, 3H, OAc); 2.11 (s, 3H, 3 × H-21); 2.55
(t, 1H, J = 8.8, H-17); 4.06 (quintet, 1H, J = 2.4, H-3); 4.91 (q, 1H,
J = 2.9, H-7). FAB MS: 399 (17%, M+Na), 317 (8%, M+1−AcOH), 299
(9%, M+1−AcOH, H2O). C23H36O4: calcd. C, 73.37; H, 9.64. Found. C,
73.70; H, 9.99.
2.1. Chemistry
2.1.1. General
Melting points were determined on a micro-melting point
apparatus Hund/Wetzlar (Germany) and are uncorrected. Optical
rotations were measured using an Autopol IV (Rudolf Research Ana-
lytical, Flanders, USA), [˛]D values are given in 10−1 ◦ cm2 g−1, IR
spectra were recorded on a Bruker IFS 88 spectrometer (wavenum-
bers in cm−1). Proton NMR spectra were measured on a FT NMR
spectrometer Varian UNITY-200 (at 200 MHz) or on a FT NMR
spectrometer Bruker AVANCE-400 (at 400 MHz) in CDCl3 with
tetramethylsilane as the internal standard. Chemical shifts are given
in ppm (ı-scale), coupling constants (J) and width of multiplets
(W) are given in Hz. Mass spectra were obtained with spectrome-
ters ZAB-EQ (at 70 eV) or LCQ Classic (Thermo Finnigan). Thin-layer
chromatography (TLC) was performed on silica gel (ICN Biochemi-
cals), preparative TLC (PLC) was carried out on 200 mm × 200 mm
plates coated with a 0.4 mm thick layer of the same material. For
column chromatography, silica gel 60–120 ␮m was used. Analyti-
cal samples were dried over phosphorus pentoxide at 50 ◦C/100 Pa.
Pyridine was dried by distillation over potassium hydroxide and
chloroform by distillation over phosphorus pentoxide.
2.1.2.4. 20-Oxo-5˛-pregnane-3˛,7˛-diyl
3-hemisuccinate
7-
acetate (5, 3˛5˛7AcHS). A mixture of compound
4
(100 mg,
0.27 mmol) and succinic anhydride (100 mg, 1 mmol) was dried
in vacuo (25 ◦C, 100 Pa) for 30 min. Dry pyridine (10 mL) and
4-dimethylaminopyridine (20 mg, 0.17 mmol) were added. The
mixture was heated for 6 h at 140 ◦C. Additional succinic anhy-
dride (200 mg, 2 mmol) and 4-dimethylaminopyridine (20 mg,
0.17 mmol) were added and the mixture was heated for 10 h at
140 ◦C. The reaction mixture was poured into water and extracted
with ethyl acetate (50 mL). Aqueous phase was extracted again
with ethyl acetate (50 mL), and the collected extracts dried. The
solvent was evaporated and the residue crystallized from hot ethyl
acetate to give hemisuccinate 5 (71 mg, 35%): m.p. 193–195 ◦C,
[˛]D + 43.3 (c 0.246, CHCl3). IR spectrum (CHCl3): 3516, 3100 broad
(COOH); 1717 (C O, acetate); 1701 (C O, ketone); 1379 (CH3); 1257,
1248 (C O, acetate). 1H NMR (200 MHz): 0.60 (s, 3H, 3 × H-18);
0.80 (s, 3H, 3 × H-19); 2.08 (s, 3H, OAc); 2.12 (s, 3H, 3 × H-21); 2.54
(t, 1H, J = 8.8, H-17); 2.60–2.71 (m, 4H, OOCCH2CH2COO); 4.92 (m,
1H, H-3); 5.06 (q, 1H, J = 2.4, H-7). FAB MS: 499 (100%, M+Na), 417
(13%, M+1−AcOH), 299 (55%, M−AcOH, C4H5O4). C27H40O7: calcd.
C, 68.04; H, 8.46. Found. C, 67.83; H, 8.46.
Whenever aqueous solution of citric acid was used, the concen-
tration was always 5%. Aqueous solution of potassium hydrogen
carbonate was used as a saturated solution. Before evaporation
on a rotary evaporator in vacuo (bath temperature 50 ◦C, pressure
1.5 kPa), solutions of organic solvents were dried over anhydrous
sodium sulfate.
2.1.2. Synthesis of 5˛-pregnane derivatives
2.1.2.1. 20-Oxo-5˛-pregnane-3˛,7˛-diol (1). This compound was
prepared according to the literature [19].
2.1.2.2. 20-Oxo-5˛-pregnane-3˛,7˛-diyl diacetate (2) and 7˛-
hydroxy-20-oxo-5˛-pregnan-3˛-yl acetate (3). To
a solution of
dihydroxy derivative 1 (100 mg, 0.3 mmol) in pyridine (5 mL),
acetic anhydride (0.42 mL, 4.4 mmol) was added and the mixture
was heated for 6 h at 50 ◦C. After standing at room temperature
for 2 days, the reaction mixture was poured into ice-water and
extracted with ethyl acetate (50 mL). Extract was washed with
solution of potassium hydrogen carbonate, water, and dried.
Solvent was evaporated and the oily residue purified by plate thin
layer chromatography (3 plates) in a mixture of acetone/petroleum
ether (1:1) to give diacetate 2 (67 mg, 54%) and monoacetate 3
(9 mg, 8%) as a side product.
2.1.2.5. 20-Oxo-5˛-pregnane-3˛,7˛-diyl 3-sulfate 7-acetate pyri-
dinium salt (6, 3˛5˛7AcS). The mixture of compound 4 (200 mg,
0.53 mmol) and
a sulfur trioxide pyridine complex (400 mg,
2.5 mmol), dried in vacuo (25 ◦C, 100 Pa) for 1 h, was dissolved
in dry chloroform (10 mL). The reaction mixture was stirred for
4 h at room temperature under argon. After standing overnight
at −20 ◦C, the undissolved sulfur trioxide pyridine complex was
filtered off. The solvent was evaporated and the residue was dis-
solved in absolute methanol (1 mL). The absolute ether (15 mL) was
added and the mixture was concentrated to almost one-half. After
standing overnight at −20 ◦C, the crystals were collected and dried
in a desiccator (over potassium hydroxide) to afford compound
6 (230 mg, 82%): m.p. 156–160 ◦C, [␣]D + 29.3 (c 0.288, CHCl3). IR
spectrum (CHCl3): 3072 (pyridinium); 1716 (C O, acetate); 1702
(C O, ketone); 1258, 1220 (C O, acetate); 1258 (S–O). 1H NMR
(400 MHz): 0.59 (s, 3H, 3 × H-18); 0.80 (s, 3H, 3 × H-19); 2.06 (s,
3H, OAc); 2.12 (s, 3H, 3 × H-21); 2.56 (t, 1H, J = 9.2, H-17); 4.78
(quintet, 1H, J = 2.6, H-3); 4.89 (q, 1H, J = 2.9, H-7); 7.98 (m, 2H,
Diacetate 2: m.p. 112–114 ◦C, [˛]D +21.8 (c 0.363, CHCl3). IR spec-
trum (CHCl3): 1726 (C O, acetate); 1702 (C O, ketone); 1259, 1241,
1030 (C O). 1H NMR (200 MHz): 0.60 (s, 3H, 3 × H-18); 0.80 (s, 3H,
3 × H-19); 2.05 (s, 3H, C(3)–OAc); 2.08 (s, 3H, C(7)–OAc); 2.12 (s, 3H,
3 × H-21); 2.54 (t, 1H, J = 8.8, H-17); 4.92 (q, 1H, J = 2.9, H-7); 5.03
(m, 1H, H-3). FAB MS: 399 (12%, M+Na), 359 (8%, M+1−H2O), 299
(80%, M+1−H2O, AcOH). C25H38O5: calcd. C, 71.74; H, 9.15. Found.
C, 71.98; H, 9.27.
Monoacetate 3: m.p. 174–178 ◦C, [˛]D + 30.6 (c 0.513, CHCl3). IR
spectrum (CHCl3): 3617 (O H); 1726 (C O, acetate); 1702 (C O,

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E. Stastna et al. / Steroids 74 (2009) 256–263
H-3 and H-5, pyridinium); 8.48 (tt, 1H, J1 = 7.8, J2 = 1.5, H-4, pyri-
dinium); 8.94 (m, 2H, H-2 and H-6, pyridinium). ESI MS: 574 (17%,
M+K), 494 (14%, M+K−pyridinium), 478 (21%, M+Na−pyridinium),
412 (15%, M−CH3CO, pyridinium), 341 (47%, M+Na−OSO3C5H6N,
CH3CO). HR-MS (+ESI) calcd. for C28H41NNaO7S [M++Na] 558.2496,
found 558.2502.
2.1.2.9. 3˛-Hydroxy-20-oxo-5˛-pregnan-7˛-yl
nicotinate
(10).
Compound 9 (120 mg, 0.21 mmol) was treated with a methanolic
solution of p-toluenesulfonic acid (0.005 M, 72 mL). After standing
at room temperature for 10 days, the mixture was neutralized with
10% potassium carbonate solution, extracted with ethyl acetate
(100 mL), organic layer was washed with water and dried. The
solvent was evaporated and the crude product purified by plate
thin layer chromatography (3 plates) in a mixture of petroleum
ether/acetone (8:2) to give 10 (83 mg, 87%): m.p. 183–187 ◦C (ace-
tone/heptane), [˛]D + 15.0 (c 0.169, CHCl3). IR spectrum (CHCl3):
3615 (O H); 1715 (C O, nicotinate); 1703 (C O, ketone); 1287
(C O, nicotinate); 1002 (C OH). 1H NMR (200 MHz): 0.72 (s, 3H,
3 × H-18); 0.86 (s, 3H, 3 × H-19); 2.25 (s, 3H, 3 × H-21); 2.53 (t,
1H, J = 8.8, H-17); 4.05 (quintet, 1H, J = 2.4, H-3); 5.25 (q, 1H, J = 2.8,
H-7); 7.44 (m, 1H, H-5, nicotinate); 8.30 (dt, 1H, J1 = 7.8, J2 = 1.9,
H-4, nicotinate); 8.78 (dd, 1H, J1 = 4.8, J2 = 1.4, H-6, nicotinate); 9.26
(m, 1H, H-2, nicotinate). ESI MS: 901 (100%, 2M+Na), 462 (93%,
M+Na), 440 (24%). C27H37NO4: calcd. C, 73.77; H, 8.48; N, 3.19.
Found. C, 73.66; H, 8.67; N, 3.04.
2.1.2.6. 3˛-(tert-Butyldimethylsilyloxy)-20-oxo-5˛-pregnan-7˛-yl
acetate (7). Alcohol 4 (528 mg, 1.4 mmol) and imidazole (570 mg,
8.37 mmol) were dissolved in N,N-dimethylformamide (11 mL)
and the mixture was cooled to 0 ◦C. Then, tert-butyldimethylsilyl
chloride was added (528 mg, 3.5 mmol). The reaction mixture
was allowed to attain room temperature and stirred. After 2 h,
the reaction was diluted with ethyl acetate (150 mL) and washed
with solution of citric acid, potassium hydrogen carbonate, and
water. The organic layer was dried and evaporated in vacuo. Crys-
tallization from ethyl acetate gave 687 mg (99%) of compound 7:
m.p. 120–121 ◦C, [˛]D + 29.0 (c 0.369, CHCl3). IR spectrum (CHCl3):
2897 ((CH3)2Si); 1715 (C O, acetate); 1701 (C O, ketone); 1472,
1463 ((CH3)3C); 1258 (C O, acetate); 1053 (C OSi). 1H NMR
(200 MHz): 0.02 (s, 6 H, 6 × (CH3)2Si); 0.59 (s, 3H, 3 × H-18); 0.76
(s, 3H, 3 × H-19); 0.89 (s, 9 H, (CH3)3C); 2.03 (s, 3H, OAc); 2.12 (s,
3H, 3 × H-21); 2.54 (t, 1H, J = 8.7, H-17); 3.97 (quintet, 1H, J = 2.9,
H-3); 4.89 (q, 1H, J = 2.4, H-7). ESI MS: 513 (83%, M+Na), 457 (20%,
M+Na−(CH3)3C), 353 (42%, M+Na−(CH3)3C(CH3)2SiO, CH3CO,
AcOH). C29H50O4Si: calcd. C, 70.97; H, 10.27. Found. C, 70.85; H,
10.41.
2.1.2.10. 20-Oxo-5˛-pregnane-3˛,7˛-diyl 3-sulfate 7-nicotinate pyri-
dinium salt (11, 3˛5˛7NicS). The method followed that described
for compound 6 but using alcohol 10 (60 mg, 0.13 mmol) and a sulfur
trioxide pyridine complex (120 mg, 0.75 mmol) in dry chloroform
(10 mL) afforded compound 11 (71 mg, 87%) as a foam: [␣]D + 6.0
(c 0.204, CHCl3). IR spectrum (CHCl3): 3457, 3139 (pyridinium);
1715 (C O, nicotinate); 1702 (C O, ketone); 1289 (C O, nicoti-
nate); 1243, 1046 (SO3). 1H NMR (400 MHz): 0.63 (s, 3H, 3 × H-18);
0.87 (s, 3H, 3 × H-19); 2.11 (s, 3H, 3 × H-21); 2.54 (t, 1H, J = 8.7, H-
17); 4.79 (quintet, J = 2.7, 1H, H-3); 5.25 (q, 1H, J = 2.5, H-7); 7.74
(dd, 1H, J1 = 7.8, J2 = 5.5, H-5, nicotinate); 7.83 (m, 2H, H-3 and H-
5, pyridinium); 8.30 (tt, 1H, J1 = 7.8, J2 = 1.5, H-4, pyridinium); 8.62
(dt, 1H, J1 = 7.8, J2 = 1.5, H-4, nicotinate); 8.84 (m, 2H, H-2 and H-6,
pyridinium); 8.93 (m, 1H, H-6, nicotinate); 9.29 (m, 1H, H-2, nicoti-
nate). FAB MS: 554 (3%, M−CH3CO), 440 (0.5%, M−2 × C5H6N), 422
(10%, M+1−OSO3C5H6N). HR-MS (+ESI) calcd. for C32H42N2NaO7S
[M++Na] 621.2605, found 621.2609.
2.1.2.7. 3˛-(tert-Butyldimethylsilyloxy)-7˛-hydroxy-5˛-pregnan-
20-one (8). The method followed that described for compound
4 but using acetate 7 (650 mg, 1.32 mmol) in benzene (50 mL)
and potassium hydroxide in ethanol (0.89 M, 60 mL) at 60 ◦C for
16 h. Compound 8 (527 mg, 88%): m.p. 135–140 ◦C (ethyl acetate),
[˛]D + 63.3 (c 0.232, CHCl3). IR spectrum (CHCl3): 3615 (O H);
1699 (C O, ketone); 1472, 1463 ((CH3)3C); 1253 ((CH3)2Si);
1052 (C OSi). 1H NMR (200 MHz): 0.02 (s, 6 H, (CH3)2Si); 0.59
(s, 3H, 3 × H-18); 0.88 (s, 12H, 3 × H-19 and (CH3)3C); 2.11
(s, 3H, 3 × H-21); 2.56 (t, 1H, J = 8.8, H-17); 3.83 (m, 1H, H-
7); 3.98 (m, 1H, H-3). ESI MS: 919 (100%, 2M+Na), 471 (50%,
M+Na). C27H48O3Si: calcd. C, 72.26; H, 10.78. Found. C, 72.20; H,
10.91.
2.1.2.11. 3˛-Hydroxy-5˛-pregnan-20-one (12). This compound was
prepared according to the literature [20].
2.1.2.12. 20-Oxo-5˛-pregnan-3˛-yl hemisuccinate (13, 3˛5˛HS).
The method followed that described for compound 5 but using
alcohol 12 (150 mg, 0.47 mmol) and succinic anhydride (150 mg,
1.5 mmol) in dry pyridine (15 mL) at reflux. After 7 h the reac-
tion was completed. Compound 13 (105 mg, 53%): m.p. 76–79 ◦C
(toluene), [˛]D + 77.7 (c 0.229, CHCl3). IR spectrum (CHCl3): 1727
(C O, ester); 1716 (C O, COOH dimer); 1700 (C O, ketone); 1188
(C O, ester). 1H NMR (400 MHz): 0.60 (s, 3H, 3 × H-18); 0.79
(s, 3H, 3 × H-19); 2.12 (s, 3H, 3 × H-21); 2.54 (t, 1H, J = 9, H-17);
2.64–2.69 (m, 4H, OOCCH2CH2COO); 5.05 (m, 1H, H-3). ESI MS: 457
(3.4% M+K), 441 (100% M+Na), 418 (7.78%, M). HR-MS (+ESI) calcd.
for C25H39O5 [M++1] 419.2792, found 419.2790. C25H38O5: calcd.,
71.74; H, 9.15. Found. C, 71.59; H, 9.32.
2.1.2.8. 3˛-(tert-Butyldimethylsilyloxy)-20-oxo-5˛-pregnan-
7˛-yl nicotinate (9). Compound
8 (450 mg, 1.0 mmol) and
4-dimethylaminopyridine (10 mg, 0.09 mmol) were dissolved
in pyridine (15 mL) and the solution was cooled to 0 ◦C. Nicotinoyl
chloride hydrochloride (900 mg, 5.0 mmol) was slowly added to
a stirred mixture in small portions. The reaction mixture was
stirred at room temperature for 3 h. Then, it was poured into water
(50 mL) and, after standing overnight at −20 ◦C, the precipitate
was separated by suction and subsequently dried in a desicca-
tor (over potassium hydroxide) overnight to yield compound 9
(498 mg, 89%): m.p. 147–151 ◦C, [˛]D + 14.5 (c 0.391, CHCl3). IR
spectrum (CHCl3): 2897 ((CH3)2Si); 1714 (C O, nicotinate); 1703
(C O, ketone); 1286 (C O, nicotinate); 1053 (C OSi). 1H NMR
(400 MHz): 0.05 (s, 6 H, (CH3)2Si); 0.64 (s, 3H, 3 × H-18); 0.72 (s,
9 H, (CH3)3C); 0.83 (s, 3H, 3 × H-19); 2.11 (s, 3H, 3 × H-21); 2.51
(t, 1H, J = 8.8, H-17); 3.96 (quintet, J = 2.4, 1H, H-3); 5.22 (q, 1H,
J = 2.6, H-7); 7.40 (ddd, 1H, J1 = 7.8, J2 = 4.8, J3 = 0.7, H-5, nicotinate);
8.29 (dt, 1H, J1 = 8, J2 = 2, H-4, nicotinate); 8.79 (dd, 1H, J1 = 4.8,
J2 = 1.8, H-6, nicotinate); 9.25 (m, 1H, H-2, nicotinate). FAB MS: 554
(36%, M+1), 496 (8%, M−(CH3)3C), 299 (4%, M−(CH3)3C(CH3)2SiO,
OCOC5H4N), 255 (79%, M−(CH3)3(CH3)2SiO, OCOC5H4N, CH3CO).
C33H51NO4Si: calcd. C, 71.56; H, 9.28; N, 2.53. Found. C, 71.40; H,
9.41; N, 2.25.
2.1.2.13. 20-Oxo-5˛-pregnan-3˛-yl sulfate pyridinium salt (14,
3˛5˛S). The method followed that described for compound 6 but
using alcohol 12 (200 mg, 0.6 mmol) and a sulfur trioxide pyridine
complex (400 mg, 2.5 mmol) in dry chloroform (5 mL). Compound
14 (254 mg, 85%): m.p. 181–183 ◦C (methanol/ether) (lit. 184 ◦C
[21]), [˛]D + 69.0 (c 0.21) (lit. + 70.0 [21]). IR spectrum (CHCl3): 3139
(pyridinium); 1699 (C O); 1261, 1253, 1237, 1194, 1171 (SO3 and
pyridinium). 1H NMR (400 MHz): 0.58 (s, 3H, 3 × H-18); 0.91 (s,
3H, 3 × H-19); 2.11 (s, 3H, 3 × H-21); 2.52 (t, 1H, J = 8.7, H-17); 4.45
(m, W = 30 Hz, H-3); 8.02 (m, 2H, H-3 and H-5, pyridinium); 8.50

E. Stastna et al. / Steroids 74 (2009) 256–263
259
(tt, 1H, J1 = 7.8, J2 = 1.5, H-4, pyridinium); 9.00 (m, 2H, H-2 and H-
0.56 mmol) in N,N-dimethylformamide (1.4 mL). After 1 h the reac-
tion was completed. Compound 19 (50 mg, 55%): m.p. 110–112 ◦C
(ethyl acetate), [˛]D + 30.5 (c 0.333, CHCl3). IR spectrum (CHCl3):
2955, 2906 ((CH3)3C); 1725 (C O, acetate); 1702 (C O, ketone);
1254, 1021 (C O, acetate); 1090, 1076 (C OSi); 853, 837 ((CH3)2Si).
1H NMR (200 MHz): 0.05 (s, 6 H, (CH3)2Si); 0.6 (s, 3H, 3 × H-18);
0.88 (s, 9 H, (CH3)3C); 0.91 (s, 3H, 3 × H-19); 2.04 (s, 3H, OAc); 2.12
(s, 3H, 3 × H-21); 2.54 (t, 1H, J = 8.7, H-17); 3.45 (m, 1H, W = 32, H-3);
3.87 (q, 1H, J = 2.4, H-7). FAB MS: 433 (2%, M−57, (CH3)3C), 373
(18%, M−(CH3)3C, AcOH), 299 (100%, M−(CH3)3(CH3)2Si, AcOH),
255 (12%, M−(CH3)3(CH3)2SiO, AcOH, CH3CO). C29H50O4Si: calcd.
C, 70.97; H, 10.27. Found. C, 70.65; H, 10.40.
6, pyridinium). FAB MS: 478 (11%, M+1). HR-MS (+ESI) calcd. for
C
26H39NNaO5S [M++Na] 500.2441, found 500.2437.
2.1.3. Synthesis of 5ˇ-pregnane derivatives
2.1.3.1. 20-Oxo-5ˇ-pregnane-3˛,7˛-diyl diacetate (15). This com-
pound was prepared according to the literature [22].
2.1.3.2. 3˛-Hydroxy-20-oxo-5ˇ-pregnan-7˛-yl acetate (16). A solu-
tion of diacetate 15 (100 mg, 0.24 mmol) in methanol (8 mL) was
treated with a solution of potassium hydrogen carbonate in water
(0.7 mL, 0.2 M) at 70 ◦C. After 5 h, the mixture was poured into
water and extracted with ethyl acetate (70 mL). The organic layer
was washed with water, dried and the solvents were evapo-
rated in vacuo. Crystallization from hot ether gave compound 16
(40 mg, 45%): m.p. 143–145 ◦C, [˛]D + 40.3 (c 0.303, CHCl3). IR spec-
trum (CHCl3): 3610, 3527 (O H); 1726 (C O, acetate); 1703 (C O,
ketone); 1252 (C O, acetate); 1047 (C O). 1H NMR (200 MHz): 0.61
(s, 3H, 3 × H-18); 0.93 (s, 3H, 3 × H-19); 2.06 (s, 3H, OAc); 2.12 (s, 3H,
3 × H-21); 2.55 (t, 1H, J = 8.8, H-17); 3.52 (m, 1H, W = 32.7, H-3); 4.89
(q, 1H, J = 2.4, H-7). FAB MS: 377 (5%, M+1), 317 (12%, M+1−AcOH),
299 (40%, M+1−AcOH, H2O), 283 (32%, M−AcOH, H2O, CH3), 255
(17%, M−AcOH, H2O, CH3CO), 159 (30%), 145 (34%), 131 (33%), 119
(37%). C23H36O4: calcd. C, 73.37; H, 9.64. Found. C, 73.49; H, 9.93.
2.1.3.6. 3˛-(tert-Butyldimethylsilyloxy)-7˛-hydroxy-5ˇ-pregnan-
20-one (20). The method followed that described for compound
4 but using acetate 19 (40 mg, 0.08 mmol) and solution of potas-
sium hydroxide in ethanol (0.89 M, 4 mL) in benzene (5 mL) at
reflux. After 3 h the reaction was completed. The residue was
purified by plate thin layer chromatography (2 plates) in a mix-
ture of petroleum ether/acetone (8:2) to give alcohol 20 (20 mg,
55%): m.p. 131–136 ◦C, [˛]D + 47.6 (c 0.29, CHCl3). IR spectrum
(CHCl3): 3621 (O H); 1699 (C O); 1472, 1463, 1385 ((CH3)3C);
1361 (Ac, (CH3)3C); 1254 ((CH3)2Si); 1098,1082 (C OSi). 1H NMR
(200 MHz): 0.05 (s, 6 H, (CH3)2Si); 0.60 (s, 3H, 3 × H-18); 0.88 (s,
12H, 3 × H-19 and (CH3)3C); 2.12 (s, 3H, 3 × H-21); 2.53 (t, 1H,
J = 9, H-17); 3.41 (m, 1H, W = 32.7, H-3); 3.86 (m, 1H, H-7). FAB
MS: 449 (11%, M+1), 317 (10%, M−(CH3)3(CH3)2SiO), 299 (26%,
M+1−(CH3)3(CH3)2SiO, H2O), 283 (13%, M+1−(CH3)3(CH3)2SiO,
H2O, O), 255 (14%, M−(CH3)3(CH3)2SiO, H2O, CH3CO). C27H48O3Si:
calcd. C, 72.26; H, 10.78. Found. C, 72.20; H, 10.84.
2.1.3.3. 20-Oxo-5ˇ-pregnane-3˛,7˛-diyl 3-hemisuccinate 7-acetate
(17, 3˛5ˇ7AcHS). The method followed that described for com-
pound 5 but using alcohol 16 (100 mg, 0.27 mmol), succinic
anhydride (200 mg, 2 mmol) and 4-dimethylaminopyridine (20 mg,
0.17 mmol) in dry pyridine (20 mL) at reflux. After 4 h the reaction
was completed. The residue purified by plate thin layer chromatog-
raphy (3 plates) in a mixture of petroleum ether/acetone (9:1) to
give compound 17 (44 mg, 35%): m.p. 131–134 ◦C, [˛]D + 50.3 (c
0.218, CHCl3). IR spectrum (CHCl3): 3517, 2676 broad (COOH); 1756
(C O, COOH); 1726 (C O, acetate); 1720 (C O, hemisuccinate);
1703 (C O, ketone); 1252 (C O, acetate); 1173 (C O, hemisuc-
cinate). 1H NMR (200 MHz): 0.61 (s, 3H, 3 × H-18); 0.94 (s, 3H,
3 × H-19); 2.06 (s, 3H, OAc); 2.12 (s, 3H, 3 × H-21); 2.54–2.69 (m,
5 H, H-17 and OOCCH2CH2COO); 4.62 (m, 1H, W = 31.8, H-3); 4.89
(q, 1H, J = 2.7, H-7). FAB MS: 499 (69%, M+Na), 417 (18%, M+1−AcOH),
299 (28%, M−AcOH, C4H5O4). HR-MS (+ESI) calcd. for C27H40NaO7
[M++Na] 499.2666, found 499.2664. C27H40O7: calcd. C, 68.04; H,
8.46. Found. C, 68.40; H, 8.60.
2.1.3.7. 3˛-(tert-Butyldimethylsilyloxy)-20-oxo-5ˇ-pregnan-7˛-
yl nicotinate (21). The method followed that described for
compound
9 but using alcohol 20 (240 mg, 0.53 mmol), 4-
dimethylaminopyridine (10 mg, 0.09 mmol) and nicotinoyl chloride
hydrochloride (720 mg, 4.0 mmol) in pyridine (10 mL). After 10 h
the reaction was completed. The crude product was purified
by plate thin layer chromatography (6 plates) in a mixture of
petroleum ether/acetone (9:1) to give compound 21 (208 mg, 70%):
m.p. 57–59 ◦C, [˛]D + 54.9 (c 0.387, CHCl3). IR spectrum (CHCl3):
2907 (CH3, (CH3)3(CH3)2SiO); 1714 (C O, nicotinate); 1703 (C O,
ketone); 1286, 1108 (C O, nicotinate); 1092 (C OSi). 1H NMR
(400 MHz): 0.05 (s, 6 H, (CH3)2Si); 0.63 (s, 3H, 3 × H-18); 0.77 (s, 9
H, (CH3)3C); 0.96 (s, 3H, 3 × H-19); 2.11 (s, 3H, 3 × H-21); 2.52 (t,
1H, J = 9.1, H-17); 3.43 (m, 1H, W = 32, H-3); 5.21 (q, 1H, J = 2.8, H-7);
7.44 (ddd, 1H, J1 = 7.8, J2 = 7.8, J3 = 0.7, H-5, nicotinate); 8.30 (dt, 1H,
J1 = 8.3, J2 = 2, H-4, nicotinate); 8.78 (dd, 1H, J1 = 4.8, J2 = 1.7, H-6,
nicotinate); 9.27 (dd, 1H, J1 = 2, J2 = 0.7, H-2, nicotinate). FAB MS:
579 (6%, M+Na), 554 (22%, M+1), 496 (7%, M−(CH3)3C), 299 (4%,
M−(CH3)3(CH3)2SiO, OCOC6H4N), 255 (79%, M−(CH3)3C(CH3)2SiO,
OCOC6H4N, CH3CO). C33H51NO4Si: calcd. C, 71.56; H, 9.28; N, 2.53.
Found. C, 71.33; H, 9.37; N, 2.31.
2.1.3.4. 20-Oxo-5ˇ-pregnan-3˛,7˛-diyl 3-sulfate 7-acetate pyri-
dinium salt (18, 3˛5ˇ7AcS). The method followed that described
for compound 6 but using alcohol 16 (110 mg, 0.3 mmol) and a
sulfur trioxide pyridine complex (220 mg, 1.4 mmol) in dry chlo-
roform (4 mL). The crystals of 18 were collected and dried in a
desiccator (over potassium hydroxide) for 5 days (155 mg, 99%):
m.p. 155–158 ◦C, [␣]D + 45.7 (c 0.285, CHCl3). IR spectrum (CHCl3):
3140 (pyridinium); 1725 (C O, acetate); 1702 (C O, ketone); 1253
(C O); 1363, 1171, 960 (O–SO3). 1H NMR (200 MHz): 0.60 (s, 3H,
3 × H-18); 0.92 (s, 3H, 3 × H-19); 2.04 (s, 3H, OAc); 2.56 (t, 1H,
J = 8.7, H-17); 4.34 (m, 1H, W = 31, H-3); 4.88 (q, 1H, J = 2.9, H-7);
7.97 (m, 2H, H-3 and H-5, pyridinium); 8.48 (tt, 1H, J1 = 7.8, J2 = 1.5,
H-4, pyridinium); 8.95 (m, 2H, H-2 and H-6, pyridinium). EI MS: 558
(0.5%, M+Na), 455 (0.5%, M−pyridinium), 256 (0.5%, M−pyridinium,
OSO3, CH3CO, AcOH), 230 (3%), 80 (100%). HR-MS (+ESI) calcd. for
2.1.3.8. 3˛-Hydroxy-20-oxo-5ˇ-pregnan-7˛-yl nicotinate (22). A
solution of protected compound 21 (100 mg, 0.18 mmol) in freshly
distilled THF (10 mL) was cooled to 0 ◦C and solution of tetrabuty-
lammonium fluoride (1 M in THF, 0.3 mL, 0.3 mmol) was added.
The mixture was stirred at room temperature. After 2 days, fur-
ther tetrabutylammonium fluoride solution (1 M in THF, 0.1 mL,
0.1 mmol) was added and the mixture was stirred for another 3
days. The mixture was diluted with ethyl acetate (100 mL), organic
layer was washed solution of citric acid, potassium hydrogen car-
bonate, water, and dried. The solvent was evaporated and the crude
product purified by plate thin layer chromatography (2 plates) in a
mixture of petroleum ether/acetone (9:1) to give 22 (58 mg, 74%):
m.p. 70–73 ◦C (acetone/heptane), [˛]D + 29.2 (c 0.279, CHCl3). IR
C
28H41NNaO7S [M++Na] 558.2496, found 558.2501.
2.1.3.5. 3˛-(tert-Butyldimethylsilyloxy)-20-oxo-5ˇ-pregnan-7˛-yl
acetate (19). The method followed that described for compound
7 but using hydroxy derivative 16 (70 mg, 0.18 mmol), imidazole
(76 mg, 1.1 mmol) and tert-butyldimethylsilyl chloride (84 mg,

260
E. Stastna et al. / Steroids 74 (2009) 256–263
spectrum (CHCl3): 3613; 3451 (O H); 1715 (C O, nicotinate); 1705
(C O, ketone); 1287 (C O, nicotinate); 1034, 1026 (C OH); 1592,
1421 (nicotinate). 1H NMR (400 MHz): 0.64 (s, 3H, 3 × H-18); 0.98
(s, 3H, 3 × H-19); 2.12 (s, 3H, 3 × H-21); 2.54 (t, 1H, J = 9.2, H-17);
3.49 (m, 1H, W = 31, H-3); 5.20 (q, 1H, J = 2.7, H-7); 7.45 (ddd, 1H,
J1 = 7.8, J2 = 4.8, J3 = 0.7, H-5, nicotinate); 8.31 (dt, 1H, J1 = 7.8, J2 = 1.7,
H-4, nicotinate); 8.80 (dd, 1H, J1 = 4.8, J2 = 1.7, H-6, nicotinate); 9.26
(dd, 1H, J1 = 2, J2 = 0.7, H-2, nicotinate). FAB MS: 440 (12%, M+1), 145
(7%), 124 (100%), 105 (35%). C27H37NO4: calcd. C, 73.77; H, 8.48; N,
3.19. Found. C, 73.67; H, 8.80; N, 2.98.
2.2. Biological assays
2.2.1. Cell culture
Primary dissociated hippocampal cultures were prepared from
1- to 2-day-old postnatal rats. Animals were decapitated and the
hippocampi dissected. Trypsin digestion, followed by mechani-
cal dissociation, was used to prepare cell suspension. Single cells
were plated at a density of 500,000 cells/cm2 on 31 mm or 12 mm
polylysine-coated glass cover slips. Neuronal cultures were main-
tained in NeurobasalTM-A (Invitrogen, Carlsbad, USA) medium
supplemented with glutamine (0.5 mM) and B-27 Serum-Free Sup-
plement (Invitrogen) at 37 ◦C and 5% CO2.
2.1.3.9. 20-Oxo-5ˇ-pregnane-3˛,7˛-diyl 3-sulfate 7-nicotinate pyri-
dinium salt (23, 3˛5ˇ7NicS). The method followed that described
for compound 6 but using alcohol 22 (45 mg, 0.1 mmol) and a sul-
fur trioxide pyridine complex (90 mg, 0.56 mmol) in freshly distilled
chloroform (5 mL). After 4 h the reaction was completed to afford
23 (40 mg, 74%) as a white foam: [˛]D + 42.0 (c 0.321, CHCl3). IR
spectrum (CHCl3): 3139, 2652 (pyridinium); 1714 (C O, nicotinate);
1703 (C O, ketone); 1287 (C O, nicotinate); 1175, 978, 958 (O–SO3).
1H NMR (400 MHz): 0.63 (s, 3H, 3 × H-18); 0.98 (s, 3H, 3 × H-19);
2.11 (s, 3H, 3 × H-21); 2.56 (t, 1H, J = 8.7, H-17); 4.32 (m, 1H, W = 32,
H-3); 5.26 (m, 1H, H-7); 7.74 (dd, 1H, J1 = 7.6, J2 = 5.3, H-5, nicoti-
nate); 7.82 (m, 2H, H-3 and H-5, pyridinium); 8.33 (t, 2H, J = 7.8,
H-4, pyridinium); 8.60 (d, 1H, J = 7.6, H-4, nicotinate); 8.88 (m,
2H, H-2 and H-6, pyridinium); 8.93 (d, 1H, J = 5, H-6, nicotinate);
9.33 (m, 1H, H-2, nicotinate). FAB MS: 564 (10%), 542 (20%), 519
(12%, M+1−pyridinium), 444 (18%), 422 (10%, M+1−OSO3C5H6N).
HR-MS (+ESI) calcd. for C32H42N2NaO7S [M++Na] 621.2605, found
621.2608.
2.2.2. Electrophysiology
For the electrophysiological experiments, neurons cultured for
5–10 days were used. Whole-cell voltage-clamp recordings were
made with a patch-clamp amplifier (Axopatch 1D; Axon Instru-
ments, Inc., Foster City, USA) after capacitance and series resistance
(<10 Mꢀ) compensation of 80–90%. Agonist-induced responses
were low-pass filtered at 1 kHz with an 8-pole Bessel filter (Fre-
quency Devices, Haverhill, USA), digitally sampled at 5 kHz and
analyzed using pClamp software version 9 (Axon Instruments).
Patch pipettes (3–6 Mꢀ) pulled from borosilicate glass were filled
with Cs+-based intracellular solution containing (in mM): 125 glu-
conic acid, 15 CsCl, 5 EGTA, 10 HEPES, 3 MgCl2, 0.5 CaCl2, and 2
ATP-Mg salt (pH-adjusted to 7.2 with CsOH). Extracellular solution
(ECS) contained (in mM): 160 NaCl, 2.5 KCl, 10 HEPES, 10 D-glucose,
0.2 EDTA and 0.7 CaCl2 (pH-adjusted to 7.3 with NaOH). Glycine
(10 ␮M) and TTX (0.5 ␮M) were present in the control and test
solutions. Steroids were dissolved in dimethyl sulfoxide (DMSO) to
make a fresh stock solution (20 mM) before each experiment. The
same concentration of DMSO was maintained in all extracellular
solutions. A microprocessor-controlled multibarrel fast perfusion
system, with a time constant of solution exchange around cells of
∼10 ms, was used to apply test and control solutions.
2.1.3.10. 3˛-Hydroxy-5ˇ-pregnan-20-one (24). This compound was
prepared according to the literature [20].
2.1.3.11. 20-Oxo-5ˇ-pregnan-3˛-yl hemisuccinate (25, 3˛5ˇHS).
The method followed that described for compound 5 but using alco-
hol 24 (100 mg, 0.27 mmol), succinic anhydride (250 mg, 2.5 mmol)
and 4-dimethylaminopyridine (25 mg, 0.21 mmol) in dry pyridine
(10 mL) at reflux. After 4 h the reaction was completed. Com-
pound 25 (63 mg, 48%): m.p. 156–159 ◦C (ether), [˛]D + 101.6 (c
0.309, CHCl3). IR spectrum (CHCl3): 1726 (C O, ester); 1717 (C O,
COOH-dimer); 1701 (C O, ketone); 1176 (C O, ester); 1175, 978,
958 (O–SO3). 1H NMR (400 MHz): 0.60 (s, 3H, 3 × H-18); 0.93
(s, 3H, 3 × H-19); 2.11 (s, 3H, 3 × H-21); 2.53 (t, 3H, J = 9, H-
17); 2.56–2.69 (m, 4H, OOCCH2CH2COO); 4.76 (m, 1H, W = 32,
H-3). EI MS: 418 (1.5%, M), 400 (12%, M−H2O), 300 (100%,
M+1−HOOC-CH2CH2–COO), 285 (19%, M−HOOC–CH2CH2–COO,
O), 267 (15%, M−HOOC–CH2CH2–COO, COCH3). HR-MS (+ESI) calcd.
for C25H38NaO5 [M++Na] 441.2611, found 441.2609. C25H38O5:
calcd. C, 71.74; H, 9.15. Found. C, 71.50; H, 9.36.
3. Results and discussion
3.1. Chemistry
The starting compound for the synthesis of 5␣-derivatives,
diol 1, was prepared from protected (20R)-pregn-5-ene-3␤,20-diol
[19]. An oxygen-containing substituent was incorporated into the
structure by allylic oxidation and subsequently, the 5(6)-double
bond was reduced by catalytic hydrogenation to afford the desired
5␣-isomer 1. Acetylation afforded diacetate 2 and by selective
hydrolysis of less stable acetate group, 7-monoacetate 4, the start-
ing compound for the synthesis of all derivatives of the 5␣-serie
was obtained in the yield of 85% (Scheme 1). Its structure was con-
firmed by comparison of 3␤-H and 7␤-H chemical shifts in 1H NMR
spectrum.
Treatment of monoacetate 4 with succinic anhydride in pyri-
dine under catalysis by 4-dimethylaminopyridine (DMAP) gave
hemisuccinate 5 in the yield of 35% (Scheme 2). 3-Sulfate 6, the next
derivative with a polar substituent in the position 3, was obtained
by treatment of monoacetate 4 with a sulfur trioxide pyridine com-
plex in chloroform. Preservation of 7-acetate group was confirmed
by 1H NMR spectrum. The synthesis of compound 10 (Scheme 2),
derivative with a nicotinate group in the position 7, involved firstly
protecting of a 3-hydroxy group as a tert-butyldimethylsilyl deriva-
tive by the reaction of monoacetate 4 with tert-butyldimethylsilyl
chloride and subsequently selective hydrolysis of acetate protecting
group by treating with methanolic solution of potassium hydroxide.
Secondly, the free 7-hydroxy group was converted into nicotinate
ester and finally, the tert-butyldimethylsilyl group of compound
2.1.3.12. 20-Oxo-5ˇ-pregnan-3˛-yl sulfate pyridinium salt (26,
3˛5ˇS). The method followed that described for compound 6 but
using alcohol 24 (200 mg, 0.6 mmol) and a sulfur trioxide pyri-
dine complex (400 mg, 2.5 mmol) in dry chloroform (5 mL). After
4 h the reaction was completed. Compound 26 (240 mg, 80%): m.p.
173–175 ◦C (methanol/ether) (lit. 170 ◦C [21]), [˛]D + 100.0 (c 0.28,
CHCl3) (lit. + 103.0 [21]). IR spectrum (CHCl3): 3139 (pyridinium);
1698 (C O, ketone); 1270, 1257, 1236, 1179, 1166 (SO3 and pyri-
dinium). 1H NMR (400 MHz): 0.60 (s, 3H, 3 × H-18); 0.78 (s, 3H,
3 × H-19); 2.11 (s, 3H, 3 × H-21); 2.52 (t, 1H, J = 8.7, H-17); 4.45 (m,
1H, W = 30); 8.03 (m, 2H, H-3 and H-5, pyridinium); 8.50 (tt, 1H,
J1 = 7.8, J2 = 1.5, H-4, pyridinium); 9.00 (m, 2H, H-2 and H-6, pyri-
dinium). FAB MS: 478 (22%, M+1), 440 (22%, M+1−C5H6N), 301
(17%, M+1−OSO3C5H6N). HR-MS (+ESI) calcd. for C26H39NNaO5S
[M++Na] 500.2441, found 500.2436.

E. Stastna et al. / Steroids 74 (2009) 256–263
261
Scheme 1. Synthesis of compounds 2–4. Reaction conditions: (a) Ac2O, pyridine, 50 ◦C; (b) KOH, MeOH, benzene.
chemical shifts in 1H NMR spectrum. Compound 10 was converted
into desired sulfate pyridinium salt 11 in the total yield of 59% from
compound 4 (Scheme 2).
For the synthesis of 5␤-derivatives, the chenodeoxycholic acid
was used, because it contains the required 5␤-configuration and
suitable substituents in positions 3 and 7. Diacetate 15 was prepared
by degradation [22] of a part (carbons 22-24) of the side chain. All
9 was selectively cleaved by solution of p-toluenesulfonic acid
in methanol. Using a solution of tetrabutylammonium fluoride,
the reagent comfortable for cleavage of the tert-butyldimethylsilyl
derivatives was not successful, because lead to cleavage both tert-
butyldimethylsilyl group and nicotinate group. On the other hand,
this reagent was successfully used in 5␤-serie to afford desired
compound 22. The structure of compound 10 was confirmed by
Scheme 2. Synthesis of compounds 5–11 and 17–23. Reaction conditions: (a) succinic anhydride, pyridine, 4-dimethylaminopyridine, 140 ◦C; (b) sulfur trioxide pyridine
complex, CHCl3; (c) tert-butyldimethylsilyl chloride, imidazol, DMF; (d) KOH, EtOH, benzene, 60 ◦C; (e) nicotinoyl chloride hydrochloride, 4-dimethylaminopyridine, pyridine;
(f) p-toluenesulfonic acid, MeOH, rt; (g) tetrabutylammonium fluoride solution, THF, rt; (h) sulfur trioxide pyridine complex, CHCl3.

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E. Stastna et al. / Steroids 74 (2009) 256–263
Scheme 3. Synthesis of compound 16. Reaction conditions: (a) NaHCO3, H2O, MeOH,
70 ◦C.
target compounds of 5␤-serie were prepared analogously to the 5␣-
serie. Partial hydrolysis of diacetate15 gave 7-acetate 16 (Scheme 3).
Hemisuccinate 17 and pyridinium salt of sulfate 18 were prepared in
the yield of 35% and 99%, respectively. Compound 23 was prepared
in the total yield of 12% from compound 16. The structures of all
derivatives in both series were confirmed by 1H NMR spectra.
For the synthesis of hemisuccinates (13, 25) and sulfates (14,
26), the 3␣-hydroxy pregnane derivatives (12, 24) were used, pre-
viously prepared by selective reduction with NaBH4 of 3-keto group
[20]. The hemisuccinates were prepared by a common procedure of
treating the steroid alcohol in pyridine with succinic anhydride and
the sulfates by treating the steroid alcohol with sulfur trioxide pyri-
dine complex (Scheme 4). Hemisuccinate 13 and 25 were prepared
in the yield of 53% and 48%, respectively. Pyridinium salt of sulfate
14 and 26 were prepared in the yield of 85% and 80%, respectively.
Fig. 1. Biological activity of the steroid—example of the text.
Table 1
Inhibition of NMDA-induced response in cultured hippocampal neurons by preg-
nanolone sulfate and its synthetic analogs.
Compound
Inhibition (%)
IC50 (␮M)
n
3␣5␤S (26)
71.3 5.0
69.2 7.0
21.4 2.7
34.1 10.2
46.9 9.7
56.8 2.3
35.1 2.6
17.4 4.3
35.2 2.7
37.8 4.7
47.2 9.9
51.9 14.2
299.9 40.9
185.8 61.1
116.0 36.5
79.8 6.1
167.6 15.5
383.5 93.5
167.2 16.3
153.8 25.0
5
6
6
5
5
6
5
5
5
6
3␣5␤HS (25)
3␣5␤7AcHS (17)
3␣5␤7AcS (18)
3␣5␤7NicS (23)
3␣5␣S (14)
3␣5␣HS (13)
3␣5␣7AcHS (5)
3␣5␣7AcS (6)
3␣5␣7NicS (11)
3.2. Biological activity
Currents elicited by 100 ␮M NMDA were recorded in cultured
hippocampal neurons voltage-clamped at a holding potential of
−60 mV. In accordance with previous results, pregnanolone sul-
fate diminished the amplitude of NMDA-induced currents (Fig. 1)
[14,16]. At 100 ␮M pregnanolone sulfate, the mean inhibitory effect
was 71.3 5.0% (n = 5). Synthetic analogs of pregnanolone sulfate
had also negative modulatory effect on NMDA-induced responses
with the mean inhibition induced by 100 ␮M steroid that ranged
from 17% (5, 3␣5␣7AcHS) to 71% (26, 3␣5␤S). The relative degree of
steroid-induced inhibition was used to estimate IC50. IC50 was cal-
culated from the logistic equation RI = 1 − (1/(1 + ([steroid]/IC50)h);
where RI is relative degree of steroid-induced inhibition and h is the
apparent Hill coefficient (1.2; see Ref. [16]). The values of relative
The first column shows relative degree of steroid (100 ␮M) inhibition of current
responses induced in cultured hippocampal neurons by fast application of 100 ␮M
NMDA. In the second column calculated IC50 values are listed. Results are expressed
as mean S.D., with n indicating the number of cells studied.
inhibition and estimated IC50 values of steroids 5, 6, 11, 13, 14, 17,
18, 25, and 26 are listed in Table 1.
Current response induced in a cultured hippocampal neuron
by 100 ␮M NMDA and its simultaneous application with 100 ␮M
3␣5␤S (duration of 3␣5␤S and NMDA application is indicated by
filled and open bar, respectively).
Scheme 4. Synthesis of compounds 13, 14, 25 and 26. Reaction conditions: (a) succinic anhydride, pyridine, 4-dimethylaminopyridine, 140 ◦C; (b) sulfur trioxide pyridine
complex, CHCl3.

E. Stastna et al. / Steroids 74 (2009) 256–263
263
4. Discussion
Research Project of the AS CR AV0Z 50110509, Z4 005 905, EC
FP6 PHOTOLYSIS (LSHM-CT-2007-037765) and Ministry of Educa-
tion, Youth and Sports of the Czech Republic (1M0002375201 and
LC554).
Pregnanolone sulfate acts as an inhibitor of NMDA receptor
channels and may function as endogenous neuromodulator [14].
Our recent results have shown that pregnanolone sulfate is a use-
dependent but voltage-independent inhibitor of NMDA receptors,
with more potent action at tonically than at phasically (synap-
tically) activated receptors [16]. In this study, we examined the
effect of newly synthesized C3 and C7-substituted analogs of preg-
nanolone sulfate on ionotropic glutamate receptors expressed in
cultured hippocampal neurons and found structural correlates that
determine steroid inhibitory efficacy at NMDA receptors.
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Acknowledgements
We are indebted to Dr. S. Vasˇícˇková for taking the IR spectra and
Dr. P. Fiedler for interpreting them. Optical rotations were measured
and elemental analyses were carried out in the Analytical Labo-
ratory (Dr. S. Mateˇjková, Head). This work was supported by the
Grant Agency of the Czech Republic (309/07/0271; 203/08/1498),
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