Suppressing aggressive behavior with analogs of allopregnanolone (epalon)

Article abstract of DOI:10.1016/S0039-128X(00)00215-4

3α-Hydroxy-20-oxo-5α-pregnan-21-yl hemisuccinate (8) was produced by partial acylation of 3α,21-dihydroxy-5α-pregnan-20-one (14). 3α-Fluoro-5α-pregnan-20-one (9) was prepared by treatment of 3β-hydroxy-5α-pregnan-20-one (11) with DAST and by solvolysis of tosylate 12 with tetrabutylammonium fluoride. A behavioral test on mice was performed using 3α-hydroxy-5α-pregnan-20-one (1) and compounds 8 and 9. Compound 8 was found to be inactive, while the fluoro derivative 9 selectively reduced aggressive behavior in mice more than the corresponding 3α-hydroxy compound 1; locomotion and other behavioral features were not affected. Copyright

Full text of DOI:10.1016/S0039-128X(00)00215-4

Steroids 66 (2001) 99–105
Suppressing aggressive behavior with analogs of allopregnanolone
(epalon)
Barbora Slav´ıkova´a, Alexander Kasala,*, Leona Uhl´ırˇova´b, Miloslav Krsˇiakb,
Hana Chodounska´a, Ladislav Kohouta
aInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Fleming Square 2, 166 10 Prague 6, Czech Republic
bDepartment of Pharmacology, 3rd Faculty of Medicine, Charles University, Ruska´ Str. 87, 100 00 Prague 10, Czech Republic
Received 20 April 2000; received in revised form 24 August 2000; accepted 29 August 2000
Abstract
3␣-Hydroxy-20-oxo-5␣-pregnan-21-yl hemisuccinate (8) was produced by partial acylation of 3␣,21-dihydroxy-5␣-pregnan-20-one (14).
3␣-Fluoro-5␣-pregnan-20-one (9) was prepared by treatment of 3␤-hydroxy-5␣-pregnan-20-one (11) with DAST and by solvolysis of tosylate 12
with tetrabutylammonium fluoride. A behavioral test on mice was performed using 3␣-hydroxy-5␣-pregnan-20-one (1) and compounds 8 and 9.
Compound 8 was found to be inactive, while the fluoro derivative 9 selectively reduced aggressive behavior in mice more than the corresponding
3␣-hydroxy compound 1; locomotion and other behavioral features were not affected. © 2001 Elsevier Science Inc. All rights reserved.
Keywords: Steroid; Allopregnanolone; Behavior; GABAA-receptor; DAST; 1H NMR spectroscopy; Epalon
1. Introduction
by its low solubility in body liquids. This property allows
the compound to pass the blood-brain barrier, but also
affects methods of its application. In the search for new
types of allopregnanolone, several water-soluble com-
pounds like 3 and 4 were discovered [8–10].
␥-Aminobutyric acid (GABA) is a major inhibitory neuro-
transmitter in the brain that is involved in controlling many
conditions ranging from anxiety to epilepsy [1–3]. The effects
of GABA can be magnified by several types of compounds
(e.g. benzodiazepines), which bind to GABAA receptors. Some
metabolites of progesterone, e.g. 3␣-hydroxy-5␣-pregnan-20-
one (1, ‘allopregnanolone,’ sometimes also ‘epalon’ see Fig.
1), also bind allosterically to the GABAA receptor and increase
inhibitory effects of GABA by opening the membrane chloride
ion channel with little tolerance [4]. The 3␣-hydroxyl of allo-
pregnanolone is considered essential for its anesthetic activity
[5]; the hydroxy group acts within the binding site as a hydro-
gen-bond donor.
The practical use of allopregnanolone 1 (see Fig. 1) is
restricted by its fast metabolism [6]: its half-life in serum is
about 16 min. One solution to this problem was found by the
introduction of a 3␤-substituent (like 2 [7]): such a com-
pound cannot be oxidized to an inactive 3-oxo derivative
and thus, acts longer in the body.
The collection of steroid compounds, obtained over the
past forty years by our Laboratory, was repeatedly searched
for new types of potential lead structures: here, Hamilton
and his colleagues [11] found good GABAA receptor li-
gands (e.g. 5 and 6), which led them to a new product 7 with
improved anesthetic properties. We picked other com-
pounds for preliminary evaluation of their binding to the
GABAA receptor using [3H]muscimol tests; some of these
compounds were developed into new products 8 and 9
whose synthesis and behavioral effects are reported in this
paper. In vitro studies of the binding of our products to the
GABAA receptor (Refs. [12–14]) will be reported else-
where.
2. Methods
The practical use of allopregnanolone 1 is also restricted
2.1. Synthesis of compounds tested
* Corresponding author. Tel.: ϩ420-2-20183-314; fax: ϩ420-2-24310-
090.
Commercial pregnenolone (10, 3␤-hydroxypregn-5-en-20-
one) was hydrogenated to the desired 5␣ dihydro product 11
E-mail address: kasal@uochb.cas.cz (A. Kasal).
0039-128X/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(00)00215-4

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B. Slav´ıkova´ et al. / Steroids 66 (2001) 99–105
Fig. 1.
(see Scheme 1), which was tosylated to yield tosylate 12. Its
treatment with sodium nitrite in hexamethylphosphoroamide
afforded alcohol 1 in which the configuration of the 3-OH
group was inverted. The change of the configuration was
apparent by the narrow width of the H-3 signal in its 1H NMR
spectrum (W1/2 ϭ 7.7 Hz while 3␤-alcohol 11 has W1/2 ϭ 24
Hz). Ketone 1 was oxidized with lead tetraacetate to yield the
21-acetoxy compound 13. With respect to the susceptibility of
this compound to oxidation in alkaline medium [13], we car-
ried out the hydrolysis in acid medium and obtained diol 14.
Partial acylation of the diol 14 with succinic anhydride yielded
the 21-hemisuccinate 8 sought. Its triethylammonium salt 15
was obtained in crystalline form by dissolving acid 8 in hot
triethylamine.
based on interactions between an aggressive or timid male
mouse housed individually and a non-aggressive group-
housed male mouse in an observational cage. In this etho-
logically oriented laboratory model, analysis of aggressive,
defensive-escape, sociable, and locomotor acts as well as
postures occurring in the individually housed males enables
the evaluation of potential antiaggressive and anxiolytic
effects of the tested drugs as well as the degree of motor
impairment and sedation they might produce [16,17].
2.2.1. Subject
Male, albino random-bred mice derived from ICR strain
(Velaz, Prague), weighing 18–20 g at the beginning of the
experimental housing, were used. All mice were housed
under room lighting (with lights on from 6 a.m. to 6 p.m.)
and with the temperature ranging from 22 to 24°C. Food
and water were available ad libitum.
Treatment of 3␤-hydroxy-5␣-pregnan-20-one 11 with
(diethylamino)sulfur trifluoride (DAST [15]) led to the 3␣-
fluoro product 9 with the reversed C-3 configuration (see
Experimental).
2.2. Behavioral testing
2.2.2. Procedure
After 4 weeks of isolation, mice were ready for testing.
Drugs were tested during a series of interactions repeated at
1 week intervals. A particular dose of tested compound or
The behavioral effects of the drugs were tested in an
experimental model of social conflict in mice. The model is

B. Slav´ıkova´ et al. / Steroids 66 (2001) 99–105
101
Scheme 1.
vehicle was given intraperitoneally (i.p.) to the singly
housed males 30 min before interactions, the order of treat-
ments was balanced according to a Latin square design.
Social interactions always involved one singly housed
and one group-housed mouse, placed as a pair in an obser-
vational cage. Each isolate mouse was paired with the same
group-housed partner throughout the experimental period.
The isolated mice were allowed 30 min of adaptation in the
observational cages before the group-housed partners were
introduced; interactions ended after 4 min.
The observational cages were transparent boxes (20 ϫ
30 ϫ 20 cm) with wood shavings on the floor and covered
with a transparent cover with apertures for air. Observations
were made under moderate room lighting from 8 a.m. to
1 p.m. The behavior of animals during the interactions was
recorded on videotape. The tapes were later analyzed by an
observer blinded with respect to the drug treatment. Anal-
ysis was performed with a key-board connected to a stan-
dard PC and special software [18].
partner often associated with biting, threat—a sideways or
an upright stance with head and forebody movements to-
ward the partner, and trying to bite the partner (also termed
Offensive Sideways or Upright Posture), tail rattle—rapid
vibrations of the tail, walk—any walking across the cage
that is not apparently related to the partner, and rear—the
mouse stands only on his hind legs and simultaneously
sniffs the air or walls.
The interobserver reliability of the recorded items was
satisfactory as determined by several observers indepen-
dently judging a videotaped record of behavior of 70 mice
in interactions lasting 4 min each. The correlations ranged
from r ϭ 0.83 to 0.97.
2.2.4. Drugs
Compound 1, 8, and 9 were dissolved in saline with two
drops of Tween 80 and injected i.p. in a volume 0.1 ml/10
g of body weight.
2.2.5. Data analysis
2.2.3. Measures
The isolate mice that exhibited attacks in the control
interaction (aggressive isolates) were selected for data anal-
ysis in the present paper. The differences between the con-
trol and experimental values were evaluated by the Paired t
test or the Wilcoxon Signed Rank test (nonparametric tests
were used when conditions for parametric tests were not
satisfied).
The frequency, total duration, and latency of a number of
aggressive, defensive-escape (timid), social, and locomotor
activities, derived from the ethogram of mice [19] and
described in detail previously [20,21], were recorded. In
short, the acts and postures evaluated in the present paper
were defined as follows: attack—a fierce lunging at the

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B. Slav´ıkova´ et al. / Steroids 66 (2001) 99–105
Fig. 3. Effects of tested compounds (1, 8 and 9) on the frequency of attacks
illustrated as a percentage of control behavior (frequency of attacks after
vehicle administration [16.9 Ϯ 2.8] ϭ 100 Ϯ 16.6%). Effects of each dose
represent mean results ( Ϯ SEM) from 15 to 23 mice. ** P Ͻ 0.01.
Fig. 2. Effects of tested compounds (1, 8 and 9) on duration of threat
postures illustrated as a percentage of control behavior (duration of threat
postures after vehicle administration [12.1 Ϯ 2.8 s] ϭ 100 Ϯ 23.1%).
Effects of each dose represent mean results ( Ϯ SEM) from 15 to 23 mice.
* P Ͻ 0.05, ** P Ͻ 0.01.
3.1. 3␣-Hydroxy-5␣-pregnan-20-one (1)
Tosylate 12 (1.5 g, 3.17 mmol) was solvolyzed with
sodium nitrite (4.5 g, 65.22 mmol) in hexamethylphosphor-
amide (20 ml) at 90°C for 2 h under a nitrogen atmosphere.
The mixture was poured into water, and the product was
extracted with chloroform. The extract was washed sequen-
tially with water, dilute hydrochloric acid, water, the solu-
tion of potassium hydrogen carbonate, and water. After
evaporation of the solvent, the product was purified by
chromatography. The silica gel column was eluted with
light petroleum:ether, 8:2. Crystallization of the major frac-
tion afforded 514 mg (51%) of compound 1. M.p. 167–
169°C (toluene), Ref. [22] gives 168–169°C. 1H NMR
spectrum: 0.60 s, 3H (H-18); 0.78 s, 3H (H-19); 2.11 s, 3H
(H-21); 2.53 t, 1H (J ϭ 8.8, H-17); 4.05 m, 1H (W1/2 ϭ 7.7,
H-3).
3. Experimental
Melting points were determined on a Boetius micro melt-
ing point apparatus (Germany) and are uncorrected. Ana-
lytical samples were dried over phosphorus pentoxide at
50°C/100 Pa. Optical rotations were measured in chloro-
form ([␣]D values are given in 10Ϫ1.deg.cm2.gϪ1), and IR
spectra of chloroform solutions were recorded on a Bruker
IFS 88 spectrometer (wavenumbers are given in cmϪ1).
NMR spectra were measured on an FT-NMR spectrometer
Varian UNITY-200 (at 200 MHz) in CDCl3 with tetra-
methylsilane as the internal reference. Chemical shifts are
given in ppm (␦-scale); coupling constants and the widths of
multiplets are given in Hz. Unless otherwise stated, the data
were interpreted as the first-order spectra. Thin-layer chro-
matography (TLC) was performed on silica gel (ICN Bio-
chemicals). Preparative TLC (PLC) was carried out on
200 ϫ 200 mm plates coated with a 0.7 mm thick layer of
the same material. For column chromatography, silica gel
(60–120 ␮m) was used. Whenever aqueous solutions of
hydrochloric acid, potassium hydrogen carbonate, and po-
tassium carbonate were used, their concentration was al-
ways 5%. Before evaporation on a rotary vacuum evapora-
tor in (bath temperature 50°C), solutions in organic solvents
were dried over anhydrous sodium sulfate.
3.2. 3␣-Hydroxy-20-oxo-5␣-pregnan-21-yl hemisuccinate
(8)
Succinic anhydride (245 mg, 2.45 mmol) was added to a
solution of diol 14 (280 mg, 0.84 mmol) in pyridine (2.8 ml)
at 0°C. After 24 h, the mixture was poured into a mixture of
ethyl acetate (50 ml), hydrochloric acid (4 ml), and ice. The
solution was washed with water, and then repeatedly
washed with the solution of potassium hydrogen carbonate.
The organic phase was separated, and the solvent was evap-

B. Slav´ıkova´ et al. / Steroids 66 (2001) 99–105
103
Fig. 5. Effects of tested compounds (1, 8 and 9) on the frequency of walks
illustrated as a percentage of control behavior (frequency of walks after
vehicle administration [32.1 Ϯ 3.7] ϭ 100 Ϯ 11.5%). Effects of each dose
represent mean results ( Ϯ SEM) from 15 to 23 mice.
Fig. 4. Effects of tested compounds (1, 8 and 9) on the duration of tail
rattling illustrated as a percentage of control behavior (duration of tail
rattling after vehicle administration [12.9 Ϯ 2.3 s] ϭ 100 Ϯ 17.8%).
Effects of each dose represent mean results ( Ϯ SEM) from 15 to 23 mice.
** P Ͻ 0.01.
by flash chromatography on a column of silica gel (50 g);
light petroleum:ether (93:7) eluted compound 9 (153 mg,
30.4%), which crystallized from acetone-heptane, m.p.
143.5–145.5°C, [␣]D ϩ93 (c 0.88). IR spectrum: 1706 (C ϭ
orated to yield the starting compound (163 mg, 66%). The
combined aqueous layers were acidified with dilute hydro-
chloric acid, the precipitate formed was filtered off, washed
with water, and dried. Yield of compound 8 107 mg (70%
with allowance for the starting material recovered). M.p.
203–206°C (acetone-toluene), [␣]D ϩ90 (c 0.99). IR spec-
trum: 3619, 3512 (OH), 1747, 1718 (C ϭ O), 1166 (C-O).
1H NMR spectrum: 0.64 s, 3H (H-18); 0.78 s, 3H (H-19);
2.76 dt, 4H (J ϭ 4, JЈ ϭ 1.2, COCH2CH2CO); 4.05 m, 1H
(W1/2 ϭ 7, H-3); 4.55 d and 4.77 d, 2H (J ϭ 17, H-21).
Anal. calc. for C24H38O6: C, 69.10; H, 8.81. Found: C,
68.93; H, 8.99.
1
O), 1155 (C-F). H NMR spectrum: 0.60 s, 3H (H-18);
0.78 s, 3H (H-19); 2.11 s, 3H (H-21); 4.81 dm, 1H (J ϭ 48,
H-3). Analysis calculated for C21H33FO: C, 78.70; H, 10.38.
Found: C, 78.56; H, 10.27.
b) Tosylate 12 ([24], 100 mg, 0.21 mmol) was treated
with a solution of tetrabutylammonium fluoride in tetrahy-
drofuran (1M, 1.3 ml) at 50°C for 4 days. The reaction was
quenched with water, and the product was extracted with
ether, washed with water, and dried. After evaporation, the
mixture was separated by PLC (4 plates, light petroleum:
ether, 9:1). The major product 9 (28 mg, 41%) was found to
be identical to the above sample.
3.3. 3␣-Fluoro-5␣-pregnan-20-one (9)
a) DAST (0.366 g, 2.27 mmol) was added to a solution
of alcohol 11 (0.5 g, 1.57 mmol) in dichloromethane (10 ml)
at room temperature. After 30 min, the reaction mixture was
poured into the potassium hydrogen carbonate solution, and
the precipitate formed was extracted with dichloromethane.
After evaporation of the solvent, the residue was separated
3.4. 3␣-Hydroxy-20-oxo-5␣-pregnan-21-yl acetate (13)
Compound 1 (0.4 g, 1.57 mmol) in benzene (16 ml),
methanol (0.8 ml), and boron trifluoride etherate (2 g, 14.1
mmol) was stirred with lead tetraacetate (0.620 g, 1.4 mmol)
for 3 h at room temperature. The mixture was diluted with

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B. Slav´ıkova´ et al. / Steroids 66 (2001) 99–105
1
3615, 3481 (OH), 1705 (C ϭ O), 1077, 1022 (C-OH). H
NMR spectrum: 0.64 s, 3H (H-18), 0.78 s, 3H (H-19),
4.05 m, 1H (W1/2 ϭ 7.2, H-3), 4.13 d and 4.23 d, 2H (J ϭ
18, H-21).
3.6. 3␣-Hydroxy-20-oxo-5␣-pregnan-21-yl hemisuccinate,
triethylammonium salt (15)
A solution of compound 8 (12 mg, 0.03 mmol) in trieth-
ylamine (1.0 ml, 7.2 mmol) was refluxed for 20 min. Evap-
oration of the base and crystallization afforded salt 15 (8
1
mg, 54%). M.p. 194–196°C (acetone-heptane). H NMR
spectrum: 0.63 s, 3H (H-18); 0.77 s, 3H (H-19); 1.25 t, 9H
(J ϭ 7, 3 x CH3-CH2); 2.70 m, 4H (W1/2 ϭ 24,
COCH2CH2CO); 3.04 q, 6 H (J ϭ 7.3, 3ϫ CH2-N); 4.04 m,
1 H (W1/2 ϭ 7.6, H-3); 4.52 d and 4.73 d, 2H (J ϭ 17,
H-21).
4. Results
Allopregnanolone 1 appeared to be only moderately ac-
tive in affecting the behavior of aggressive mice; this com-
pound produced some reduction of aggressive activities
(Attacks, Threats, and Tail Rattles, Figs. 2–4), but only the
reduction of Threats was significant at the higher dose of
compound 1 (15 mg/kg, P ϭ 0.0479, Fig. 2). Its fluoro
analog (compound 9) was more active in reducing aggres-
sive behavior; the higher dose (15 mg/kg) more markedly
and significantly reduced all aggressive activities (Attacks,
P ϭ 0.0017; Fig. 3, Threats, P ϭ 0.0067, Fig 2; Tail Rattles,
P ϭ 0.0065, Fig. 4). This reduction did not seem to be due
to motor impairment or general sedation because walking
and rearing were not affected significantly by these com-
pounds (Figs. 5 and 6). Allopregnanolone 1 and its fluoro
analog 9 did not affect other behavioral activities (social
investigation, defensive-escape behavior) in aggressive
mice (data not shown). Compound 8 was quite inactive; it
did not produce any significant behavioral changes in ag-
gressive mice, even at a relatively high dose (25 mg/kg,
Figs. 1–6).
Fig. 6. Effects of tested compounds (1, 8 and 9) on the frequency of rears
illustrated as a percentage of control behavior (frequency of rears after
vehicle administration [16.8 Ϯ 2.7] ϭ 100 Ϯ 16.1%). Effects of each dose
represent mean results ( Ϯ SEM) from 15 to 23 mice.
water, and the product was extracted with ether and washed
successively with the solution of potassium hydrogen car-
bonate, and water. Evaporation of the solvent afforded
0.310 mg (66%) of 13. M.p. 215–216°C (ethanol), Ref. [23]
gives 216–218°C. IR spectrum: 3615 (OH), 1747, 1722
1
(C ϭ O), 1002 (C-OH). H NMR spectrum: 0.64 s, 3H
(H-18), 0.78 s, 3H (H-19), 2.17 s, 3H (CH3CO), 4.05 m, 1H
(W1/2 ϭ 7.2, H-3), 4.52 d and 4.71 d, 2H (J ϭ 17, H-21).
5. Discussion
3.5. 3␣,21-Dihydroxy-5␣-pregnan-20-one (14)
The effects of compound 9 (the 3␣-fluoro analog of
allopregnanolone) on aggressive mice in the present study
resembled those of diazepam and other benzodiazepines
[17,20,21]. The present results suggest that the fluoro ana-
log 9 may possess stronger tranquilizing activity than allo-
pregnanolone 1, which is probably due to the greater sta-
bility of its C-F bond at position 3.
The low activity or inactivity of the 3␣-hydroxy com-
pounds 1 and 8 in the present studies may be due to the
rather long interval (30 min) between the injection of these
compounds and the measurement of behavior (the half-life
A solution of acetoxy compound 13 (300 mg, 0.80
mmol) in chloroform (3 ml) was treated with a mixture of
methanol (9 ml), water (1 ml), and hydrochloric acid (0.6
ml). After 18 h, the mixture was concentrated to a quarter of
its volume and poured into water. The resulting product was
extracted with chloroform, washed sequentially with water,
the solution of potassium hydrogen carbonate, and water
and then dried. After evaporation of the solvent, the residue
crystallized from acetone-heptane. Yield: 234 mg (88%).
M.p. 162–163°C, Ref. [23] gives 163–166°C. IR spectrum:

B. Slav´ıkova´ et al. / Steroids 66 (2001) 99–105
105
Upasani RB. Substituted 3␤-phenylethynyl derivatives of 3␣-hy-
droxy-5␣-pregnan-20-one: remarkably potent neuroactive steroid
modulators of ␥-aminobutyric acidA receptors. J Pharmacol Exp Ther
1998;287:198–207.
of the compound 1 is only several minutes). When com-
pound 1 was administered 10 min before measurement, it
reportedly showed significant anxiolytic effects in a widely
used plus-maze test in mice [25]. The interval (30 min) for
adaptation of animals to the observational cages was de-
rived from experiments with nonsteroidal modulators of the
GABAA receptor, such as diazepam, which are not metab-
olized as quickly as the compounds used in the present
studies.
Compound 9 reduced aggressive behavior of aggressive
mice without causing overall sedation or motor impairment,
which is a common effect of higher doses of benzodiaz-
epines and other anxiolytics [20,21]. Social contacts (sniff-
ing, climbing and following partners) and locomotion
(walking across cage, rearing) were not affected by com-
pound 9.
The present results show that the 3␣-hydroxy group of
allopregnanolone like that of fluoro glucose can be replaced
by fluorine which elicits strong inductive effects but does
not yield a proton for a hydrogen bond. These results con-
trast the generally accepted belief that the presence of a
3␣-hydroxy group is essential for GABA-like activity. Ap-
parently, the fluorine atom, which also can participate in a
hydrogen bond though in a reversed manner, can substitute
for the hydroxyl. The slightly reduced GABAA-receptor
binding ability of the fluoro analog, when compared with
the 3␣-hydroxy compound, is more than compensated for
by its metabolic stability [26,27].
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This work was supported by the Ministry of Health of the
Czech Republic (grant No. 4668–3).
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