Neuroactive steroids with perfluorobenzoyl group

Article abstract of DOI:10.1016/j.steroids.2012.07.004

During an initial study in searching for the alternative derivatives suitable for photolabeling of neuroactive steroids, perfluorobenzoates and perfluorobenzamides in position 17 of 5β-androstan-3α-ol were synthesized from the corresponding 17-hydroxy and

Full text of DOI:10.1016/j.steroids.2012.07.004

Steroids 77 (2012) 1233–1241
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Neuroactive steroids with perfluorobenzoyl group
a,
a
a
Ivan Cerny , Miloš Budešínsky , Vladimír Pouzar , Vojtech Vyklicky b,c, Barbora Krausová b,
⇑
ˇ
´
ˇ
´
ˇ
´
b
´
Ladislav Vyklicky Jr.
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, Czech Republic
c 2nd Faculty of Medicine, Charles University, 150 06 Prague, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 May 2012
Received in revised form 15 June 2012
Accepted 3 July 2012
Available online 25 July 2012
During an initial study in searching for the alternative derivatives suitable for photolabeling of neuroac-
tive steroids, perfluorobenzoates and perfluorobenzamides in position 17 of 5b-androstan-3
synthesized from the corresponding 17-hydroxy and 17-amino derivatives. After transformation into
glutamates or sulfates, 17 -epimers had comparable inhibitory activity at NMDA receptors to the natural
a-ol were
a
neurosteroid (20-oxo-5b-pregnan-3b-yl sulfate), however, were more potent (2- to 36-fold) than their
17b-substituted analogs. In one case, fluorine in position 40 of perfluorobenzoate group was substituted
with azide and activity of the final glutamate was retained comparing with the corresponding perflu-
orobenzoate. The series was expanded with perfluorobenzoyl derivatives of pregnanolone: Perfluoroben-
zamide of glutamate and perfluorobenzoate of 11a-hydroxy pregnanolone were prepared and tested.
From nine tested compounds, four of them exhibit very good inhibition activity and can serve as prom-
Keywords:
Neuroactive steroids
Synthesis
Perfluorobenzoates
NMDA receptor
ising leads for photolabeling experiments.
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
were measured at 25 °C on a AUTOPOL IV polarimeter (Rudolph
Research Analytical, USA), and [
a]
values are given in 10ꢀ1
D
Azidoperfluorophenyl group has been designed as a source of
nitrenes for photoaffinity labeling and crosslinking due to their
favorable photochemical properties, suitable storage stability,
and ease of preparation [1,2]. The reagents for introducing this
group in form of substituted benzoate are commercially available
[3]. In the steroidal field, use of diazirine label is the most frequent
[4-7]. Iodine substituted azidobenzoates, however, were used for
labeling of DHEA [8]. During a process of searching for suitable
photoaffinity label for neuroactive steroids, we have decided to
prepare several models with perfluorobenzoyl group attached to
various parts of base skeleton of neuroactive steroids and study
the activity of modified compounds in inhibition tests on N-
deg cm2 gꢀ1. Infrared spectra (wavenumbers in cmꢀ1) were re-
corded on a Bruker IFS 88 spectrometer in chloroform. 1H, 13C,
and 19F NMR spectra were taken on Bruker AVANCE-400 instru-
ment at 23 °C and referenced to tetramethylsilane as the internal
standard. For referencing of 19F spectra, hexafluorobenzene
(d = 162.9) was used. 13C NMR spectra for selected derivatives (Ta-
bles 1a and b) were measured on Bruker AVANCE-600 instrument
under the above conditions, secondary referencing was performed
using the solvent signal at position d(CDCl3) = 77.0. In addition to
1D proton and carbon NMR spectra, homonuclear 2D-spectra
(H,H-PFG-COSY, and ROESY) together with heteronuclear 2D-spec-
tra (H,C-PFG-HSQC and H,C-PFG-HMBC) were used for complete
structural assignment of signals. Chemical shifts are given in
ppm (d-scale); coupling constants (J) are given in Hz.
Methyl-D-aspartate (NMDA) receptors. We were encouraged with
our previous findings [9] which proved the possibility of replacing
20-keto pregnane side chain of pregnanolone with azide without
disturbing the inhibitory activity on the NMDA receptor.
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 lm) was used. Prior to evaporation on a
2. Experimental
rotary evaporator in vacuo (0.25 kPa, bath temperature 40 °C),
solutions in organic solvents were dried over anhydrous sodium
sulfate.
2.1. General
17b-Hydroxy-5b-androstan-3-one (1a) [10], 17
androstan-3-one (1b) [11,12], 3 -hydroxy-5b-androstan-17-one
oxime (9) [13], 17 -azido-5b-androstan-3 -ol (11) [9], and 11
a-hydroxy-5b-
Melting points were determined on a Boetius micro melting
point apparatus (Germany) and are uncorrected. Optical rotations
a
a
a
a-
hydroxy-5b-pregnane-3,20-dione (18) [14,15] are known com-
pounds and were prepared according to literature procedures.
⇑
Corresponding author. Tel.: +420 220 183 385; fax: +420 220 183 578.
ˇ
E-mail address: cerny@uochb.cas.cz (I. Cerny´).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.07.004

ˇ
´
I. Cerny et al. / Steroids 77 (2012) 1233–1241
1234
Table 1a
2.2.2. 3-Oxo-5b-androstan-17
a
-yl pentafluorobenzoate (2b)
-hydroxy-5b-androstan-3-one (1b)
Carbon-13 chemical shifts of selected perfluorobenzoyl steroidsa: Skeletal carbon
atoms.
To a stirred solution of 17
a
(960 mg, 3.31 mmol) in dichloromethane (8 mL) under argon
atmosphere, pentafluorobenzoic acid (770 mg, 3.63 mmol) and 4-
dimethylaminopyridine (100 mg, 0.82 mmol) were added. Then,
Carbon
5a
6b
8
14a
14bb
17
34.83
21
37.79
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
34.88
26.46
76.74
31.92
41.85
26.77
25.81
40.42
35.55
34.63
20.25
36.82
42.98
50.42
23.55
27.53
85.67
12.11
23.22
-
35.31
27.59
79.52
33.09
41.97
26.78
26.57
40.19
35.87
34.45
20.17
31.92
45.09
50.15
24.49
30.12
85.28
16.49
23.11
-
34.85
26.44
76.95
31.89
41.82
26.74
25.79
40.43
35.54
34.62
20.24
36.82
42.96
50.42
23.54
27.54
85.49
12.11
23.20
-
34.85
26.37
76.52
31.96
41.81
26.77
25.93
40.51
35.92
34.61
20.34
37.07
43.93
52.44
23.58
27.58
59.84
11.92
23.14
-
35.81
27.56
78.52
33.23
43.30
28.05
27.93
41.78
37.58
35.99
21.54
33.94
46.64
51.58
25.85
30.17
60.66
18.42
23.75
-
N,N0-diisopropylcarbodiimide (570
lL, 3.68 mmol) was added
26.43
76.65
31.97
41.78
26.80
26.22
40.39
35.73
34.57
20.83
39.08
44.32
56.61
24.38
22.86
63.82
13.41
23.21
209.90
31.53
27.89
79.00
33.62
43.26
27.05
26.32
34.96
43.67
35.67
74.92
45.18
43.86
55.15
24.32
23.00
63.10
14.05
23.49
208.52
31.39
dropwise and stirring continued for 1 h. The reaction mixture
was cooled with ice bath, solids were filtered off and the solution
was applied on the column of silica gel (50 mL) in petroleum ether.
The product was eluted with a mixture of petroleum ether/acetone
(10:1) yielding 298 mg (81%) of ester 2b. Analytical sample crystal-
lized from ethanol, m.p. 144-145 °C, [a] -12 (c 0.29, chloroform).
D
IR: 1730 (C@O, ester); 1710 (C@O, ketone); 1382 (CH3); 1245,
1238, 1107 (C–O). 1H NMR (CDCl3): 5.23 bd, 1H, J = 6.2 (H-17b);
2.81 dd, 1H, J = 15.0, J0 = 13.4 (H-4
a); 1.04 s, 3H (3 ꢁ H-19); 0.83
s, 3H (3 ꢁ H-18). Calcd. for C26H29F5O3 (484.5): C, 64.45; H, 6.03;
F, 19.61. Found: C, 64.38; H, 6.11; F, 19.39.
2.2.3. 3
a-Hydroxy-5b-androstan-17b-yl pentafluorobenzoate (3a)
A
solution of cerium(III) chloride heptahydrate (713 mg,
1.91 mmol) in methanol (10 mL) was added to a stirred solution
of ketone 2a (845 mg, 1.74 mmol) in ethyl acetate (4 mL), and
starting immediately, sodium borohydride (66 mg, 1.74 mmol)
was added in portions over 5 min at room temperature. After addi-
tional stirring for 10 min, the reaction mixture was poured into ice-
cold 5% hydrochloric acid, extracted with ethyl acetate (100 mL),
and organic layer was washed with saturated aqueous potassium
hydrogen carbonate (2ꢁ), water, and dried. Solvents were evapo-
rated and crude product (820 mg, 97%) was crystallized from ace-
tone. The yield of ester 3a was 490 mg (58%); additional 140 mg
(16%) were obtained by column chromatography of the mother li-
quors on silica gel (70 mL) in a mixture of petroleum ether/acetone
-
-
-
-
-
a
Measured in CDCl3 or in a mixture of CDCl3/CD3OD (10:1) (5a, 8, and 14a).
Hydrochloride.
b
Table 1b
Carbon-13 chemical shifts of selected perfluorobenzoyl steroids: Substituents.
Carbon 5a
6ba
8
14a
14bb
17a
21
Perfluorobenzoyl group
1
158.56 158.61 159.31 157.71 158.80 157.36 158.51
108.56 109.00 108.14 112.23 113.94 110.85 108.66
145.33 145.12 145.57 143.78 145.02 142.53 143.12
137.62 137.69 141.41 141.89 138.87 137.66 137.60
142.99 142.96 123.02 137.44 143.20 144.24 143.00
10
20,60
(10:1). M.p. 163-164 °C, [a]D + 40 (c 0.24, chloroform). IR: 3609
30,50
40
(OH); 1732 (C@O); 1389, 1377 (CH3); 1243, 1009, 998 (C-O);
1
1033 (C-OH). H NMR (CDCl3): 4.87 dd, 1H, J = 9.0, J0 = 7.7 (H-
Glutamate
17a
); 3.64 tt, 1H, J = 10.9, J0 = 4.6 (H-3b); 0.94 s, 3H (3ꢁ H-19);
1
2
3
4
5
170.26
53.05
26.38
33.04
177.65
-
-
-
-
-
169.92 171.34 169.81 170.43
-
-
-
-
-
52.86
26.09
32.09
59.84
27.41
32.67
53.39
26.73
30.24
52.52
26.96
29.57
0.83 s, 3H (3ꢁ H-18). 19F NMR (CDCl3): ꢀ139.5 m, 2 F; ꢀ150.4 m,
1 F; ꢀ161.7 m, 2 F. Calcd. for C26H31F5O3 (486.5): C, 64.19; H,
6.42; F, 19.53. Found: C, 64.21; H, 6.30; F, 19.80.
176.93 176.96 175.40 176.99
a
Data for sulfates (pyridinium nucleus) - 6b: 142.23 (2,6), 127.14 (3,5), 145.72
2.2.4. 3a-Hydroxy-5b-androstan-17a-yl pentafluorobenzoate (3b)
(4); 21: 142.20 (2,6), 127.12 (3,5), 145.74 (4).
b
Ketone 2b (1.3 g, 2.68 mmol), ethyl acetate (5 mL), cerium(III)
chloride heptahydrate (1.1 g, 2.95 mmol) in methanol (15 mL),
and sodium borohydride (100 mg, 2.67 mmol) were processed as
described in Section 2.2.3. Crude product (1.3 g) was chromato-
graphed on a silica gel column (100 mL) in a mixture of petroleum
ether/acetone from (20:1) to (10:1). The main fractions contained
foamy hydroxy derivative 3b (1.2 g, 92%), which did not crystallize.
Data for hydrochloride.
2.2. Chemical synthesis
2.2.1. 3-Oxo-5b-androstan-17b-yl pentafluorobenzoate (2a)
Pentafluorobenzoyl chloride (1 mL, 6.94 mmol) was added
dropwise to a stirred solution of 17b-Hydroxy-5b-androstan-3-
one (1a) (1 g, 3.44 mmol) in pyridine (10 mL) cooled with ice-bath.
The mixture was allowed to attain the room temperature and the
stirring continued for 1 h. Then, after pouring into water with
ice, the product was extracted with ethyl acetate (200 mL). The
ethyl acetate extract was washed with 5% hydrochloric acid, satu-
rated aqueous potassium hydrogen carbonate (2ꢁ), water, and
dried. Solvents were evaporated and the crude pentafluorobenzo-
ate 2a (1.7 g) was chromatographed on a silica gel column
(20 mL) in a mixture of petroleum ether/acetone (20:1). Resulting
foamy product 2a (1.56 g, 93%) was used without further purifica-
tion. Analytical sample crystallized after addition of ether, m.p.
[a] -16 (c 0.33, chloroform). IR: 3609 (OH); 1730 (C@O); 1381
D
(CH3); 1245, 1108 (C–O); 1032 (C–OH). 1H NMR (CDCl3): 5.08 bd,
1H, J = 5.7 (H-17b); 3.65 tt, 1H, J = 11.0, J0 = 4.7 (H-3b); 0.93 s, 3H
(3ꢁ H-19); 0.79 s, 3H (3 ꢁ H-18). Calcd. for C26H31F5O3 (486.5):
C, 64.19; H, 6.42; F 19.53. Found: C, 64.23; H, 6.51; F, 19.64.
2.2.5. 5b-Androstane-3
a
,17b-diyl 17-(pentafluorobenzoate)
3-[O5-tert-butyl-N-(tert-butoxycarbonyl)-
L
-glutamate] (4a)
To a stirred solution of ester 3a (200 mg, 0.41 mmol) in dichlo-
romethane (2 mL), O5-tert-butyl hydrogen N-(tert-butoxycarbonyl)
-L
-glutamate (136 mg, 0.45 mmol) and 4-dimethylaminopyridine
(20 mg, 0.16 mmol) were added. Then, N,N0-diisopropylcarbodiim-
ide (64 L, 0.41 mmol) was added dropwise under argon atmo-
126-128 °C, [
1709 (C@O, ketone); 1390, 1381 (CH3); 1242, 1010, 1000 (C–O).
1H NMR (CDCl3): 4.89 dd, 1H, J = 9.0, J0 = 7.8 (H-17
); 2.69 dd,
1H, J = 14.6, J0 = 13.7 (H-4
a]D + 49 (c 0.30, chloroform). IR: 1732 (C@O, ester);
l
sphere and stirring continued for 1 h. The reaction mixture was
cooled with ice bath, solids were filtered off and the solution was
applied on the column of silica gel (10 mL) in petroleum ether.
The product was eluted with a mixture of petroleum ether/acetone
(20:1) yielding 298 mg (94%) of foamy ester 4a, which was used
a
a
); 1.04 s, 3H 3ꢁ H-19); 0.87 s, 3H
(3 ꢁ H-18). Calcd. for C26H29F5O3 (484.5): C, 64.45; H, 6.03; F,
19.61. Found: C, 64.34; H, 5.88; F, 19.89.

ˇ
´
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I. Cerny et al. / Steroids 77 (2012) 1233–1241
further without additional purification. IR: 3437 (NH); 2980, 1475,
1392, 1369, 1158 (CH3 tert-butyl); 1728 (C@O, ester); 1710 (C@O,
amide); 1381 (CH3); 1230, 1008 (C–O).1H NMR (CDCl3): 5.08 d, 1H,
2.2.10. 17
pyridinium salt (6b)
Hydroxy derivative 3b (200 mg, 0.41 mmol) was processed as
described in Section 2.2.9. After chromatography, the sulfate 6b
(240 mg) was crystallized from a mixture of ethanol/diethyl ether.
a-(Pentafluorobenzoyl)oxy-5b-androstan-3a-yl sulfate
J = 8.5 (NH); 4.88 dd, 1H, J = 9.0, J0 = 7.7 (H-17
a); 4.78 tt, 1H,
J = 11.0, J0 = 4.6 (H-3b); 4.25 m, 1H (CH-NH); 1.45 s, 9H and 1.44
s, 9H (2ꢁ (CH3)3C); 0.95 s, 3H (3ꢁ H-19); 0.84 s, 3H (3 ꢁ H-18).
The yield was 175 mg (66%), m.p. 164-166 °C, [a]D -4 (c 0.28, chlo-
roform). IR: 3200-1950 (N+–H); 1638, 1548, 1490 (pyridine ring);
1382 (CH3); 1238, 1171, 1049 (SO3). 1H NMR (CDCl3): 8.94 m, 2
H (H-20, H-60); 8.48 tt, 1H, J = 1.6, J0 = 8.0 (H-40); 7.99 m, 2 H (H-
30, H-50); 5.07 bd, 1H, J = 5.9 (H-17b); 4.48 tt, 1H, J = 4.9, J0 = 11.2
(H-3b); 0.92 s, 3H (3ꢁ H-19); 0.78 s, 3H (3ꢁ H-18). Calcd. for C31-
O36F5NO6S (645.7): C, 57.67; H, 5.62; F, 14.71; N, 2.17; S, 4.97.
Found: C, 57.37; H, 5.59; F, 14.56; N, 2.11; S, 4.78.
2.2.6. 5b-Androstane-3
3-[O5-tert-butyl-N-(tert-butoxycarbonyl)-
Ester 3b (300 mg, 0.61 mmol) in dichloromethane (2 mL) was
treated with O5-tert-butyl hydrogen N-(tert-butoxycarbonyl)-
glutamate (204 mg, 0.67 mmol), 4-dimethylaminopyridine
(30 mg, 0.25), and (96 L,
N,N0-diisopropylcarbodiimide
a
,17a-diyl 17-(pentafluorobenzoate)
L-glutamate] (4b)
L
-
l
0.62 mmol) as described in Section 2.2.5. The crude product
(430 mg, 90%) was chromatographed on a silica gel column
(80 mL) in a mixture of petroleum ether/acetone from (50:1) to
(20:1) to give foamy ester 4b (340 mg, 71%). IR: 3437 (NH);
2980, 1469, 1393, 1369, 1165 (CH3 tert-butyl); 1726 (C@O, ester);
1710 (C@O, amide); 1382 (CH3); 1238, 1006 (C-O). 1H NMR
(CDCl3): 5.08 d, 1H, J = 8.8 (H-17b); 5.06 bd, 1H (NH); 4.77 tt, 1H,
J = 11.0, J0 = 4.6 (H-3b); 4.23 m, 1H (CH-NH); 1.44 s, 9H and 1.43
s, 9H (2ꢁ (CH3)3C); 0.95 s, 3H (3 ꢁ H-19); 0.80 s, 3H (3 ꢁ H-18).
2.2.11. 3a-Hydroxy-5b-androstan-17b-yl 4-azidotetrafluorobenzoate
(7)
Ester 3a (300 mg, 0.62 mmol) in acetone (1.5 mL) was stirred
and heated to 55 °C with water (75 L) and sodium azide (80 mg,
l
0.69 mmol) for 5 h. Then, the reaction mixture was poured into
water with ice, extracted with ethyl acetate (40 mL), and organic
layer was washed with saturated aqueous potassium hydrogen
carbonate (2ꢁ), water, and dried. Solvents were evaporated and
the crude product (300 mg) was chromatographed on silica gel
(50 mL) in a mixture of petroleum ether/acetone (10:1). The yield
of foamy azido derivative 7 was 240 mg (76%). IR: 3609 (OH);
2131 (N3); 1728 (C@O); 1389, 1378 (CH3); 1261, 1008 (C-O);
2.2.7. 5b-Androstane-3
3-( -glutamate) (5a)
Ester 4a (240 mg, 0.31 mmol) was treated with trifluoroacetic
a,17b-diyl 17-(pentafluorobenzoate)
L
1
1032 (C–OH). H NMR (CDCl3): 4.85 dd, 1H, J = 10.9, J0 = 7.8 (H-
acid (0.85 mL) at room temperature for 10 min. Solvent was evap-
orated, the residue was dissolved in a solution of pyridine (0.6 mL)
in methanol (4 mL) and the mixture was poured into ice cold water
(50 mL). After overnight standing at 5 °C, the product was filtered
off and dried. The yield of glutamate 5a was 177 mg (93%). 1H
17a
); 3.64 tt, 1H, J = 10.9, J0 = 4.7 (H-3b); 0.94 s, 3H (3ꢁ H-19);
0.83 s, 3H (3ꢁ H-18). 19F NMR (CDCl3): ꢀ139.9 m, 2 F; ꢀ152.3 m,
2 F. Calcd. for C26H31F4N3O3 (509.5): C, 61.29; H, 6.13; F, 14.91;
N, 8.25. Found: C, 61.53; H, 6.30; F, 14.80.
NMR (CDCl3/CD3OD, 10:1): 4.88 dd, 1H, J = 8.7, J0 = 8.0 (H-17
a);
2.2.12. 5b-Androstane-3
3-( -glutamate) (8)
Compound 7 (200 mg, 0.39 mmol) was processed in dichloro-
methane (2 mL) with O5-tert-butyl hydrogen N-(tert-butoxycar-
bonyl)- -glutamate (131 mg, 0.43 mmol), 4-
dimethylaminopyridine (20 mg, 0.16 mmol), and N,N0-diisopropyl-
carbodiimide (62 L, 0.39 mmol) as described in Section 2.2.5. The
a,17b-diyl 17-(4-azidotetrafluorobenzoate)
4.83 tt, 1H, J = 11.5, J0 = 5.0 (H-3b); 4.00 bdd, 1H, J = 8.4, J0 = 5.0
(CH-NH2); 0.96 s, 3H (3ꢁ H-19); 0.84 s, 3H (3ꢁ H-18). For 13C
NMR data see Tables 1a and b. Calcd. for C31H38F5NO6.1/2 H2O
(624.6): C, 59.61; H, 6.29; F, 15.21; N, 2.24. Found: C, 59.51; H,
6.44; F, 15.36; N, 2.07.
L
L
l
2.2.8. 5b-Androstane-3
3-( -glutamate) (5b)
Ester 4b (200 mg, 0.26 mmol) was treated with trifluoroacetic
a,17a-diyl 17-(pentafluorobenzoate)
same work-up gave 290 mg (93%) of protected glutamate and
deprotection with trifluoroacetic acid (Section 2.2.7) gave 190 mg
(76% overall from 7) of glutamate 8. 1H NMR (CDCl3/CD3OD,
L
10:1): 4.86 dd, 1H, J = 8.7, J0 = 8.0 (H-17
a); 4.84 m (H-3b); 4.02 m
acid (0.80 mL) as described in Section 2.2.7. The yield of glutamate
5b was 153 mg (96%). 1H NMR (CDCl3/CD3OD, 10:1): 5.08 d, 1H,
1H, (CH–NH2); 0.96 s, 3H (3ꢁ H-19); 0.84 s, 3H (3ꢁ H-18). For
13C NMR data see Tables 1a and b. Calcd. for C31H38F4N4O6.H2O
(656.7): C, 56.70; H, 6.14; F, 11.57; N, 8.53. Found: C, 56.52; H,
5.93; F, 11.77; N, 8.29.
J = 6.2 (H-17a
); 4.82 tt, 1H, J = 11.4, J0 = 4.6 (H-3b); 3.85 bdd, 1H,
J = 7.8, J0 = 3.2 (CH-NH2); 0.96 s, 3H (3ꢁ H-19); 0.80 s, 3H (3ꢁ H-
18). Calcd. for C31H38F5NO6.H2O (633.6): C, 58.76; H, 6.36; F,
14.99; N, 2.21. Found: C, 58.79; H, 6.13; F, 14.83; N, 2.02.
2.2.13. N-(3
(12a)
a
-Hydroxy-5b-androstan-17b-yl)-pentafluorobenzamide
-hydroxy-5b-androstan-17-one oxime
2.2.9. 17b-(Pentafluorobenzoyl)oxy-5b-androstan-3
a
-yl sulfate
pyridinium salt (6a)
To a stirred solution of 3
a
To
a
stirred solution of hydroxy derivative 3a (200 mg,
(9) (1 g, 3.27 mmol) in methanol (73 mL), nickel(II) chloride hexa-
hydrate (1.55 g, 6.52 mmol) was added and after dissolution, so-
dium borohydride (1.24 g, 32.80 mmol) was added in portions
over 30 min at room temperature. After additional 15 min stirring,
the reaction mixture was cooled in ice bath and 1 M sulfuric acid
(40 mL) was added under stirring. The mixture was poured into
25% aqueous ammonia, diluted with ice water (1:1, 150 mL), and
extracted with chloroform (150 mL). The extract was washed with
ice water, dried and the solvent was evaporated. The crude mixture
of amines 10a and 10b (1 g) was dissolved in dichloromethane
(12 mL) while stirring under argon and N,N-diisopropylethylamine
(1.2 mL, 6.89 mmol) was added. Active ester solution was added to
this mixture, prepared separately from pentafluorobenzoic acid
(763 mg, 3.60 mmol), 1-hydroxybenzotriazole (620 mg, 3.96
0.41 mmol) in pyridine (3 mL), sulfur trioxide - pyridine complex
(196 mg, 1.23 mmol) was added and stirring continued at room
temperature for 30 min. Then, the solvent was evaporated and
the residue was dissolved in methanol (0.5 mL) and water
(1.5 mL) was added and the solution was applied to semi-prepara-
tive column of Lichrosorb RP-18 (75 mL) pretreated with water.
Polar impurities were washed out with water and the product
was eluted with 80% aqueous methanol. The main fraction con-
tained oily sulfate 6a, 120 mg, (45%), which did not crystallize.
1H NMR (CDCl3): 8.92 m, 2 H (H-20, H-60); 8.49 tt, 1H, J = 1.6,
J0 = 7.8 (H-40); 8.00 m, 2H (H-30, H-50); 4.85 dd, 1H, J = 7.9, J0 = 8.9
(H-17
a
); 4.50 tt, 1H, J = 4.8, J0 = 11.1 (H-3b); 0.94 s, 3H (3ꢁ H-
19); 0.83 s, 3H (3 ꢁ H-18). MS (negESI): Calcd. for [C26H30F5O6S]-:
565.57. Found: 565.2.
mmol), and N,N0-diisopropylcarbodiimide (557
lL, 3.60 mmol) in

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dichloromethane (15 mL) by 20 min stirring at room temperature,
cooling with ice bath, and filtering into syringe. After 30 min stir-
ring at room temperature, the reaction mixture was diluted with
chloroform (100 mL), washed with 5% hydrochloric acid, saturated
aqueous potassium hydrogen carbonate (2ꢁ), water, and dried.
Solvents were evaporated and the crude mixture of perflu-
orobenzoates (1.9 g) was chromatographed on a silica gel column
(200 mL) in a mixture of petroleum ether/acetone from (20:1) to
protected glutamate 13b was 370 mg (89%). 1H NMR (CDCl3):
5.80 d, 1H, J = 9.1 (NH); 5.06 d, 1H, J = 8.4 (NH Boc); 4.76 tt, 1H,
J = 11.2, J0 = 4.6 (H-3b); 4.23 m, 2H (CH-NH Boc, H-17
a); 1.44 s,
9H and 1.43 s, 9H (2ꢁ (CH3)3C); 0.95 s, 3H (3ꢁ H-19); 0.86 s, 3H
(3ꢁ H-18).
2.2.17. 17b-(Pentafluorobenzoyl)amino-5b-androstan-3
-glutamate (14a)
Protected glutamate 13a (400 mg, 0.52 mmol) was dissolved in
a-yl
L
(10:1). Less polar fractions contained 17a-isomer 12b (275 mg,
17% from oxime 9), see below. Further elution gave 1.04 g of amide
12a (66% from oxime 9). Analytical sample was crystallized from
methanol, m.p. 243–244 °C, [a]D + 16 (c 0.22, chloroform). IR:
dioxane (0.5 mL) and hydrogen chloride in dioxane (4.0 M solution,
1.5 mL, 6 mmol) was added while stirring at room temperature.
After 24 h standing, the solvent was evaporated, the residue was
coevaporated twice with ethanol and triturated with ether. Solid
product was filtered off, dissolved in a mixture of methanol
3609 (OH); 3432 (NH); 1685, 1505 (CONH); 1388, 1377 (CH3);
1033 (C-OH). 1H NMR (CDCl3): 5.72 d, 1H, J = 8.9 (NH); 4.09 q,
1H, J = 9.2 (H-17
a
); 3.63 m, 1H (H-3b); 0.94 s, 3H (3ꢁ H-19); 0.73
(4 mL) and pyridine (40 lL, 0.49 mmol) and added dropwise into
s, 3H (3ꢁ H-18). 19F NMR (CDCl3): ꢀ142.1 m, 2 F; ꢀ152.8 tt, 1 F,
J = 20.7, J0 = 2.8; ꢀ161.7 m, 2 F. Calcd. for C26H32F5NO2 (485.5): C,
64.32; H, 6.64; F, 19.56; N, 2.88. Found: C, 64.36; H, 6.72; F,
19.79; N, 2.74.
water with ice (20 mL). After standing at 5 °C overnight, the solid
glutamate 14a was filtered of and dried. The yield was 190 mg
1
(60%). H NMR (CDCl3/CD3OD, 10:1): 4.83 tt, 1H, J = 11.2, J0 = 4.5
(H-3b); 4.07 t, 1H, J = 9.4 (H-17a); 3.73 dd, 1H (CH–NH2); 0.96 s,
3H (3ꢁ H-19); 0.73 s, 3H (3ꢁ H-18). For 13C NMR data see Tables
1a and b. Calcd. for C31H39F5N2O5.H2O (632.7): C, 58.85; H, 6.53;
F, 15.01; N, 4.43. Found: C, 58.94; H, 6.22; F, 15.20; N, 4.38.
2.2.14. N-(3
a-Hydroxy-5b-androstan-17
a-yl)-pentafluorobenzamide
(12b)
To a stirred solution of 17
a-azido-5b-androstan-3a-ol (11)
(500 mg, 1.57 mmol) in a mixture of dioxane (5 mL) and ethanol
(15 mL), sodium borohydride (200 mg, 5.29 mmol) was added fol-
lowed by a solution of nickel(II) chloride hexahydrate in methanol
(0.1 mol.Lꢀ1, 1.5 mL, 0.15 mmol). Stirring continued for 45 min at
room temperature. Then, the reaction mixture was cooled in ice
bath and 1 M sulfuric acid (9 mL) was added under stirring. The
mixture was poured into 25% aqueous ammonia diluted with ice
water, (1:1, 90 mL) and extracted with chloroform (60 mL). The ex-
tract was washed with ice water, dried and the solvent was evap-
orated. The crude amine (490 mg) was processed as described in
Section 2.2.13. The yield was 627 mg (82% to azide 11) of foamy
2.2.18. 17
-glutamate (14b)
Protected glutamate 13b (350 mg, 0.46 mmol) was processed as
a-(Pentafluorobenzoyl)amino-5b-androstan-3a-yl
L
described in Section 2.2.17. Trituration with ether gave crystalline
hydrochloride, 250 mg (83%). 1H NMR (CD3OD): 4.87 tt, 1H,
J = 11.2, J0 = 4.5 (H-3b); 4.08 dd, 1H, J = 6.2, J’ = 1.5 (H-17b); 4.07 t,
1H, J = 6.8 (CH–NH2); 1.01 s, 3H (3ꢁ H-19); 0.88 s, 3H (3ꢁ H-18).
19F NMR (CDCl3): ꢀ141.4 m, 2 F; ꢀ153.6 m, 1 F; ꢀ161.7 m, 2 F.
For 13C NMR data see Tables 1a and b. Solid product was filtered
off and processed as described in Section 2.2.17. The glutamate
14b had rather geloid structure and the yield was 90 mg (32% from
13b) due to mass loss during filtration. Calcd. for C31H39F5N2O5.H2-
O (632.7): C, 58.85; H, 6.53; F, 15.01; N, 4.43. Found: C, 58.52; H,
6.53; F, 14.97; N, 4.20.
amide 12b, [a]D + 8 (c 0.24, chloroform). IR: 3609 (OH); 3431
(NH); 1682, 1504 (CONH); 1383 (CH3); 1038 (C-OH). 1H NMR
(CDCl3): 5.76 d, 1H, J = 8.4 (NH); 4.20 ddd, 1H, J = 8.9, J0 = 7.8,
J00 = 1.4 (H-17b); 3.65 tt, 1H, J = 10.9, J0 = 4.8, (H-3b); 0.93 s, 3H
(3ꢁ H-19); 0.85 s, 3H (3ꢁ H-18). 19F NMR (CDCl3): ꢀ141.4 m, 2
F; ꢀ152.4 tt, 1 F, J = 20.8, J0 = 2.7; ꢀ161.1 m, 2 F. Calcd. for C26H32-
F5NO2 (485.5): C, 64.32; H, 6.64; F, 19.56; N, 2.88. Found: C, 64.58;
H, 6.70; F, 19.78; N, 2.81. The product was identical with the less
polar product from the chromatography of amide 12a
(Section 2.2.13).
2.2.19. 20-Oxo-5b-pregnan-3
-Hydroxy-5b-pregnan-20-one (15) (500 mg, 1.57 mmol) in
dichloromethane (5 mL) was processed with O5-tert-butyl hydro-
gen N-(tert-butoxycarbonyl)- -glutamate (524 mg, 1.72 mmol),
4-dimethylaminopyridine (50 mg, 0.41 mmol), and N,N0-diisopro-
pylcarbodiimide (250 L, 1.61 mmol) as described in Section 2.2.5.
a-yl L-glutamate hydrochloride (16)
3a
L
l
Crude foamy protected glutamate (935 mg) was treated in dioxane
(1.5 mL) with hydrogen chloride in dioxane (4.0 M solution, 3.0 mL,
12 mmol) overnight. The solvents were evaporated, the residue
was coevaporated twice with ethanol and crystallized from mini-
mum ethanol by adding of ether. The yield of hydrochloride 16
was 460 mg (61% from pregnanolone 15). Calcd. for C26H42ClNO5
(484.1): C, 64.51; H, 8.75; Cl, 7.32; N, 2.89. Found: C, 64.24; H,
8.77; Cl, 7.07; N, 2.73.
2.2.15. 17b-(Pentafluorobenzoyl)amino-5b-androstan-3
a-yl
O5-tert-butyl-N-(tert-butoxycarbonyl)-
L-glutamate (13a)
Amide 12a (400 mg, 0.82 mmol) in dichloromethane (4 mL) was
processed as described in Section 2.2.5 with O5-tert-butyl hydro-
gen N-(tert-butoxycarbonyl)-
L-glutamate (272 mg, 0.90 mmol),
4-dimethylaminopyridine (40 mg, 0.33 mmol), and N,N0-diisopro-
pylcarbodiimide (128 lL, 0.83 mmol). The yield of foamy protected
glutamate 13a was 630 mg (99%). 1H NMR (CDCl3): 5.74 d, 1H,
J = 8.9 (NH); 5.08 d, 1H, J = 8.4 (NH Boc); 4.77 tt, 1H, J = 11.0,
J0 = 4.6 (H-3b); 4.25 m, 1H (CH–NH Boc); 4.11 q, 1H, J = 9.2 (H-
2.2.20. 20-Oxo-5b-pregnan-3
-glutamate (17)
Hydrochloride 16 (250 mg, 0.52 mmol) was suspended in
a-yl N-(pentafluorobenzoyl)-
L
17a
); 1.45 s, 9H and 1.44 s, 9H (2ꢁ (CH3)3C); 0.95 s, 3H (3ꢁ H-
19); 0.73 s, 3H (3ꢁ H-18). 19F NMR (CDCl3): ꢀ141.7 m, 2 F;
ꢀ152.3 m, 1 F; ꢀ161.2 m, 2 F.
dichloromethane (2 mL) and while stirring under argon N,N-diiso-
propylethylamine (0.4 mL, 2.30 mmol) was added. Active ester
solution was added to this mixture, prepared separately from
pentafluorobenzoic acid (120 mg, 0.57 mmol), 1-hydroxybenzotri-
azole (89 mg, 0.57 mmol), and N,N0-diisopropylcarbodiimide
2.2.16. 17
a
-(Pentafluorobenzoyl)amino-5b-androstan-3
a-yl
O5-tert-butyl-N-(tert-butoxycarbonyl)-
L-glutamate (13b)
Amide 12b (264 mg, 0.54 mmol) in dichloromethane (2.5 mL)
was processed as described in Section 2.2.5. with O5-tert-butyl
(90 lL, 0.58 mmol) in dichloromethane (1.5 mL) by 20 min stirring
at room temperature, cooling with ice bath, and filtering into syr-
inge. After 1 h stirring at room temperature, the reaction mixture
was diluted with chloroform (100 mL), washed with 10% sulfuric
acid, water (2 ꢁ), and dried. Solvents were evaporated and the
hydrogen N-(tert-butoxycarbonyl)-
mmol), 4-dimethylaminopyridine (25 mg, 0.20 mmol), and N,N0-
diisopropylcarbodiimide (85 L, 0.55 mmol). The yield of foamy
L-glutamate (182 mg, 0.60
l

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crude product (380 mg) was chromatographed on a silica gel col-
t, 1H, J = 8.9 (H-17
a
); 2.51 dd, 1H, J = 5.3, J0 = 11.7 (H-12
a); 2.11
umn (75 mL) in a mixture of benzene/ethyl acetate from (10:1)
to (5:1). The yield of foamy perfluorobenzoyl glutamate 17 was
300 mg (91%), [a]D + 66 (c 0.32, chloroform). IR: 3513 (OH);
s, 3H (3ꢁ H-21); 0.92 s, 3H (3ꢁ H-19); 0.78 s, 3H (3ꢁ H-18). Calcd.
for C33H38F5NO7S (687.7): C, 57.63; H, 5.57; F, 13.81; N, 2.04; S,
4.66. Found: C, 57.46; H, 5.53; F, 14.04; N, 1.97; S, 4.79.
3091, 2649 (OH, dimer); 3407 (NH); 1728 (C@O, ester); 1713
(C@O, dimer); 1695, 1505 (CONH); 1386, 1357 (CH3). 1H NMR
(CDCl3): 6.90 d, 1H, J = 7.5 (NH); 4.83 tt, 1H, J = 11.2, J0 = 4.5 (H-
3b); 4.79 dt, 1H, J = 7.5, J0 = 4.8 (CH–NH); 2.12 s, 3H (3ꢁ H-21);
0.95 s, 3H (3ꢁ H-19); 0.61 s, 3H (3ꢁ H-18). 19F NMR (CDCl3):
ꢀ141.3 m, 2 F; ꢀ151.1 tt, 1 F, J = 20.9, J0 = 3.2; ꢀ161.0 m, 2 F. Calcd.
for C33H40F5NO6 (641.7): C, 61.77; H, 6.28; F, 14.80; N, 2.18;.
Found: C, 61.89; H, 6.06; F, 15.10; N, 1.95.
2.3. Biological assays
For the electrophysiological experiments on NMDA receptors,
human embryonic kidney cells (HEK293) were transfected with
GluN1–1a/GluN2B/GFP plasmids as described previously [16].
Experiments were performed 24–48 h after the end of transfection.
HEK293 cells were voltage-clamped at a holding potential of
ꢀ60 mV and currents elicited were recorded. Whole-cell voltage-
clamp recordings were made with a patch-clamp amplifier (Axo-
patch 200B; Molecular Devices, Sunnyvate, CA). Patch pipettes
2.2.21. 3,20-Dioxo-5b-pregnan-11a-yl pentafluorobenzoate (19)
A
solution of 11 -hydroxy-5b-pregnane-3,20-dione (18)
a
(900 mg, 2.71 mmol) in dichloromethane (9 mL) was stirred with
4-dimethylaminopyridine (90 mg, 0.74 mmol) and pentafluoro-
benzoic acid (630 mg, 2.97 mmol) at room temperature and N,N0-
(3–5 M
X
) 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). Extracellular
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
diisopropylcarbodiimide (465 lL, 3.00 mmol) was added dropwise.
After 1 h stirring, the reaction mixture was cooled with ice bath, fil-
tered into syringe and applied to silica gel column (50 mL) pre-
treated with petroleum ether. The product was eluted with a
mixture of petroleum ether/acetone (10:1); the yield was 1.3 g
(91%). Analytical sample of 19 was crystallized from a mixture of
to 7.3 with NaOH). Glycine (10 lM), 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 sulfox-
ide (DMSO). The same concentration of DMSO was added in all
extracellular solutions (see [17] for details).
acetone/hexanes, m.p. 156-157 °C, [a]D + 67 (c 0.37, chloroform).
IR: 1732 (C@O, ester); 1706 (C@O, ketone); 1391, 1382, 1357
(CH3); 1234, 1109 (C–O). 1H NMR (CDCl3): 5.50 dt 1H, J = 5.2,
J0 = 10.5 (H-11b); 2.13 s, 3H (3ꢁ H-21); 1.19 s, 3H (3ꢁ H-19);
0.75 s, 3H (3ꢁ H-18). Calcd. for C28H31F5O4 (526.5): C, 63.87; H,
5.93; F, 18.04. Found: C, 64.34; H, 5.88; F, 19.89.
3. Results and discussion
3.1. Syntheses of perfluorobenzoylated compounds
The initial experiments were performed on the 5b-androstan-
2.2.22. 3a-Hydroxy-20-oxo-5b-pregnan-11a-yl pentafluorobenzoate
(20)
Diketone 19 (830 mg, 1.58 mmol) was dissolved in tetrahydro-
furan (33 mL), stirred and cooled to -70 °C and lithium tri(tert-but-
oxy)aluminum hydride (600 mg, 2.36 mmol) was added during
5 min. The reaction mixture was stirred 30 min at ꢀ70 °C, then al-
lowed to attain ꢀ30 °C during 45 min and stirred additional 1 h.
Saturated aqueous solution of ammonium chloride (2 mL) was
added and about one half of the solvent was evaporated. The mix-
ture was poured into 5% hydrochloric acid and extracted with ethyl
acetate (80 mL). The extract was washed with saturated aqueous
potassium hydrogen carbonate (2ꢁ), water, and dried. The solvents
were evaporated and the crude product (860 mg) was chromato-
graphed on a silica gel column (160 mL) in a mixture of petroleum
ether/acetone from (10:1) to (5:1). The main fractions contained
hydroxy derivative 20 (580 mg, 70%) with spots of less polar 3b-
isomer. Crystallization from a mixture of acetone/hexanes gave
3a-ol skeleton with perfluorobenzoyl group attached in position
17 (Scheme 1). Starting compound was easily available 17b-hydro-
xy-5b-androstan-3-one (1a) [10], which was reacted with perflu-
orobenzoyl chloride in pyridine and resulting ester 2a was
selectively reduced with sodium borohydride in presence of ceriu-
m(III) chloride heptahydrate [18]. Thus obtained 3a-hydroxy
derivative 3a was transformed both into a glutamate 5a and to a
sulfate 6a. For a preparation of glutamate, an indirect method
was used. In the first step, protected glutamate 4a was prepared
by the reaction of 3a with O5-tert-butyl hydrogen N-(tert-butoxy-
carbonyl)-L
-glutamate and N,N0-diisopropylcarbodiimide in pres-
ence of 4-dimethylaminopyridine. Then, both protective groups
were removed with trifluoroacetic acid. Corresponding sulfate
was prepared in form of pyridinium salt by reaction 3a with sul-
fur(VI) oxide - pyridine complex.
pure compound (450 mg, 55%), m.p. 174–175 °C, [
a
]
+77 (c 0.26,
The preparation of 17
step, where the reaction of 17
a
-isomer 3b was similar, except the first
-hydroxy-5b-androstan-3-one
D
chloroform). IR: 3610 (OH); 1731 (C@O, ester); 1703 (C@O, ke-
tone); 1390, 1360 (CH3); 1245, 1235, 1107 (C–O); 1037 (C-OH).
1H NMR (CDCl3): 5.42 dt 1H, J = 5.4, J0 = 10.5 (H-11b); 3.63 m, 1H
(H-3b); 2.12 s, 3H (3ꢁ H-21); 1.09 s, 3H (3ꢁ H-19); 0.70 s, 3H
(3ꢁ H-18). Calcd. for C28H33F5O4 (528.6): C, 63.63; H, 6.29; F,
17.97. Found: C, 63.52; H, 6.48; F, 17.81.
a
(1b) [11,12] with perfluorobenzoic acid, N,N0-diisopropylcarbodi-
imide, and 4-dimethylaminopyridine was used. The yield of perflu-
orobenzoate 2b was slightly lower than for 17b-isomer (81%), but
the acid is cheaper and more easily handled in comparison with
corresponding acid chloride. The ketone reduction to 3b, prepara-
tion of glutamate 5b via protected intermediate 4b and prepara-
tion of sulfate 6b were identical to procedures used previously.
In the case of more accessible 17b ester 3a, conversion into 4-
azidoperfluorobenzoate was performed (Scheme 2). Nucleophilic
substitution with sodium azide in acetone at 55 °C took 5 h and
it was accompanied in some extent with thermal degradation.
Nevertheless, it was easy way to azide 7, which was then trans-
formed into glutamate 8 by the indirect procedure mentioned
previously.
2.2.23. 20-Oxo-11a-(pentafluorobenzoyl)oxy-5b-pregnan-3a-yl
sulfate pyridinium salt (21)
Hydroxy derivative 20 (200 mg, 0.38 mmol) in pyridine (3 mL),
sulfur trioxide - pyridine complex (180 mg, 1.13 mmol) was pro-
cessed as described in Section 2.2.9. The crude sulfate after chro-
matography (250 mg) was crystallized from a mixture of ethanol/
diethyl ether. The yield of 21 was 145 mg (51%), m.p. 187-189 °C,
[a]
+ 78 (c 0.23, chloroform). 1H NMR (CDCl3): 8.92 m, 2 H (H-
D
20, H-60); 8.48 tt, 1H, J = 1.6, J0 = 7.9 (H-40); 7.98 m, 2 H (H-30, H-
50); 5.42 dt, 1H, J = 5.3, J0 = 10.4 (H-11b); 4.45 m, 1H, (H-3b); 2.59
For the synthesis of analogous amides, firstly the corresponding
amino derivatives were prepared (Scheme 3). As a starting com-

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1238
Scheme 1. Synthesis of 17-perfluorobenzoyl derivatives. (i) PfbCl / Py, r.t., 1 h, 93%; (ii) PfbOH, DMAP, DIC / DCM, r.t., 1 h, 81%; (iii) NaBH4, CeCl3.7H2O / EtOAc, MeOH, r.t.,
15 min, 17b: 74%, 17 : 92%; (iv) Boc-L-Glu(t-Bu)OH, DMAP, DIC / DCM, r.t., 1 h, 17b: 94%, 17 : 71%; and (v) 1. TFA, r.t., 10 min, 2. Py / MeOH, 17b: 93%, 17 : 96%; (vi) SO3-Py /
Py, r.t., 30 min, 17b: 45%, 17 : 66%.
a
a
a
a
pound, 3
a
-hydroxy-5b-androstan-17-one oxime (9) [13] was used.
deprotection, hydrogen chloride in dioxane was used and in the
case of derivative 14b, corresponding hydrochloride could be
isolated.
Reduction with sodium borohydride – nickel(II) chloride [19,20]
gave preferently 17b-amino derivative 10a with an admixture of
17a-isomer 10b. The crude mixture of amines 10a and 10b reacted
The series was expanded with derivatives of different type
with active ester, prepared from 1-hydroxybenzotriazole and per-
fluorobenzoic acid. The resulting mixture of amides 12a and 12b
was separated by column chromatography. From the isolated
yields, ratio of isomers can be estimated roughly as 4:1 in favor
(Scheme 4). From 3a-hydroxy-5b-pregnan-20-one 15 (pregnano-
lone), the corresponding glutamate [21] was prepared in form of
hydrochloride 16 and condensation with active ester of perfluoro-
benzoic acid with procedure mentioned previously gave
of 12a. For the preparation of 17
a-isomer 12b more suitable syn-
perfluorobenzamide 17. Finally, from 11a-hydroxy-5b-pregnane-
thesis can be alternatively used. 17
a
-Azido-5b-androstan-3 -ol
a
3,20-dione (18) [14,15], perfluorobenzoate 19 was prepared by
the method used for 17-hydroxy derivative 3b and subsequent
selective reduction of 3-keto group with lithium tri(tert-but-
oxy)aluminum hydride [22] gave 3a-hydroxy derivative 20. For
transformation into corresponding sulfate 21, reaction with sul-
(11) [9] gave with before-mentioned reducing agent pure amine
10b and subsequently amide 12b in the 82% overall yield. Both
amides 12a and 12b were transformed into corresponding gluta-
mates 14a and 14b via protected derivatives 13a and 13b in the
analogous way, as described for the esters 5a and 5b. For the final
fur(VI) oxide - pyridine complex was used.

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Scheme 2. Synthesis of 4-azidoperfluorobenzoate. (i) NaN3 / aq. acetone, 55 °C, 5 h, 76%; and (ii) 1. Boc-L-Glu(t-Bu)OH, DMAP, DIC / DCM, r.t., 1 h, 2. TFA, r.t., 10 min, 3. Py /
MeOH, 76%.
Scheme 3. Synthesis of 17-perfluorobenzamido derivatives. (i) NaBH4, NiCl2.6H2O / MeOH, r.t., 45 min; (ii) NaBH4, NiCl2.6H2O / dioxane - EtOH, r.t., 45 min; (iii) 1. PfbOH,
HOBt, DIC / DCM, r.t. 20 min, 2. DIPEA / DCM, r.t., 30 min, 17b: 66% (i) + (ii), 17a: 82% (i) + (iii); (iv) Boc-L-Glu(t-Bu)OH, DMAP, DIC / DCM, r.t., 1 h, 17b: 99%, 17a: 89%; and (v)
1. HCl / dioxane, r.t., 24 h, 2. Py / MeOH, 17b: 60%, 17 : 32%.
a

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I. Cerny et al. / Steroids 77 (2012) 1233–1241
1240
Scheme 4. Syntheses of perfluorobenzoyl pregnanolone derivatives. (i) 1. Boc-L-Glu(t-Bu)OH, DMAP, DIC / DCM, r.t., 1 h, 2. HCl / dioxane, r.t., o.n., 61%; (ii) PfbOH, HOBt, DIC /
DCM, r.t. 20 min, 2. DIPEA, / DCM, r.t., 1 h, 91%; (iii) PfbOH, DMAP, DIC / DCM, r.t., 1 h, 91%; (iv) Li(tri-t-BuO)AlH / THF, -70 to -30 °C, 2.25 h, 55%; and (v) SO3-Py / Py, r.t.,
30 min, 51%.
Fig. 1. Example of traces obtained from HEK293 cells transfected with GluN1/GluN2B receptor. Perfluorobenzoates 5a (17bOPfbAG, 100
lM) and 5b (17aOPfbAG, 30 lM)
were applied simultaneously with 1 mM glutamate (duration of steroid and glutamate application is indicated by filled and open bars, respectively).

ˇ
´
1241
I. Cerny et al. / Steroids 77 (2012) 1233–1241
Table 2
Acknowledgments
Inhibition of Glu-induced response in recombinant NMDA receptors cells by
perfluorobenzoate substituted steroids.
ˇ
We thank Mrs. L. Kloucková and Mrs. M. Kuntošová for excel-
lent technical assistance. Dr. S. Vašícková was taking the IR spectra
Compounda
Conc. (
l
M)
% Inhib. S.D.b
IC50
(l
M) S.D.c
nd
ˇ
and Dr. P. Fiedler was interpreting them. Mass spectra were mea-
sured by the staff of the Laboratory of Mass Spectrometry (Dr. J.
17bOPfbAG 5a
100
30
100
10
100
100
50
6.4 3.3
39.5 7.0
76.6 2.0
76.7 4.9
6.0 1.8
1580 1105
44.1 11.9
37.3 3.5
3.7 0.8
1365 565
718 175
45.5 4.9
22.2 1.7
15.7 2.7
5
4
4
5
5
5
5
5
5
17
17bOPfbAS 6a
17 OPfbAS 6b
aOPfbAG 5b
ˇ
Cvacka, Head). Elemental analyses were carried out in the Analyt-
a
ˇ
ical Laboratory (Dr. S. Matejková, Head). This work was supported
17bOApfbAG 8
17bNHPfbAG 14a
by the GACR (P304/12/G069; P303/12/1464), TACR (TE01020028),
and Research Projects of the AS CR (RVO: 61388963 and 67985823.
9.0 2.2
17
NHPfbPG 17
11 OPfbPS 21
aNHPfbAG 14b
53.0 3.2
73.2 1.9
82.2 7.8
50
50
a
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