Synthesis and acetylcholinesterase inhibitory activity of 2β,3α-disulfoxy-5α-cholestan-6-one

Article abstract of DOI:10.1016/j.steroids.2011.05.005

Disodium 2β,3α-dihydroxy-5α-cholestan-6-one disulfate (8) has been synthesized using cholesterol (1) as starting material. Sulfation was performed using trimethylamine-sulfur trioxide complex in dimethylformamide as the sulfating agent. The acetylcholinesterase inhibitory activity of compound 8 was evaluated and compared to that of disodium 2β,3α-dihydroxy- 5α-cholestane disulfate (10) and diols 7 and 9. Compounds 8 and 10 were active with IC₅₀ values of 14.59 and 59.65 μM, respectively. Diols 7 and 9 showed no inhibitory activity (IC₅₀ > 500 μM).

Full text of DOI:10.1016/j.steroids.2011.05.005

Steroids 76 (2011) 1160–1165
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and acetylcholinesterase inhibitory activity of
2␤,3␣-disulfoxy-5␣-cholestan-6-one
a
a
b
a,∗
Victoria Richmond , Gustavo A. Garrido Santos , Ana P. Murray , Marta S. Maier
a
UMYMFOR (CONICET-UBA) and Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, 1428
Buenos Aires, Argentina
INQUISUR, Departamento de Química, Universidad Nacional del Sur, Avda. Alem 1253, 8000 Bahía Blanca, Buenos Aires, Argentina
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Disodium 2␤,3␣-dihydroxy-5␣-cholestan-6-one disulfate (8) has been synthesized using cholesterol (1)
as starting material. Sulfation was performed using trimethylamine–sulfur trioxide complex in dimethyl-
formamide as the sulfating agent. The acetylcholinesterase inhibitory activity of compound 8 was
evaluated and compared to that of disodium 2␤,3␣-dihydroxy-5␣-cholestane disulfate (10) and diols
Received 1 March 2011
Received in revised form 10 May 2011
Accepted 13 May 2011
Available online 26 May 2011
7
and 9. Compounds 8 and 10 were active with IC50 values of 14.59 and 59.65 ␮M, respectively. Diols 7
and 9 showed no inhibitory activity (IC50 > 500 ␮M).
Keywords:
©
2011 Elsevier Inc. All rights reserved.
Sulfated steroids
Synthesis
Acetylcholinesterase activity
1
. Introduction
tive. These results were indicative of the role of the sulfate groups
in the antiviral activity of 10 and prompted us to synthesize a
new disulfated steroid, disodium 2␤,3␣-dihydroxy-5␣-cholestan-
6-one disulfate (8), which differs from 10 in the presence of a
carbonyl group at C-6. The aim of this work was the synthesis of this
new disulfated analog and the evaluation of its acetylcholinesterase
activity.
Sulfated polyhydroxysteroids have been described from marine
sources, especially from Porifera and Ophiuroidea (Echinodermata)
1–4]. These polar compounds have received considerable attention
[
due to their broad spectrum of biological activities, such as anti-HIV
effects [5], inhibition of protein tyrosine kinases [6,7], and antiviral
activities [8]. In 2000, six new sulfated antimicrobial aminosterols
were isolated from the dogfish shark Squalus acanthias [9]. Recently,
two new disulfated steroids were isolated from the sea lamprey
Petromyzon marinus migratory pheromone [10].
Due to the low concentration of these natural products in marine
organisms and the constant need for new bioactive compounds, the
synthesis of analogs poses an alternative for the development of
compounds with interesting biological activities. In 2006, Murphy
et al. [11] synthesized 2␤,3␣,6␣-cholestanetrisulfate, an analog of
the natural sokotrasterol sulfate [12] and demonstrated the impor-
tance of the sulfate groups for the angiogenic activity. Recently,
three new cytotoxic disulfated steroids with sulfate groups located
at C-3 and C-6 of ring A and B were prepared [13]. In a previous work
Acetylcholine serves as a neurotransmitter in the central and
peripheral nervous system. Acetylcholinesterase (AChE) stops the
function of acetylcholine by its hydrolytic destruction in the cholin-
ergic synapses [15]. According to the cholinergic hypothesis, the
selective and irreversible deficiency of cholinergic functions leads
to memory impairment in Alzheimer’s disease (AD) [16]. Enhance-
ment of acetylcholine level in the brain is considered one of the
most promising approaches for treating AD [17]. Retaining the
neurotransmitters especially at the synaptic terminals via the inhi-
bition of the hydrolytic enzymes, i.e., the cholinesterases, in order
to compensate the deficiency of the cholinergic neurotransmit-
ters would lead to the improved cognitive activities of the patient
[18]. Recently, we have found that the sulfation of calenduladiol, a
pentacyclic triterpene isolated from Chuquiraga erinacea, enhanced
the inhibitory activity against AChE from 31% for calenduladiol
(0.5 mM) to 94% for disodium calenduladiol disulfate (0.5 mM)
[19]. These results prompted us to test the acetylcholinesterase
inhibitory activity of sulfated compounds 8 and 10 and compare
it to their corresponding diols (7 and 9).
[
14], we have synthesized five new sulfated and acetylated deriva-
tives of 2␤,3␣-dihydroxy-5␣-cholestane and tested their antiviral
activity against herpes simplex virus type 2 (HSV-2). Disodium
2
␤,3␣-dihydroxy-5␣-cholestane disulfate (10) showed the best
selectivity index (CC50/IC50) while its desulfated analog was inac-
Molecular docking studies of an active steroid into the periph-
eral cavity of AChE were performed in order to explain the
inhibitory activity the steroids showed and to corroborate the
∗ Corresponding author. Tel.: +54 11 4576 3346; fax: +54 11 4576 3385.
E-mail address: maier@qo.fcen.uba.ar (M.S. Maier).
0
039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.05.005

V. Richmond et al. / Steroids 76 (2011) 1160–1165
1161
inhibition kinetic data. The determination of the inhibition type
is critical for identification of the mechanism of inhibition and the
site of inhibitor binding.
was added and the organic layer was separated and then washed
with saturated NaCl solution. It was dried over anhydrous MgSO4
and evaporated to dryness. The reaction product was purified by
dry column flash chromatography on silica gel using hexane/AcOEt
(97:3) to give 1.83 g (73%) of 3 as a white crystalline solid, m.p.
2
. Experimental
◦
5–66 C (acetone–H O). H NMR ı (CDCl ): 0.29 (dd, J = 4.9 Hz,
2 3
1
6
2
.1. General methods
8.1 Hz, 1H, H-4␣), 0.52 (t, J = 4.5 Hz, 4.5 Hz, 1H, H-4␤), 0.72 (s, 3H, H-
8), 0.86 (d, J = 6.6 Hz, 6H, H-26, H-27), 0.91 (d, J = 6.6 Hz, 3H, H-21),
1
13
Melting points (m.p.) were determined on a Fisher Johns appa-
1.05 (s, 3H, H-19), 1.08 (m, 1H, H-3␣), 3.25 (m, 1H, H-6␣). C NMR
ı (CDCl3): 33.34 (C-1), 25.17 (C-2), 24.35 (C-3), 11.72 (C-4), 39.03
(C-5), 73.95 (C-6), 37.31 (C-7), 29.99 (C-8), 47.80 (C-9), 43.03 (C-10),
22.96 (C-11), 40.35 (C-12), 42.87 (C-13), 56.64 (C-14), 24.39 (C-15),
28.45 (C-16), 56.50 (C-17), 12.33 (C-18), 20.36 (C-19), 35.94 (C-20),
18.85 (C-21), 36.31 (C-22), 24.00 (C-23), 39.65 (C-24), 28.16 (C-
25). 22.71 (C-26), 22.85 (C-27). Anal. calcd for C27H46O, C 83.87, H
11.99, O 4.14. Found, C 83.61, H 11.83. HREIMS (ESI+), calculated for
ratus and are uncorrected. 1H NMR, C NMR, HSQC-DEPT, HMBC,
COSY and NOESY spectra were recorded on a Bruker AM 500 spec-
trometer. Chemical shifts (ı) are given in ppm downfield from
TMS as the internal standard. 2D NMR spectra were obtained using
standard Bruker software. High resolution mass spectra were deter-
mined on a Bruker micrOTOF-Q II mass spectrometer with ESI
as ionization source. IR spectra were acquired on a FT-IR Nicolet
Magna 550 spectrometer. UV spectra were recorded on a GBC Spec-
tral UV–VIS spectrophotometer. Elemental analysis was performed
on an EAI Exeter Analytical, Inc. CE-440 apparatus. Microwave
assisted reactions were carried out in a CEM Discover reactor.
Analytical thin layer chromatography (TLC) was performed on
pre-coated silica plates (Merck F254, 0.2 mm thickness); TLC of sul-
13
+
C27H46NaO [M+Na] : 409.3441, found m/z = 409.3427. FT-IR (NaBr,
−
1
film, cm ) 3431 (ꢀ O–H), 1493 (ıas CH3), 1377 (ıs CH3).
2.4. 3˛,5-cyclo-cholestan-6-one (4)
Jones reagent (4 mL) was added dropwise to a stirred and cooled
solution of 1.83 g of 3 (4.73 mmol) in 46 mL of MEK until the mix-
ture remained reddish. Then, a solution of diluted NaHSO3 was
added until the solution remained green. The organic layer was
separated and then washed with 15% NaCl solution, dried over
anhydrous MgSO4 and evaporated to dryness. The reaction product
was purified by dry column flash chromatography on silica gel using
hexane/AcOEt (95:5) to give 1.36 g (75%) of 4 as a white crystalline
fated steroids was performed on silica gel F254 (n-BuOH/AcOH/H O
2
(
12:3:5)) and C18 reversed-phase plates (MeOH/H O (80:20 v/v))
2
and detected by spraying with sulfuric acid (10% EtOH). Flash col-
umn chromatography was performed with silica gel Merck 60 G
(
90% < 45 ␮m). Solid phase extraction tubes of silica gel (55 ␮m)
were purchased from Phenomenex. Reversed-phase chromatogra-
phy was carried out on octadecyl-functionalized silica gel (Aldrich).
All chemicals and solvents were analytical grade and solvents were
purified by general methods before being used.
The commercially available trimethylamine–sulfur trioxide
complex and cholesterol (1) were purchased from Aldrich. Choles-
◦
1
solid, m.p. 96.5–97 C (acetone–H2O). H NMR ı (CDCl3): 0.72 (s, 3H,
H-18), 0.73 (t, J = 5.1 Hz, H-4␣), 0.86 (d, J = 6.6 Hz, 6H, H-26, H-27),
0.91 (d, J = 6.6 Hz, 3H, H-21), 1.05 (s, 3H, H-19), 1.52 (m, 1H, H-3␣),
1.69 (t, H-4␤), 1.88 (m, H-7␣), 2.43 (d, J = 12.3 Hz, H-7␤). 13C NMR
ı (CDCl3): 33.60 (C-1), 26.05 (C-2), 35.45 (C-3), 11.79 (C-4), 46.47
(C-5), 209.97 (C-6), 44.96 (C-7), 34.94 (C-8), 46.22 (C-9), 46.90 (C-
10), 22.70 (C-11), 39.88 (C-12), 42.90 (C-13), 57.14 (C-14), 24.20
(C-15), 28.30 (C-16), 56.25 (C-17), 12.18 (C-18), 19.83 (C-19), 35.84
(C-20), 18.82 (C-21), 36.25 (C-22), 23.97 (C-23), 39.62 (C-24), 28.15
(C-25). 22.70 (C-26), 22.96 (C-27). Anal. calcd for C27H44O, C 84.31,
H 11.53, O 4.16. Found, C 84.51, H 11.36. HREIMS (ESI+), calculated
terol was used as starting material for the synthesis of compounds
2
1
-8. Compound 1: H NMR ı (CDCl ): 0.68 (s, 3H, H-18), 0.87
3
(
d, J = 6.9 Hz, 6H, H-26, H-27), 0.92 (d, J = 6.9 Hz, 3H, H-21), 1.01
(
s, 3H, H-19), 3.52 (m, 1H, H-3␤), 5.34 (dd, J = 1.9, 3.3 Hz, 1H, H-
ꢀ
). Acetylcholinesterase (electric eel), 5,5 -dithiobis(2-nitrobenzoic
6
acid) (DTNB), acetylthiocholine iodide (ATCI) and eserine were pur-
chased from Sigma.
+
for C27H44NaO [M+Na] : 407.32844, found m/z = 407.32837. FT-IR
−
1
2
.2. Cholesterol mesylate (2)
(NaBr, film, cm ) 1679 (ꢀ C O), 1443 (ıas CH3), 1363 (ıs CH3).
A solution of 1.16 mL (15 mmol) of methanesulfonyl chloride in
0 mL of MEK was added dropwise over 30 min to a solution of
2.5. 5˛-cholest-2-en-6-one (5)
3
cholesterol (1) (3.8683 g, 10 mmol) and triethylamine (2.8 mL) in
MEK (70 mL) at 5 C and then stirred for 30 min at room tempera-
ture. The solution was washed successively with 15% NaCl solution,
p-Toluensulfonic acid (146.7 mg) and sodium bromide
(172.6 mg) were added to a solution of 1.31 g (3.41 mmol) of
4 in 14 mL of dry dimethylformamide. The mixture was stirred
and heated under reflux for 4 h. Ethyl acetate (40 mL) was added
to the cooled reaction mixture, the organic layer was washed
successively with water (3 × 40 mL), then dried over anhydrous
MgSO4 and evaporated to dryness. The reaction product was
isolated by dry column flash chromatography on silica gel using
hexane/AcOEt (96:4) to give 1.04 g (79%) of 5 as a white crystalline
solid, m.p. 99.5–100.5 (acetone–H2O). 1H NMR ı (CDCl3): 0.66 (s,
3H, H-18), 0.70 (s, 3H, H-19), 0.86 (d, J = 6.6 Hz, 6H, H-26, H-27),
0.91 (d, J = 6.5 Hz, 3H, Me-21), 1.97 (m, H-7␣), 2.35 (m, H-7␤), 5.56
◦
saturated NaHCO solution and 15% NaCl solution, dried over anhy-
3
drous MgSO and evaporated in vacuo to give 4.51 g (97%) of 2,
4
◦
1
m.p. 120 C (acetone–H O). H NMR ı (CDCl ): 0.67 (s, 3H, H-18),
2
3
0
1
5
8
.86 (d, J = 6.6 Hz, 6H, H-26, H-27), 0.91 (d, J = 6.6 Hz, 3H, H-21),
.02 (s, 3H, H-19), 3.01 (s, 3H, CH –SO –), 4.52 (m, 1H, H-3␤),
3
3
1
3
.41 (m, 1H, H-6). C NMR (CDCl ): 138.64 (C-5), 123.82 (C-6),
3
2.06 (C-3), 38.75 (–OSO –Me), 22.80 (C-26), 22.54 (C-27), 18.69
2
(
Me-21), 19.18 (Me-19), 11.8 (Me-18). HREIMS (ESI+), calculated
+
for C28H52NO S [M+NH ] : 482.3662, found m/z = 482.3679. FT-IR
(
3
4
−1
13
NaBr, film, cm ) 3050 (ı C = C–H), 1665 (ı C C), 1465 (ıas CH ),
(m, H-2), 5.68 (m, H-3). C NMR ı (CDCl3): 39.61 (C-1), 124.68
3
1
352 (ıs CH ), 1177 (ıs O
S
O).
(C-2), 125.01 (C-3), 21.87 (C-4), 53.99 (C-5), 212.24 (C-6), 47.17
3
(
C-7), 37.87 (C-8), 53.58 (C-9), 39.65 (C-10), 21.26 (C-11), 39.51
(C-12), 42.96 (C-13), 56.91 (C-14), 24.10 (C-15), 28.17 (C-16), 56.26
C-17), 12.07 (C-18), 13.83 (C-19), 35.85 (C-20), 18.80 (C-21), 36.24
2.3. 3˛,5-cyclo-cholestan-6ˇ-ol (3)
(
A solution of 3.02 g (6.49 mmol) of the mesyl ester 2 in ace-
tone (30 mL) was added to a solution of NaHCO3 (6.15 g) in water
(C-22), 23.95 (C-23), 39.51 (C-24), 28.14 (C-25). 22.69 (C-26), 22.95
(C-27). Anal. calcd for C27H44O, C 84.31, H 11.53, O 4.16. Found, C
+
(
8
12.1 mL). It was stirred vigorously and heated under reflux at
84.37, H 11.61. HREIMS (ESI+), calculated for C27H44NaO [M+Na] :
◦
−1
5–95 C for 7 h. Then, a mixture of ethyl acetate and water (1:1)
407.32844, found m/z = 407.32861. FT-IR (NaBr, film, cm ) 3025

1
162
V. Richmond et al. / Steroids 76 (2011) 1160–1165
◦
(
ꢀ C C–H), 1710 (ꢀ C O), 1657 (ꢀ C C), 1459 (ıas CH ), 1380 (ıs
was irradiated and stirred at 150 C for 7 min in a sealed tube
3
CH ).
in a microwave reactor and then quenched with water (0.5 mL).
After evaporation to dryness the residue was eluted through
Amberlite CG-120 (sodium form) with methanol, evaporated under
reduced pressure and purified by solid phase extraction over silica
gel (55 ␮m). Fractions eluted with CH Cl /MeOH (98:2) afforded
3
2.6. 2,3˛-epoxy-5˛-cholestan-6-one (6)
To
a
solution of 5␣-cholest-2-ene-6-one (5) (246 mg,
2
2
0
.64 mmol) in CH Cl2 (8.6 mL) were added 10% Na CO3 solu-
pure disodium 2␤,3␣-dihydroxy-5␣-cholestan-6-one disulfate (8)
2
2
◦
1
tion (9.8 mL). The reaction mixture was stirred vigorously and
(19 mg, 92%), m.p. 184.5-185 C (decomp). H NMR ı (CD OD):
3
m-chloroperbenzoic acid (359 mg, 2.08 mmol) in 3.7 mL of CH Cl
was added slowly at 5 C. The mixture was stirred for 4 h at 5 C,
0.71 (s, 3H, H-18), 0.88 (d, J = 6.7 Hz, 3H, H-27), 0.89 (d, J = 6.7 Hz,
3H, H-26), 0.94 (s, 3H, H-19), 0.95 (d, J = 6.7 Hz, 3H, Me-21), 2.12
(m, H-7␣), 2.19 (dd, J = 13.3 Hz, 5.1 Hz, H-7␤), 4.73 (m, 1H, H-2␣),
2
2
◦
◦
and then the aqueous layer was extracted with CH Cl (3 × 20 mL).
2
2
The combined CH Cl extracts were washed successively with
4.77 (m, 1-H, H-3␤). 13C NMR ı (CD OD): 38.35 (C-1), 75.17 (C-
2
2
3
5
% Na SO solution, saturated NaHCO3 solution and water, dried
2), 75.11 (C-3), 22.45 (C-4), 52.65 (C-5), 214.83 (C-6), 47.32 (C-7),
38.89 (C-8), 55.63 (C-9), 41.81 (C-10), 24.88 (C-11), 40.84 (C-12),
44.16 (C-13), 57.82 (C-14), 24.93 (C-15), 29.14 (C-16), 57.42 (C-17),
12.43 (C-18), 15.24 (C-19), 37.03 (C-20), 19.14 (C-21), 37.29 (C-22),
22.25 (C-23), 40.66 (C-24), 29.14 (C-25), 23.17 (C-26), 22.92 (C-
2
3
over anhydrous MgSO4 and evaporated to dryness. The reaction
product was purified by dry column flash chromatography on
silica gel using hexane/AcOEt (95:5) to give 169.2 mg (66%) of 6
◦
1
as a white powder, m.p. 141-142 C (acetone). H NMR ı (CDCl ):
3
−
0
0
.64 (s, 3H, H-18), 0.70 (s, 3H, H-19), 0.85 (d, J = 6.6 Hz, 3H, H-26),
.86 (d, J = 6.6 Hz, 3H, H-27), 0.90 (d, J = 6.5 Hz, 3H, Me-21), 1.92
27). HREIMS (ESI+), calculated for C27H44NaO9S2 [M − 2H + Na] :
−
1
599.23299, found m/z = 599.23265. FT-IR (KBr, cm ) 1699 (ꢀ C O),
1463(ıas CH ), 1382 (ıs CH ), 1254 and 1221 (ıs S O).
(m, H-7␣), 2.31 (dd, J = 13.2 Hz, 4.1 Hz, H-7␤), 3.11 (dd, J = 5.7 Hz,
3
3
4
.1 Hz, 1H H-2␤), 3.26 (m, 1H, H-3␤). 13C NMR ı (CDCl ): 38.00
3
(
(
(
2
1
2
C-1), 50.31 (C-2), 52.53(C-3), 21.00 (C-4)*, 50.02 (C-5), 211.68
C-6), 47.07 (C-7), 37.63 (C-8), 53.25 (C-9), 38.41 (C-10), 21.03
C-11)*, 39.50 (C-12), 42.87 (C-13), 56.68 (C-14), 24.06 (C-15),
8.13 (C-16), 56.17 (C-17), 12.02 (C-18), 15.15 (C-19), 35.81 (C-20),
8.77 (C-21), 36.19 (C-22), 23.91 (C-23), 39.59 (C-24), 28.13 (C-25),
2.68 (C-26), 22.94 (C-27). (*) Signals might be interchangeable.
2.9. Acetylcholinesterase inhibition assay
Electric eel (Torpedo californica) AChE activity was measured
in vitro by a modified spectrophotometric method developed by
Ellman et al. [20]. The lyophilized enzyme (425 U/mg solid) was
prepared in buffer phosphate (8 mM K HPO , 2.3 mM NaH PO ) to
2
4
2
4
Anal. calcd for C27H44O ·1/2H O, C 79.16, H 11.07, O 9.77. Found, C
obtain 5 U/mL stock solution. Further enzyme dilution was carried
out with buffer phosphate (8 mM K HPO , 2.3 mM NaH PO , 0.15 M
2
2
+
7
8.88, H 10.71. HREIMS (ESI+), calculated for C27H NaO [M+Na] :
44 2
2
4
2
4
−
1
4
23.32335, found m/z = 423.32413. FT-IR (NaBr, film, cm ) 1704
NaCl, 0.05% Tween 20, pH 7.6) to produce 0.126 U/mL enzyme solu-
tion. Samples were dissolved in the same buffer. AChE solution
(300 ␮L) and 300 ␮L of sample solution were mixed in a test tube
and incubated for 30 min at room temperature. The reaction was
started by adding 600 ␮L of the substrate solution (0.1 M Na HPO ,
(
ꢀ C O), 1469 (ıas CH ), 1382 (ıs CH ), 1254 (ꢀs C–O–C), 802 (ꢀas
3 3
C–O–C).
2.7. 2ˇ,3˛-dihydroxy-5˛-cholestan-6-one (7)
2
4
0
.5 mM (DTNB, 0.6 mM ATCI, pH 7.5). The absorbance was read at
◦
A solution of epoxide 6 (85.7 g, 0.21 mmol) in THF (4 mL)
405 nm for 180 s at 27 C. Enzyme activity was calculated by com-
paring reaction rates for the sample to the blank. All the reactions
were performed in triplicate. IC50 values were determined with
probit analysis (EPA Probit 1.4). Eserine (99%) was used as the ref-
erence AChE inhibitor.
was treated with 1 N H SO4 (0.20 mL) and stirred for 24 h at
2
room temperature. After neutralization with saturated NaHCO3
solution the mixture was evaporated to fifth initial volume,
diluted with water (10 mL), and extracted with ethyl acetate
(
3 × 10 mL). The combined organic extracts were washed with
water, dried over anhydrous MgSO , filtered and evaporated to
2.10. Graphic determination of inhibitor type
4
dryness. The crude diol 7 was submitted to column chromatog-
raphy on silica gel (0.063–0.200 mm), eluted with CH Cl /MeOH
The enzyme reaction was carried out at three fixed inhibitor
(compound 8) concentrations (0, 52 and 104 ␮M). In each case the
initial velocity measurements were obtained at varying substrate
(S) (acetylthiocholine) concentrations and the reciprocal of the ini-
tial velocity (1/v) was plotted as a function of the reciprocal of [S].
The double-reciprocal (Lineweaver–Burk) plot showed a pattern of
parallels lines with the same slopes, characteristic of an uncom-
petitive inhibitor. The data of the enzyme activity at different fixed
substrate concentrations with increasing inhibitor concentrations
were analyzed with GraphPad Prism 5. The nonlinear regression of
2
2
(
95:5 v/v) and evaporated under reduced pressure to afford pure
2
1
1
␤,3␣-dihydroxy-5␣-cholestan-6-one (7) (66.82 mg, 76%), m.p.
◦
1
81-182 C (acetone–H O). H NMR ı (CD OD): 0.71 (s, 3H, H-
2
3
8), 0.88 (d, J = 6.6 Hz, 6H, H-26, H-27), 0.94 (s, 3H, H-19), 0.95 (d,
J = 6.6 Hz, 3H, Me-21), 2.09 (m, H-7␣), 2.20 (dd, J = 13.1 Hz, 4.8 Hz,
H-7␤), 3.81 (m, 1H, H-2␣), 3.85 (m, 1-H, H-3␤). 13C NMR ı (CD OD):
3
4
(
1
0.07 (C-1), 70.94 (C-2), 70.09 (C-3), 24.00 (C-4), 52.77 (C-5), 215.73
C-6), 47.47 (C-7), 38.82 (C-8), 55.78 (C-9), 42.16 (C-10), 24.93 (C-
1), 40.88 (C-12), 44.17 (C-13), 57.87 (C-14), 24.93 (C-15), 29.15
2
(
(
(
C-16), 57.46 (C-17), 12.46 (C-18), 15.56 (C-19), 37.04 (C-20), 19.18
C-21), 37.30 (C-22), 22.27 (C-23), 40.67 (C-24), 29.15 (C-25), 23.20
C-26), 22.95 (C-27). Anal. calcd for C27H46O ·1/2H O, C 75.83,
these data fitted with uncompetitive inhibition with a R = 0.9703.
The calculated ˛Ki was 121.1 ␮M.
3
2
H 11.07, O 13.10. Found, C 75.72, H 11.07. HREIMS (ESI+), cal-
culated for C27H47O3 [M+H ]: 419.35197, found m/z = 419.35174.
FT-IR (NaBr, film, cm ) 3431 (ꢀ O–H), 1690 (ꢀ C O), 1450 (ıas
2.11. Molecular docking determinations
+
−1
Structure of Protein Data Bank (PDB) entry 2ACE [21], T. cali-
fornica AChE crystal structure, complexed with acetylcholine, was
used for the docking simulations of disodium 2␤,3␣-dihydroxy-
5␣-cholestan-6-one disulfate (8). Geometry optimization was
performed with the Hartree-Fock [22] method and the 6–31 + G (d)
basis set and semiempirical calculations (AM1) [23] incorporated
in the Gaussian 03 program [24]. The charges of the ligands were
obtained using the standard RESP procedure.
CH ).
3
2.8. Disodium 2ˇ,3˛-dihydroxy-5˛-cholestan-6-one disulfate (8)
Trimethylamine–sulfur trioxide complex (36 mg, 0.26 mmol)
was added to a solution of 2␤,3␣-dihydroxy-5␣-cholestan-6-one
7) (14 mg, 0.033 mmol) in DMF (0.45 mL). The reaction mixture
(

V. Richmond et al. / Steroids 76 (2011) 1160–1165
1163
Docking studies were performed with version 4.0 of the program
AutoDock [25], using the implemented empirical free energy func-
tion. The graphical user interface program AutoDock Tools was used
to prepare, run and analyze the docking simulations. The simula-
tion space was defined as a 21.28 A˚ × 31.24 A˚ × 20.37 A˚ box which
included the active site and the peripheral site. Atomic interac-
tion energy on a 0.375 A˚ grid was calculated with the auxiliary
program Autogrid 4 using probes corresponding to each map type
found in the inhibitor. All rotatable dihedrals in the steroids were
allowed to rotate freely but those of the side chain. The starting
positions of the steroids were outside the grid on a random posi-
tion.
Fig. 1. Chemical structure of diols (7, 9) and sulfated steroids (8, 10) tested against
AChE.
The steroids were docked by the Lamarckian genetic algorithm
protocol. A total of 256 independent simulations with a population
size of 150 members were run for the steroid using Autodock 4.0
with default parameters (random starting position and conforma-
tion, translation step of 2.0 A˚ , mutation rate of 0.02, crossover rate of
Table 1
Summary of the in vitro antiacetylcholinesterase activities.
Compound
7
8
9
10
IC50 (␮M)
>500
14.59 ± 0.88
>500
59.65 ± 2.30
0.8, local search rate 0.06 and 2,500,000 energy evaluations). After
docking, the 256 conformers generated for each compound were
assigned to clusters based on a tolerance of 2.0 A˚ all atom root-
mean-square deviation (rmsd) in position from the lowest-energy
solution. The clusters were also ranked according to the energies of
their representative conformations, which were the lowest-energy
solutions within each cluster.
The acetylcholinesterase inhibitory activity of compounds 7 and
8 was evaluated and compared to that of analogs 9 and 10 (Fig. 1)
previously synthesized by our group [14]. From the data shown
in Table 1, disulfated compounds 8 and 10 were the most active
with IC50 values of 14.59 and 59.65 ␮M, respectively. Diols 7 and
9
showed no inhibitory activity (IC50 > 500 ␮M). These results are
indicative of the role of the sulfate groups in the AChE inhibitory
activity of 8 and 10 and suggest that the presence of a carbonyl
group at C-6 in 8 enhances its activity.
3
. Results and discussion
The first reported synthesis of disulfated steroid 8 (Scheme 1)
Disodium 2␤,3␣-dihydroxy-5␣-cholestan-6-one disulfate (8),
the most active compound in the tested set, was chosen for the
determination of the inhibitor type kinetic study. Enzyme activ-
ity was evaluated at different fixed substrate concentrations and
increasing inhibitor concentrations and the data obtained were
used to elucidate the enzyme inhibition mechanism. The results are
illustrated in the form of Lineweaver–Burk plots (Graphic 1). The
double-reciprocal plots show that with increasing concentration
of 8 the values of both Km and Vmax are enhanced, but the ratio of
Km/Vmax is still unchanged. The slopes are independent of the con-
centration of the inhibitor, which indicate that this compound is an
uncompetitive inhibitor of the enzyme. Compound 8 does not bind
to the free enzyme but binds reversibly to the enzyme–substrate
complex, yielding an inactive complex. This inhibition is an exam-
ple of the sequential order of binding of two ligands to the enzyme
in an obligate order. The substrate binding is considered neces-
sary to produce conformational changes in the enzyme, which
creates or opens the inhibitor binding site [28]. Therefore, molecu-
lar docking studies were performed with the AChE complexed with
acetylcholine. Table 2 summarizes the docking results of 2␤,3␣-
starts from commercially available cholesterol (1). This reacted
with methanesulfonyl chloride to give cholesterol mesylate (2).
Following the procedure of Mori et al. [26] a rearrangement and
subsequent oxidation with Jones reagent converted the mesylate
steroid 2 to the 3␣,5-cyclo-6-one steroid 4. The 1H NMR spectrum
of 4 showed two signals at ı 0.73 (H-4␣) and 1.69 (H-4␤) ppm
ascribed to the methylene protons of the cyclopropane ring. This is
in accordance with the chemical shift observed at ı 11.79 ppm in
13
the C NMR spectrum of 4 and assigned to C-4 on the basis of HSQC
and HMBC spectra. Rearrangement of compound 4 generated the
2
ꢁ
double bond in 5. Multiplets at ı 5.56 (H-2) and 5.68 (H-3) ppm
1
in the H NMR spectrum together with the corresponding signals at
ı 124.68 (C-2) and 125.68 (C-3) ppm as well as the carbonyl signal at
12.24 (C-6) ppm confirmed the structure of compound 6. Further
epoxidation and acid aperture of epoxide 6 [14] rendered diol 7. The
H NMR spectrum of 7 showed two multiplets at ı 3.81 (H-2␤) and
2
1
3
.85 (H-3␣) ppm, geminal to hydroxyl groups at C-2 (70.94) and
13
C-3 (70.09), as determined by analysis of C NMR, HSQC, HMBC
and NOESY spectra.
Compound 7 was purified by column chromatography on silica
gel and used as starting material for the synthesis of compound 8.
Trimethylamine–sulfur trioxide complex was selected as the sul-
fating reagent instead of the triethyl complex used in our previous
work [14] owing to the better yields obtained. A microwave based
protocol was chosen to enhance the rate of sulfation, dramatically
decreasing the reaction time from 13 h to 7 min [27]. Treatment of
diol 7 with 8 equiv. of trimethylamine–sulfur trioxide complex for
◦
7
min at 150 C at a microwave reactor afforded the ammonium
sulfate of compound 8, which was transformed via ion exchange
into the disodium salt 8 (Scheme 1). Disodium 2␤,3␣-dihydroxy-
5
␣-cholestan-6-one (8) showed two methine signals at ı 4.73 (H-2)
and 4.77 (H-3), characteristic of the presence of two sulfate groups
at C-2 and C-3 [14]. This is in accordance with the chemical shifts
observed for C-2 (72.0 ppm) and C-3 (71.8 ppm) in the 13C NMR
Graphic 1. Lineweaver–Burk plots of the inhibition of AChE by 2b,3a-dihydroxy-
5a-cholestane-6-one disulfate (8) with acetylthiocholine (S) as
a substrate.
spectrum, as determined from the HSQC spectrum. Both methine
signals are broad singlets according to their equatorial position in
the ring supporting a 2␤,3␣ configuration, as determined by NOESY.
Concentrations of compound 8: (circle): 0 ␮M; (square): 52 ␮M; (triangle): 104 ␮M.
2
Linear regression equations: y = 0.3004x + 0.0014 (R = 0.9953); y = 0.3066x + 0.0023
2
2
(R = 0.9959); y = 0.3142x + 0.0031 (R = 0.9963) for 0, 52 and 104 ␮M respectively.

1
164
V. Richmond et al. / Steroids 76 (2011) 1160–1165
Scheme 1. Conditions: (a) MsCl, TEA, MEK, (b) NaHCO3, H2O, MEK, (c) CrO3, H2SO4, MEK, (d) LiBr, p-TsOH, DMF (e) m-ClPBA, Na2CO3, H2O–Cl2CH2, (f) H2SO4, THF and (g)
equiv. Et3N.SO3, DMF, Amberlite CG-120 (MeOH).
8
dihydroxy-5␣-cholestan-6-one disulfate (8). 256 docking runs with
generated six clusters. All of them showed the steroid buried into
The docking studies allowed us to establish the binding mode
of steroid 8 to the enzyme–substrate complex and identify
hydrophobic interactions inside the aromatic gorge as well as
hydrogen bonding interactions as the stabilizing factors in the
enzyme–substrate–inhibitor complex. Further molecular dynam-
ics studies of this complex as starting point are necessary to check
the complex inhibitor–enzyme stability and to determinate if the
enzyme undergoes structural rearrangements.
8
the aromatic gorge and five of the six clusters showed that the
steroid penetrates the peripheral site through the side chain, which
might be due to its aliphatic character in comparison with that of
the disulfated six-membered ring A (Fig. 2). The main hydrophobic
interactions between the hydrocarbon skeleton of the inhibitor and
the protein were observed with the residues: TYR 70, ASP 72, TRP
8
4, TYR 124, TYR 121, TRP 279, PHE 330 and TYR 334 (Fig. 3). These
Taking into account the docking results and the AChE inhibitory
activity of steroid 8, this disulfated compound is a good model
for a rationale design of analogs by introducing additional func-
tional groups at the steroidal skeleton or its side chain in order to
evaluate structure–activity correlations in the acetylcholinesterase
inhibitory activity.
results are consistent with molecular modeling studies of natural
cholinesterase-inhibiting steroidal alkaloids isolated from Sarco-
cocca saligna [28]. As for steroid 8, the steroidal alkaloids penetrate
the aromatic gorge of the enzyme through the most hydrophobic
side of the molecule [29].
The docking studies also showed that the affinity of steroid 8 for
the complex enzyme–substrate is favoured by hydrogen bonding
interactions, which involve both sulfates at ring A. Sulfate groups
at C-2 and C-3 come close to TYR 70 and TRP 279, respectively.
Hydrogen bonding between the sulfate group at C-3 of the inhibitor
and the amidic hydrogen of TRP 279, and between the sulfate group
at C-2 and the hydroxyl of TYR 70 may occur as the distances and
angles observed are within a suitable range.
Table 2
Summary of the docking results of 2(,3(-dihydroxy-5(-cholestan-6-one disulfate (8).
Compound
Total number
of clusters
Docking statistics
Cluster rank
Number of runs
in the cluster
8
6
1
2
3
4
5
6
33
140
15
1
65
2
Fig. 2. Docking result for disodium 2␤,3␣-dihydroxy-5␣-cholestan-6-one disulfate
(
8).

V. Richmond et al. / Steroids 76 (2011) 1160–1165
1165
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Acknowledgments
We thank the University of Buenos Aires for financial support
Grant X124) of this work. V.R. thanks the National Research Council
of Argentina (CONICET) for a Doctoral fellowship. A.P.M. and M.S.M.
are Research Members of CONICET.
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