Synthesis and acetylcholinesterase inhibitory activity of polyhydroxylated sulfated steroids: Structure/activity studies

Article abstract of DOI:10.1016/j.steroids.2013.08.003

Disulfated and trisulfated steroids have been synthesized from cholesterol and their acetylcholinesterase inhibitory activity has been evaluated. In our studies we have found that the activity was not only dependent on the location of the sulfate groups but on their configurations. 2β,3α,6α- trihydroxy-5α-cholestan-6-one trisulfate (18) was the most active steroid with an IC₅₀ value of 15.48 μM comparable to that of 2β,3α-dihydroxy-5α-cholestan-6-one disulfate (1). Both compounds were found to be less active than the reference compound eserine. The butyrylcholinesterase activity of 1 and 18 was one magnitude lower than that against acetylcholinesterase revealing a selective inhibitor profile.

Full text of DOI:10.1016/j.steroids.2013.08.003

Steroids 78 (2013) 1141–1147
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and acetylcholinesterase inhibitory activity of
polyhydroxylated sulfated steroids: Structure/activity studies
Victoria Richmond a, Ana P. Murray b, Marta S. Maier a,
⇑
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
b INQUISUR, Departamento de Química, Universidad Nacional del Sur, Avda. Alem 1253, 8000 Bahía Blanca, Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 June 2013
Received in revised form 25 July 2013
Accepted 6 August 2013
Available online 20 August 2013
Disulfated and trisulfated steroids have been synthesized from cholesterol and their acetylcholinesterase
inhibitory activity has been evaluated. In our studies we have found that the activity was not only
dependent on the location of the sulfate groups but on their configurations. 2b,3
cholestan-6-one trisulfate (18) was the most active steroid with an IC50 value of 15.48
to that of 2b,3 -dihydroxy-5 -cholestan-6-one disulfate (1). Both compounds were found to be less
a
,6a
-trihydroxy-5
a-
l
M comparable
a
a
active than the reference compound eserine. The butyrylcholinesterase activity of 1 and 18 was one mag-
nitude lower than that against acetylcholinesterase revealing a selective inhibitor profile.
Ó 2013 Elsevier Inc. All rights reserved.
Keywords:
Sulfated steroids
Synthesis
Acetylcholinesterase activity
1. Introduction
in order to compensate the deficiency of the cholinergic neuro-
transmitters would lead to the improved cognitive activities of
Alzheimer’s disease (AD) is an age-related neurodegenerative
process characterized by a progressive memory loss, decline in lan-
guage skills and other cognitive impairments [1]. According to the
cholinergic hypothesis, the selective and irreversible deficiency of
cholinergic functions leads to memory impairment in AD [2]. Ace-
tylcholine serves as a neurotransmitter in the central and periphe-
ral nervous system. Acetylcholinesterase (AChE), located either in
peripheral (muscles) and/or central nervous (cholinergic) system,
stops the function of acetylcholine by its hydrolytic destruction
in the cholinergic synapses [3]. Another cholinesterase, namely
butyrylcholinesterase (BChE), is involved in the metabolic degrada-
tion of acetylcholine and differs from AChE in tissue distribution
and sensitivity to substrates and inhibitors. The relative propor-
tions of AChE to BChE depend on the brain regions and the stage
of disease progression in AD patients [4]. BChE activity increases
as AD progresses, which suggests that BChE may also play an
important role in cholinergic dysfunction, particularly at the latter
stages of AD. For this reason, the administration of inhibitors with
different AChE/BChE selectivity should be very useful as AD
progresses [5].
the patient and related conditions such as Lewy Body dementia,
subcortical vascular dementia and Parkinson’s disease [7,8].
The enhancement of the inhibitory activity against AChE by
sulfation of calenduladiol, a pentacyclic triterpene isolated from
Chuquiraga erinacea, (IC50 from 94 to 0.190 mM) [9] prompted us
to test the acetylcholinesterase inhibitory activity of two sulfated
compounds, disodium 2b,3a-dihydroxy-5a-cholestan-6-one disulfate
(1) and disodium 2b,3 -dihydroxy-5
a
a-cholestane disulfate (2),
and compare it to their corresponding diols (3 and 4) (Fig. 1)
[10,11]. The results were indicative of the role of the sulfate groups
in the AChE inhibitory activity of 1 and 2 and showed that the pres-
ence of a carbonyl group at C-6 in 1 increases the activity [11]. In
this report, we synthesized disulfated and trisulfated steroids with
sulfate groups at C-2, C-3 and C-6 (a/b configurations) on ring A
and B and determined their acetylcholinesterase inhibitory activity.
Interestingly, we found that the activity of the sulfated steroids is
not only dependent on the location of the sulfate groups but on their
configurations. Our results provide new evidence for the relationship
between chemical structure and acetylcholinesterase inhibitory
activity.
Enhancement of acetylcholine level in the brain is considered
one of the most promising approaches for treating AD [6]. Retain-
ing the neurotransmitters especially at the synaptic terminals via
the inhibition of the hydrolytic enzymes, i.e., the cholinesterases,
2. Experimental
2.1. General methods
Melting points (m.p.) were determined on a Fisher Johns
apparatus and are uncorrected. 1H NMR, 13C NMR, HSQC-DEPT,
HMBC, COSY and NOESY spectra were recorded on a Bruker AM
⇑
Corresponding author. Tel./fax: +54 11 4576 3385.
E-mail address: maier@qo.fcen.uba.ar (M.S. Maier).
0039-128X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.08.003

1142
V. Richmond et al. / Steroids 78 (2013) 1141–1147
500 spectrometer. Chemical shifts (d) 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
determined 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 and on a Perkin Elmer Spectrum 400 with
an ATR accessory. UV spectra were recorded on a JASCO V-630BIO
spectrophotometer. Elemental analysis was performed on an EAI
Exeter Analytical, Inc. CE-440 apparatus. Microwave assisted reac-
tions 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 sulfated steroids
was performed on silica gel F254 (n-BuOH/AcOH/H2O (12:3:5))
and detected by spraying with sulfuric acid (10% H2O). Flash col-
umn chromatography was performed with silica gel Merck 60 G
(35.4 mg, 65%), m.p. 203–204 °C (acetone–H2O) and 2
a,3a-dihy-
droxy-5
a
-cholestan-6-one (8) (19 mg, 35%). 1H NMR d (CDCl3):
0.66 (s, 3H, H-18), 0.86 (d, J = 6.6 Hz, 6H, H-26, H-27), 0.91 (d,
J = 6.6 Hz, 3H, Me-21), 0.98 (s, 3H, H-19), 3.63 (dt, J = 11.7 Hz,
4.2 Hz, H-3a), 4.03 (m, 1H, H-2a
). 13C NMR d (CDCl3): 42.6 (C-1),
69.3 (C-2), 72.0 (C-3), 24.4 (C-4), 57.3 (C-5), 210.8 (C-6), 46.6 (C-
7), 37.4 (C-8), 54.9 (C-9), 40.7 (C-10), 21.7 (C-11), 39.7 (C-12),
43.2 (C-13), 56.8 (C-14), 24.1 (C-15), 28.2 (C-16), 56.3 (C-17),
12.2 (C-18), 15.4 (C-19), 35.8 (C-20), 18.8 (C-21), 36.2 (C-22),
24.0 (C-23), 39.6 (C-24), 28.2 (C-25), 22.7 (C-26, C-27). Anal. calcd
for C27H46O3.½H2O, C 75.82, H 11.08, O 13.10. Found, C 75.46, H
11.29. HREIMS (ESI+), calculated for
441.3339, found m/z = 441.3322. FT-IR (NaBr, film, cmꢁ1) 3525,
3464 ( O–H), 1696 ( C@O), 1454 (das CH3), 1363 (ds CH3).
C
27H46NaO3 [M + Na+]:
m
m
(90% < 45
lm). Solid phase extraction tubes of silica gel (55
lm)
2.3. Disodium 2b,3b-dihydroxy-5a-cholestan-6-one disulfate (7)
were purchased from Phenomenex. Preparative HPLC was carried
out on an SP liquid chromatograph equipped with a Spectra Series
P100 solvent delivery system, a Rheodyne manual injector and a
Trimethylamine-sulfur trioxide complex (25.7 mg, 0.19 mmol)
was added to a solution of 2b,3b-dihydroxy-5 -cholestan-6-one (6)
a
refractive index detector using a Phenomenex AQUA 5
l
C18
(10 mg, 0.024 mmol)inDMF (1 ml). Thereactionmixturewas irradi-
ated and stirred at 150 °C for 7 min in a sealed tube in a microwave
reactor and then quenched with water (1 ml). After evaporation to
dryness the residue was eluted through Amberlite CG-120 (sodium
form) with methanol, evaporated under reduced pressure and puri-
125A column. All chemicals and solvents were analytical grade
and solvents were purified by general methods before being used.
The commercially available trimethylamine-sulfur trioxide com-
plex was purchased from Aldrich. 2b,3
one (3) was used as starting material for the synthesis of compounds
10–14 and 5 -cholest-2-en-6-one (5), to obtain the rest of the
a-Dihydroxy-5a-cholestan6-
fiedbysolidphaseextractionoversilicagel(55
lm). Fractionseluted
a
with CH2Cl2/MeOH (85:15) afforded pure disodium 2b,3b-dihy-
compounds (i.e. 6–9 and 15–18). Compounds 3 and5 were obtained
following the procedure described in our previous work [11]. Acetyl-
cholinesterase (electric eel), 5,50-dithiobis(2-nitrobenzoic acid)
(DTNB), acetylthiocholine iodide (ATCI), butyrylthiocholine iodide
(BTCI) and eserine were purchased from Sigma. Butyrylcholinester-
ase (horse serum) was purchased from MP Biomedicals.
droxy-5a-cholestan-6-one disulfate (7) (11.8 mg, 79.4%), m.p. 210–
215 °C (decomp). 1H NMR d (CDOD3): 0.71 (s, 3H, H-18), 0.88 (d,
J = 6.6 Hz, 6H, H-26, H-27,), 0.95 (d, J = 6.4 Hz, 3H, Me-21), 1.00 (s,
3H, H-19), 4.31 (dt, J = 12.1 Hz, 3.7 Hz 1-H, H-3
a), 4.84 (m, 1H, H-
2a
). 13C NMR d (CDOD3): 41.7 (C-1), 76.5 (C-2), 78.3 (C-3), 23.6 (C-
4), 58.3 (C-5), 213.2 (C-6), 47.1 (C-7), 38.8 (C-8), 55.5 (C-9), 42.0 (C-
10), 25.0 (C-11), 40.9 (C-12), 44.2 (C-13), 57.7 (C-14), 24.9 (C-15),
29.2 (C-16), 57.5 (C-17), 12.4 (C-18), 15.6 (C-19), 37.1 (C-20), 19.1
(C-21), 37.3 (C-22), 22.7 (C-23), 40.7 (C-24), 29.2 (C-25), 22.9 (C-
26), 23.2 (C-27). HREIMS (ESIꢁ), calculated for C27H44NaO9S2 [M–
Na]ꢁ: 599.2330, found m/z = 599.2355. ATR FT-IR (ZnSe, cmꢁ1) 1699
2.2. 2b,3b-Dihydroxy-5a-cholestan-6-one (6)
Asymmetric dihydroxylation reactions were performed on
5a-cholest-2-en-6-one (5) using the experimental conditions re-
(m
C@O), 1463 (das CH3), 1376 (ds CH3), 1220 (ds S@O).
ported by Sharpless [12]. A solution of 5 (50 mg, 0.13 mmol) in
tert-butyl alcohol (2 ml) was added to a solution of AD-mix-
a
2.4. 2 ,3 -Dihydroxy-5 -cholestan-6-one (8)
a
a
a
(181.7 mg) in water (2 ml). The mixture was stirred for few min-
utes at room temperature until two clear phases were observed.
Methanesulphonamide (12.5 mg, 0.13 mmol) was added and the
mixture was stirred at room temperature for two weeks. Then, a
saturated sodium bisulphite solution (1.7 ml) was added to the
cold reaction mixture and the suspension was stirred for 30 min
at room temperature. After extraction with EtOAc (3 ꢀ 5 ml), the
combined organic extracts were washed successively with 2 N
KOH solution (3 ꢀ 5 ml) and water, dried over anhydrous MgSO4
and evaporated to dryness. The reaction product was submitted
to column chromatography on silica gel (0.063–0.200 mm), eluted
with cyclohexane/EtOAc (60:40 v/v) and evaporated under reduced
Asymmetric dihydroxylation reactions were performed on
cholest-2-en-6-one (5) using the experimental conditions reported
by Sharpless [12]. A solution of 5 (200 mg, 0.52 mmol) in tert-butyl
alcohol (5.8 ml) was added to a solution of AD-mix-b (732 mg) in
water (5.8 ml). The mixture was stirred for few minutes at room
temperature until two clear phases were observed. Methanesulph-
onamide (49.3 mg, 0.51 mmol) was added and the mixture was
stirred at room temperature for two weeks. Then, a saturated
sodium bisulphite solution (6.6 ml) was added to the cold reaction
mixture and the suspension was stirred for 30 min at room
temperature. After extraction with EtOAc (3 ꢀ 15 ml), the
combined organic extracts were washed successively with 2 N
KOH solution (3 ꢀ 15 ml) and water, dried over anhydrous MgSO4
and evaporated to dryness. The reaction product was submitted to
column chromatography on silica gel (0.063–0.200 mm), eluted
with cyclohexane/EtOAc (60:40 v/v) and evaporated under
pressure to afford pure 2b,3b-dihydroxy-5a-cholestan-6-one (6)
reduced pressure to afford pure 2
6-one (8) (123.4 mg, 65%), m.p. 199–200 °C (acetone–H2O) and
2b,3b-dihydroxy-5 -cholestan-6-one (6) (67.3 mg, 35%), together
a,3a-dihydroxy-5a-cholestan-
a
with 12.4 mg of 5. 1H NMR d (CDCl3): 0.66 (s, 3H, H-18), 0.74 (s,
3H, H-19), 0.85 (d, J = 6.7 Hz, 3H, H-26), 0.86 (d, J = 6.8 Hz, 3H, H-
27), 0.91 (d, J = 6.5 Hz, 3H, Me-21), 3.75 (ddd, J = 11.8 Hz, 4.7 Hz,
3.2 Hz, H-2b), 4.03 (m, 1H, H-3b).13C NMR d (CD3OD): 40.3 (C-1),
68.4 (C-2), 68.5 (C-3), 26.4 (C-4), 50.9 (C-5), 212.5 (C-6), 46.9 (C-7),
37.8 (C-8), 53.9 (C-9), 42.7 (C-10), 21.3 (C-11), 39.5 (C-12), 43.1
Fig. 1. Chemical structures of acetylcholinesterase inhibitors (1, 2) and inactive
diols (3, 4).

V. Richmond et al. / Steroids 78 (2013) 1141–1147
1143
(C-13), 56.8 (C-14), 24.1 (C-15), 28.2 (C-16), 56.2 (C-17), 12.2 (C-
18), 13.7 (C-19), 35.8 (C-20), 18.8 (C-21), 36.2 (C-22), 24.0 (C-23),
39.6 (C-24), 28.1 (C-25), 22.7 (C-26), 23.0 (C-27). Anal. calcd for
(10) (7.8 mg, 0.019 mmol) in DMF (0.4 ml). The reaction mixture
was irradiated and stirred at 150 °C for 7 min in a sealed tube in
a microwave reactor and then quenched with water (1 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
C
27H46O3.½H2O, C 75.83, H 11.07, O 13.10. Found, C 75.86, H
11.14. HREIMS (ESI+), calculated for
27H46NaO3 [M + Na+]:
441.3339, found m/z = 441.3323. FT-IR (NaBr, film, cmꢁ1) 3348,
3523 ( O–H), 1710 ( C@O), 1463 (das CH3), 1380 (ds CH3).
C
m
m
(55
lm). Fractions eluted with CH2Cl2/MeOH (85:15) afforded pure
disodium 2b,3
a
-dihydroxycholest-5-ene disulfate (11) (4.4 mg,
2.5. Disodium 2
a,3
a-dihydroxy-5a-cholestan-6-one disulfate (9)
38%), m.p. >250 °C (MeOH). 1H NMR d (DMSO-d6): 0.63 (s, 3H, H-
18), 0.83 (d, J = 6.6 Hz, 6H, H-26, H-27), 0.89 (d, J = 6.3 Hz, 3H,
Trimethylamine-sulfur trioxide complex (25.7 mg, 0.19 mmol)
was added to a solution of 2 ,3 -dihydroxy-5 -cholestan-6-one
Me-21), 1.05 (s, 3H, H-19), 4.32 (m, 1H, H-2a), 4.40 (m, 1H, H-
a
a
a
3b), 5.17 (m, 1H, H-6). 13C NMR d (DMSO-d6): 32.4 (C-1), 72.7 (C-
2), 72.2 (C-3), 37.1 (C-4), 139.4 (C-5), 120.6 (C-6), 31.2 (C-7), 30.7
(C-8), 49.8 (C-9), 36.2 (C-10), 20.2 (C-11), 39.2 (C-12), 42.3 (C-
13), 56.1 (C-14), 23.7 (C-15), 27.6 (C-16), 55.3 (C-17), 11.15 (C-
18), 18.3 (C-19), 35.0 (C-20), 18.5 (C-21), 35.5 (C-22), 23.0 (C-23),
38.7 (C-24), 27.2 (C-25), 22.1 (C-26), 22.6 (C-27). HREIMS (ESIꢁ),
(8) (10 mg, 0.024 mm ol) in DMF (1 ml). The reaction mixture
was irradiated and stirred at 150 °C for 7 min in a sealed tube in
a microwave reactor and then quenched with water (1 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
calculated for
C
27H44NaO8S2 [M–Na]ꢁ: 583.2381, found m/
(55
disodium
l
m). Fractions eluted with CH2Cl2/MeOH (85:15) afforded pure
,3 -dihydroxy-5 -cholestan-6-one disulfate (9)
z = 599.2361.
2a
a
a
(11.6 mg, 77.6%), m.p. 195–208 °C (decomp). 1H NMR d (CDOD3):
0.69 (s, 3H, H-18), 0.82 (s, 3H, H-19), 0.86 (d, J = 6.6 Hz, 3H, H-
26), 0.87 (d, J = 6.6 Hz, 3H, H-27), 0.93 (d, J = 6.5 Hz, 3H, Me-21),
4.44 (ddd, J = 12.3, 4.7, 3 Hz, 1H, H-2b), 4.86 (m, 1-H, H-3b). 13C
NMR d (CDOD3): 39.6 (C-1), 75.4 (C-2), 76.4 (C-3), 25.9 (C-4),
52.3 (C-5), 214.3 (C-6), 47.4 (C-7), 39.2 (C-8), 55.0 (C-9), 43.7 (C-
10), 24.9 (C-11), 40.8 (C-12), 44.2 (C-13), 57.8 (C-14), 25.0 (C-15),
29.2 (C-16), 57.4 (C-17), 12.4 (C-18), 14.0 (C-19), 37.0 (C-20),
19.1 (C-21), 37.3 (C-22), 22.4 (C-23), 40.7 (C-24), 29.2 (C-25),
22.9 (C-26), 23.2 (C-27). HREIMS (ESIꢁ), calculated for C27H44NaO9-
S2 [M–Na]ꢁ: 599.2330, found m/z = 599.2349. ATR FT-IR (ZnSe,
2.8. Disodium 2b,3a,6b-trihydroxy-5a-cholestan-2b,3a-disulfate (12)
Two portions of 7 mg of sodium borohydride were added to a
solution of 8.6 mg (0.014 mmol) of disodium 2b,3 -dihydroxy-
-cholestan-6-one disulfate (1) in 0.40 ml of methanol at room
a
5a
temperature for 4 h. After acidification with 0.5 M HCl solution,
methanol was added and the mixture was evaporated to eliminate
the B(OMe)3. The reaction product was purified by solid phase
extraction over silica gel (55
methane/methanol (85:15) afforded pure disodium 2b,3
l
m). Fractions eluted with dichloro-
,6b-tri-
hydroxy-5a-cholestan-2b,3a-disulfate (12) (8.4 mg, 97%), m.p.
a
cmꢁ1) 1702 (
m
C@O), 1463(das CH3), 1379 (ds CH3), 1218 (ds S@O).
>250 °C (decomp) (acetone–H2O). 1H NMR d (DMSO-d6): 0.64 (s,
3H, H-18), 0.83 (d, J = 6.6 Hz, 3H, H-26), 0.84 (d, J = 6.6 Hz, 3H, H-
27), 0.89 (d, J = 6.6 Hz, 3H, H-21), 1.03 (s, 3H, H-19), 3.53 (m, 1H,
2.6. 2b,3 ,6b-Trihydroxy-5
a
a
-cholestane (10)
H-6a), 4.30 (m, 1H, H-2a
), 4.37 (m, 1-H, H-3b). 13C NMR (DMSO-
Three portions of 0.5 ml of a solution of 4.5 mg/ml of sodium
borohydride in methanol were added to a solution of 19.5 mg
(0.046 mmol) of 2b,3a-dihydroxy-5a-cholestan-6-one (3) in
1.25 ml of methanol and the mixture was stirred at room temper-
ature overnight. After acidification with 0.5 M HCl solution, meth-
anol was added and the mixture was evaporated to eliminate the
B(OMe)3. Then, water was added and the steroid was extracted
with EtOAc (3 ꢀ 10 ml). The combined organic extracts were
washed with water, dried over anhydrous MgSO4, filtered and
evaporated to dryness. The crude triol 10 was purified by solid
d6): 39.0 (C-1), 73.2 (C-2), 73.4 (C-3), 27.0 (C-4), 41.5 (C-5), 69.7
(C-6), 39.9 (C-7), 29.7 (C-8), 54.8 (C-9), 35.1 (C-10), 20.3 (C-11),
39.6 (C-12), 42.2 (C-13), 55.9 (C-14), 23.9 (C-15), 27.8 (C-16),
55.7 (C-17), 11.9 (C-18), 17.2 (C-19), 35.2 (C-20), 18.6 (C-21),
35.7 (C-22), 23.2 (C-23), 38.8 (C-24), 27.4 (C-25), 22.7 (C-26),
22.4 (C-27). HREIMS (ESIꢁ), calculated for C27H46NaO9S2 [M–
Na]ꢁ: 601.2486, found m/z = 601.2486. ATR FT-IR (ZnSe, cmꢁ1
)
3317 (
m
O–H), 1462 (das CH3), 1373 (ds CH3), 1212 (ds S@O).
2.9. Trisodium 2b,3
a
,6b-trihydroxy-5 -cholestane trisulfate (13)
a
phase extraction over silica gel (55
cyclohexane/EtOAc (70:30) afforded pure 2b,3
l
m). Fractions eluted with
,6b-trihydroxy-
a
Trimethylamine-sulfur trioxide complex (13.5 mg, 0.097 mmol)
was added to a solution of disodium 2b,3 ,6b-trihydroxy-5
cholestan-2b,3 -disulfate (12) (12.5 mg, 0.020 mmol) in DMF
(0.25 ml). The reaction mixture was irradiated and stirred at
60 °C for an hour in a sealed tube 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. Preparative HPLC
(methanol/water (75:25 v/v)) of the crude product afforded pure
5
a
-cholestane (10) (16.8 mg, 87%), m.p. >250 °C (acetone–H2O).
a
a-
1H NMR d (CD3OD): 0.72 (s, 3H, H-18), 0.88 (d, J = 6.6 Hz, 6H, H-
26, H-27), 0.94 (d, J = 6.5 Hz, 3H, Me-21), 1.18 (s, 3H, H-19), 1.62
a
(m, 1H, H-5a), 3.79 (m, 1H, H-2a), 3.71 (m, H-6a), 3.86 (m, 1H,
H-3b). 13C NMR d (CD3OD): 42.6 (C-1), 72.2 (C-2), 71.4 (C-3), 30.1
(C-4), 43.1 (C-5), 72.9 (C-6), 40.7 (C-7), 31.3 (C-8), 57.7 (C-9),
37.1 (C-10), 24.9 (C-11), 40.4 (C-12), 43.9 (C-13), 56.7 (C-14)⁄,
25.3 (C-15), 29.3 (C-16), 57.7 (C-17)⁄, 12.6 (C-18), 18.3 (C-19),
37.0 (C-20), 19.2 (C-21), 37.4 (C-22), 21.8 (C-23), 40.7 (C-24),
29.1 (C-25), 22.9 (C-26), 23.2 (C-27). Anal. calcd for C27H48O3.3/4
H2O, C 74.69, H 11.49, O 13.82. Found, C 74.26, H 11.58. HREIMS
(ESI+), calculated for C27H48NaO3 [M + Na+]: 443.3496, found m/
trisodium 2b,3a,6b-trihydroxy-5a-cholestane trisulfate (13)
(12.8 mg, 88%), m.p. >250 °C (decomp. at 85 °C). 1H NMR d
(DMSO-d6): 0.65 (s, 3H, H-18), 0.84 (d, J = 6.6 Hz, 6H, H-26, H-
27), 0.89 (d, J = 6.4 Hz, 3H, Me-21), 0.95 (s, 3H, H-19), 4.06 (m,
z = 443.3479. ATR FT-IR (ZnSe, cmꢁ1) 3302 (
CH3), 1379 (ds CH3).
m
O–H), 1463 (das
1H, H-6a), 4.30 (m, 1H, H-2a
), 4.37 (m, 1H, H-3b). 13C NMR d
(DMSO-d6): 36.6 (C-1), 73.0 (C-2), 73.2 (C-3), 26.7 (C-4), 41.2 (C-
5), 75.6 (C-6), 38.9 (C-7), 30.0 (C-8), 54.5 (C-9), 35.0 (C-10), 20.3
(C-11), 39.8 (C-12), 42.2 (C-13), 55.9 (C-14), 23.9 (C-15), 27.8 (C-
16), 55.7 (C-17), 12.0 (C-18), 16.9 (C-19), 35.2 (C-20), 18.6 (C-21),
35.7 (C-22), 23.1 (C-23), 38.8 (C-24), 27.4 (C-25), 22.4 (C-26),
2.7. Disodium 2b,3a-dihydroxycholest-5-ene disulfate (11)
Trimethylamine-sulfur trioxide complex (52.6 mg, 0.38 mmol)
was added to a solution of 2b,3 ,6b-trihydroxy-5 -cholestane
a
a
22.7 (C-27). HREIMS (ESI-), calculated for C27H45Na2O12S3

1144
V. Richmond et al. / Steroids 78 (2013) 1141–1147
[M–Na]ꢁ: 703.1874, found m/z = 703.1894. ATR FT-IR (ZnSe, cmꢁ1
1463(das CH3), 1382 (ds CH3), 1203 (ds S@O).
)
tions eluted with cyclohexane/EtOAc (90:10) afforded pure 2
a,3a-
epoxy-5 -cholestan-6 -ol (16) (37.2 mg, 71%), as a colorless syrup.
a
a
1H NMR d (CDCl3): 0.64 (s, 3H, H-18), 0.77 (s, 3H, H-19), 0.86 (d,
J = 6.6 Hz, 3H, H-26), 0.86 (d, J = 6.7 Hz, 3H, H-27), 0.90 (d,
J = 6.6 Hz, 3H, Me-21), 3.12 (dd, J = 6.0 Hz, 4.0 Hz, 1H H-2b), 3.24
(m, 1H, H-3b), 3.38 (dt, J = 11.1 Hz, 4.7 Hz, H-6b). 13C NMR d
(CDCl3): 38.7 (C-1), 50.7 (C-2), 52.4 (C-3), 24.6 (C-4), 44.1 (C-5),
71.1 (C-6), 41.8 (C-7), 34.2 (C-8), 53.4 (C-9), 34.6 (C-10), 20.9 (C-
11), 39.8 (C-12), 42.5 (C-13), 56.1 (C-14), 24.3 (C-15), 28.3 (C-16),
56.3 (C-17), 12.0 (C-18), 14.5 (C-19), 35.9 (C-20), 18.8 (C-21),
36.3 (C-22), 23.9 (C-23), 39.6 (C-24), 28.2 (C-25), 22.7 (C-26),
23.0 (C-27). HREIMS (ESI+), calculated for C27H46NaO2 [M + Na]+:
2.10. 2b,3a-Dihydroxy-5b-cholestan-6-one (14)
To
(8.1 mg, 0.019 mmol) in 1.7 ml of ethanol under reflux, small
pieces of Na were added until no 2b,3 -dihydroxy-5 -cholestan-
a solution of 2b,3a-dihydroxy-5a-cholestan-6-one (3)
a
a
6-one was detected by TLC. Then, 10 ml of 15% NaCl solution was
added and the steroid was extracted with EtOAc (3 ꢀ 10 ml). The
combined organic extracts were dried over anhydrous MgSO4
and evaporated to dryness. The reaction mixture was purified by
solid phase extraction over silica gel (55
lm) using cyclohexane/
425.3390, found m/z = 425.3396. FT-IR (NaBr, film, cmꢁ1) 3406 (
m
EtOAc (60:40) to give 6.6 mg (81.5%) of 14 as a white crystalline so-
lid, m.p. 157–158 °C (acetone–H2O). 1H NMR d (CDCl3): 0.65 (s, 3H,
H-18), 0.86 (d, J = 6.7 Hz, 6H, H-26, H-27), 0.89 (s, 3H, H-19), 0.91
(d, J = 6.5 Hz, 3H, Me-21), 2.19 (m, 1-H, H-5b), 3.43 (m, 1-H, H-
O–H), 1469 (das CH3), 1377 (ds CH3), 1255 (ms C–O–C), 808 (mas C–
O–C).
2.13. 2b,3a,6a-Trihydroxy-5a-cholestane (17)
3b), 3.65 (ddd,J = 12.5 Hz, 8.7 Hz, 4.0 Hz 1H, H-2a
). 13C NMR d
(CDCl3): 32.7 (C-1), 70.5 (C-2), 75.3 (C-3), 41.9 (C-4), 59.0 (C-5),
213.2 (C-6), 42.7 (C-7), 36.9 (C-8), 41.1 (C-9), 42.0 (C-10), 20.8
(C-11), 39.4 (C-12), 42.9 (C-13), 56.6 (C-14), 23.7 (C-15), 27.9 (C-
16), 55.9 (C-17), 11.7 (C-18), 23.0 (C-19), 35.5 (C-20), 18.4 (C-21),
35.8 (C-22), 23.6 (C-23), 39.3 (C-24), 27.8 (C-25), 23.5 (C-26),
22.3 (C-27). Anal. calcd for C27H46O3. 1/4 H2O, C 76.64, H 11.07,
O 12.29. Found, C 76.38, H 10.58. HREIMS (ESI+), calculated for C27-
H46NaO3 [M + Na]+: 441.3339, found m/z = 441.3340. ATR FT-IR
A solution of epoxide 16 (34.8 mg, 0.086 mmol) in THF (1.77 ml)
was treated with 1 N H2SO4 (0.05 ml) and stirred for 24 h at room
temperature. After neutralization with saturated NaHCO3 solution
the mixture was evaporated to fifth initial volume, diluted with
water (5 ml), and extracted with EtOAc (3 ꢀ 5 ml). The combined
organic extracts were washed with water, dried over anhydrous
MgSO4, filtered and evaporated to dryness. The reaction product
was purified by solid phase extraction over silica gel (55
lm). Frac-
(ZnSe, cmꢁ1) 3454, 3376(
m O–H), 1690 (m C@O), 1457 (das CH3),
tions eluted with cyclohexane/EtOAc (40:60) afforded pure
1376 (ds CH3).
2b,3a,6a-trihydroxy-5a-cholestane (17). (35.6 mg, 98%), m.p.
249–250 °C (acetone–H2O). 1H NMR d (CDCl3/CD3OD; 4:1): 0.66
(s, 3H, H-18), 0.87 (d, J = 6.6 Hz, 6H, H-26, H-27), 0.98 (s, 3H, H-
2.11. 5
a
-Cholest-2-en-6
a
-ol (15)
-cholest-2-en-6-one (5) (79.9 mg,
19), 0.91 (d, J = 6.5 Hz, 3H, Me-21), 3.81 (m, 1H, H-2a), 3.38 (dt,
To
a
solution of
5a
J = 11.0 Hz, 4.5 Hz, H-6b), 3.84 (m, 1H, H-3b). 13C NMR d (CDCl3/
CD3OD; 4:1): 40.4 (C-1), 70.5 (C-2), 69.9 (C-3), 25.4 (C-4), 45.9
(C-5), 69.3 (C-6), 41.7 (C-7), 34.0 (C-8), 54.9 (C-9), 36.7 (C-10),
24.0 (C-11), 40.1 (C-12), 42.8 (C-13), 56.5 (C-14)⁄, 24.3 (C-15),
28.4 (C-16), 56.5 (C-17)⁄, 12.2 (C-18), 15.2 (C-19), 36.0 (C-20),
18.8 (C-21), 36.4 (C-22), 21.0 (C-23), 39.7 (C-24), 28.2 (C-25),
22.6 (C-26), 22.9 (C-27). (⁄) Signals might be interchangeable. Anal.
calcd for C27H48O3, C 77.09, H 11.50, O 11.41. Found, C 77.34, H
0.21 mmol) in 16.5 ml of n-propanol under reflux, small pieces of
Na were added until no cholest-2-en-6-one was detected by TLC.
Then, 10 ml of 15% NaCl solution was added and the steroid was
extracted with AcOEt (3 x 20 ml). The combined AcOEt extracts
were dried over anhydrous MgSO4 and evaporated to dryness.
The reaction product was purified by dry column flash chromatog-
raphy on silica gel using hexane/EtOAc (98:2) to give 72.3 mg (90%)
of 15 as a white crystalline solid, m.p. 120–121 °C (acetone–H2O).
1H NMR d (CDCl3): 0.66 (s, 3H, H-18), 0.76 (s, 3H, H-19), 0.86 (d,
J = 6.6 Hz, 6H, H-26, H-27), 0.91 (d, J = 6.5 Hz, 3H, H-21), 3.44 (dt,
J = 10.9 Hz, 4.6 Hz, H-6b), 5.59 (m, 1H, H-2), 5.67 (m, 1H, H-3).
13C NMR d (CDCl3): 40.1 (C-1), 125.7 (C-2), 125.3 (C-3), 25.9 (C-
4), 48.8 (C-5), 71.8 (C-6), 41.5 (C-7), 34.4 (C-8), 53.7 (C-9), 35.6
(C-10), 21.0 (C-11), 40.0 (C-12), 42.7 (C-13), 53.4 (C-14), 24.3 (C-
15), 28.3 (C-16), 56.3 (C-17), 12.1 (C-18), 13.2 (C-19), 35.9 (C-20),
18.8 (C-21), 36.3 (C-22), 24.0 (C-23), 39.7 (C-24), 28.2 (C-25).
22.7 (C-26), 23.0 (C-27). Anal. calcd for C27H46O, C 83.87, H
11.99, O 4.14. Found, C 83.50, H 12.27. HREIMS (ESI+), calculated
for C27H46NaO [M + Na]+: 409.3441, found m/z = 409.3442. FT-IR
11.95. HREIMS (ESI+), calculated for
C
27H48NaO3 [M + Na+]:
443.3496, found m/z = 443.3494. FT-IR (NaBr, film, cmꢁ1) 3262 (
O–H), 1460 (das CH3), 1377 (ds CH3).
m
2.14. Trisodium 2b,3a,6a-trihydroxy-5a-cholestane trisulfate (18)
Trimethylamine-sulfur trioxide complex (23.8 mg, 0.17 mmol)
was added to a solution of 2b,3 ,6 -trihydroxy-5 -cholestane
a
a
a
(17) (7.2 mg, 0.017 mmol) in DMF (0.61 ml). The reaction mixture
was irradiated and stirred at 150 °C for 9 min in a sealed tube 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
presure and purified by solid phase extraction over silica gel
(NaBr, film, cmꢁ1) 3270 (
m O–H), 3017 (m C@C-H), 1468 (das CH3),
1382 (ds CH3).
2.12. 2
a
,3a
-Epoxy-5a-cholestan-6a-ol (16)
(55
lm). Fractions eluted with CH2Cl2/MeOH (85:15) afforded pure
trisodium 2b,3
a,6a-trihydroxy-5a-cholestane trisulfate (18)
A 10% Na2CO3 solution (2.02 ml) was added to 5
a-cholest-2-
(9.4 mg, 76%), m.p. 189–192 °C (decomp.). 1H NMR d (DMSO-d6):
0.62 (s, 3H, H-18), 0.84 (d, J = 6.6 Hz, 6H, H-26, H-27), 0.87 (s, 3H,
H-19), 0.88 (d, J = 6.6 Hz, 3H, Me-21), 3.82 (dt, J = 10.9 Hz, 4.3 Hz,
ene-6 -ol (15) (51.2 mg, 0.13 mmol) in CH2Cl2 (1.8 ml). The reac-
a
tion mixture was stirred vigorously at 5 °C and m-chloroperben-
zoic acid (74.3 mg, 0.43 mmol) in 0.8 ml of CH2Cl2 was added
slowly. The mixture was stirred for 4 h at room temperature, and
then the aqueous layer was extracted with CH2Cl2 (3 ꢀ 10 ml).
The combined CH2Cl2 extracts were washed successively with 5%
Na2SO3 solution, saturated NaHCO3 solution and water, dried over
anhydrous MgSO4 and evaporated to dryness. The reaction product
H-6b), 4.37 (m, 1H, H-3b), 4.45 (m, 1H, H-2a
). 13C NMR d
(DMSO-d6): 37.5 (C-1), 72.4 (C-2), 72.3 (C-3), 23.7 (C-4), 43.8 (C-
5), 74.0 (C-6), 38.9 (C-7), 33.4 (C-8), 54.1 (C-9), 35.9 (C-10), 20.4
(C-11), 39.5 (C-12), 42.2 (C-13), 55.9 (C-14), 23.8 (C-15), 27.8 (C-
16), 55.6 (C-17), 11.9 (C-18), 14.7 (C-19), 35.2 (C-20), 18.5 (C-21),
35.6 (C-22), 23.1 (C-23), 38.9 (C-24), 27.4 (C-25), 22.4 (C-26),
was purified by solid phase extraction over silica gel (55
lm). Frac-
22.7 (C-27). HREIMS (ESIꢁ), calculated for
C27H45Na2O12S3

V. Richmond et al. / Steroids 78 (2013) 1141–1147
1145
mined with GraphPad Prism 5. Eserine (99%) was used as the ref-
erence AChE/BChE inhibitor.
3. Results and discussion
3.1. Chemistry
3.1.1. Synthesis of disodium (2
cholestan-6-one disulfate
a,3a)- and (2b,3b)-dihydroxy-5a-
Isomeric disulfated steroids 7 and 9 (Fig. 2) were synthesized
according to the following sequence. First, intermediate 5 was ob-
tained from cholesterol in four steps as described previously [11].
Osmium-catalized asymmetric dihydroxylation [12] of double
bond of 5 using K3Fe(CN)6 as cooxidant and hydroquinine-1,4-
phthalazinediyl diether [(DHQ)2-PHAL] as chiral ligand gave, after
Fig. 2. Chemical structures of compounds 6 and 8 and their sulfated analogs (7, 9).
[M–Na]ꢁ: 703.1874, found m/z = 703.1865. ATR FT-IR (cmꢁ1
1485(das CH3), 1380 (ds CH3), 1210 (ds S@O).
)
purification, 65% yield of the 2b,3b diol (6) and 35% of the 2
diastereomer (8). When hydroquinidine-1,4-phthalazinediyl
diether [(DHQD)2-PHAL] was used as the chiral ligand, the 2 ,3
a,3a
2.15. Cholinesterase inhibition assays
a
a
Electric eel (Torpedo californica) AChE and horse serum BChE
were used as source of both cholinesterases. AChE and BChE inhib-
iting activities were measured in vitro by the spectrophotometric
method developed by Ellman with slight modification [13]. The
lyophilized enzyme, 500U AChE/300U BChE, was prepared in buf-
fer phosphate A (8 mM K2HPO4, 2.3 mM NaH2PO4) to obtain 5/
3 U/mL stock solution. Further enzyme dilution was carried out
with buffer phosphateB (8 mM K2HPO4, 2.3 mM NaH2PO4, 0.15 M
NaCl, 0.05% Tween 20, pH 7.6) to produce 0.126/0.06 U/mL enzyme
solution. Samples were dissolved in buffer B. Compounds 6 and 8
diastereomer (8) was obtained in 51% yield together with 6 (28%)
and starting material 5 (12%), which was recycled. These results
are in accordance with reported synthesis of cis-diols using these
hydroxylation reagents [14,15]. Subsequent treatment of diols 6
and 8 with 8 equiv. of trimethylamine-sulfur trioxide complex
for 7 min at 150 °C at a microwave reactor [11] afforded the corre-
sponding ammonium sulfates, which were transformed via ion ex-
change into the disodium salts 7 and 9. The structures of 7 and 9
were confirmed by analysis of the proton and carbon NMR chem-
ical shifts at C-2 and C-3. Resonances showing H-2
a at 4.84 ppm
required 2.5% of MeOH as cosolvent. Enzyme solution (300
and 300 L of sample solution were mixed in a test tube and incu-
bated for 60/120 min at room temperature. The reaction was
started by adding 600 L of the substrate solution (0.5 mM DTNB,
0.6 mM ATCI/BTCI, 0.1 M Na2HPO4, pH 7.5). The absorbance was
read at 405 nm for 180 s at 27 °C. Enzyme activity was calculated
by comparing reaction rates for the sample to the blank. All the
reactions were performed in triplicate. IC50 values were deter-
l
L)
(m) and H-3 at 4.31 ppm (dt, J = 12.1, 3.7 Hz) for compound 7
a
l
and H-2b at 4.44 ppm (ddd, J = 12.3, 4.7, 3 Hz) and H-3b at
4.86 ppm (m) for isomer 9 were characteristic of the presence of
two sulfate groups at C-2 and C-3 in cis position. This was in accor-
dance with the chemical shifts observed for C-2 (76.47 ppm (iso-
mer 7) and 75.44 ppm (isomer 9)) and C-3 (78.30 ppm (isomer 7)
and 76.40 ppm (isomer 9)) in the 13C NMR spectra, as determined
from the HSQC and HMBC spectra [16].
l
Scheme 1. Conditions: (a) NaBH4, methanol, room temperature (b) 20 equiv. Me3N.SO3, DMF, 7 min, 150 °C, microwave (c) Amberlite CG-120 (MeOH) (d) 8 equiv. Me3N.SO3,
DMF, 7 min, 150 °C, microwave (e) NaBH4, methanol, room temperature (f) 5 equiv. Me3N.SO3, DMF, 1 h, 60 °C, microwave.
Scheme 2. Conditions: (a) Sodium, ethanol, reflux (b) 8 equiv. Me3N.SO3, DMF, 7 min, 150 °C, microwave (c) Amberlite CG-120 (MeOH).

1146
V. Richmond et al. / Steroids 78 (2013) 1141–1147
Scheme 3. Conditions: (a) Sodium, n-propanol, reflux (b) m-ClPBA, Na2CO3, H2O-Cl2CH2, room temperature, 4 h (c) H2SO4, THF room temperature, 24 h (d) 15 equiv.
Me3N.SO3, DMF, 150 °C, microwave (e) Amberlite CG-120 (MeOH).
3.1.3. Synthesis of trisodium (2b,3a,6a)-trihydroxy-5a-cholestane
trisulfate
Table 1
Reduction of 3 with sodium in ethanol under reflux gave the epi-
mer at C-5 (14) instead of the expected 2b,3 ,6 -triol (Scheme 2).
Summary of the in vitro antiacetylcholinesterase
activities.
a
a
The 13C NMR spectrum of 14 showed a C-19 methyl signal at
23.0 ppm indicative of a cis conformation for rings A and B [17]. This
was confirmed by NOESY correlations of the signal of H-5b at
2.19 ppm with CH3-19 and H-3b. Evidently, epimerization at C-5
via enolate formation was favored over reduction of the carbonyl
group due to the pseudo-equatorial position of both hydroxyl
groups in 14. On the other hand, sulfation of 14 in the same condi-
tions as for 3 gave the disulfated steroid 1 in 84.1% yield.
Compound
IC50 (lM)
1
3
6
7
8
9
14.59 0.88
59.65 2.30
>200
40.94 8.62
>200
30.66 6.91
>200
>200
>200
>200
>200
15.48 0.88
0.0113 0.0011
10
11
12
13
17
18
Eserine
Looking forward to prepare the 2b,3a,6a-triol 17 we followed
the strategy of Murphy et al. [18] for the synthesis of trisulfated
analog 18 of sokotrasterol sulfate, a natural product isolated from
the sponge Topsentia ophirhaphidites (Scheme 3). Reduction of the
ketone 5 with sodium in n-propanol (reflux) gave 5
en-6 -ol (15). Further epoxidation and acid-catalyzed epoxide ring
opening of 16 gave triol 17. The 1H NMR spectrum of 17 showed
two multiplets at d 3.81 (H-2 ) and 3.84 (H-3b) ppm, geminal to
hydroxyl groups at C-2 (70.47 ppm) and C-3 (69.93 ppm) and a
double triplet at 3.38 ppm (H-6b) (J = 11.0 Hz, 4.5 Hz), as deter-
mined by analysis of 13C NMR, HSQC, HMBC and NOESY spectra.
Sulfation of 17 with 15 equiv. of trimethylamine-sulfur trioxide
complex for 9 min at 150° in microwave reactor and transforma-
a-cholest-2-
a
3.1.2. Synthesis of trisodium (2b,3a,6b)-trihydroxy-5a-cholestane
trisulfate
The strategy followed to obtain compound 13 is shown in
Scheme 1. The diol 3 was prepared from cholesterol in 6 steps as
described previously [11]. Reduction of the carbonyl group with
a
NaBH4/MeOH from the
the 2b,3 ,6b-triol (10) in 87% yield. The b configuration of the hy-
droxyl group at C-6 was inferred from NOE interactions between
signals at 1.62 ppm (H-5 ) and 3.71 (H-6 ) ppm. Further sulfation
a less hindered face of the steroid gave
a
tion via ion exchange afforded trisodium 2b,3a,6a-trihydroxy-
5a-cholestane trisulfate (18) in 76% yield (Scheme 3).
a
a
of 10 with 20 equiv. of trimethylamine-sulfur trioxide complex for
7 min at 150 °C at a microwave reactor and elution through
Amberlite CG-120 (sodium form) afforded a mixture of products,
as observed by TLC. Purification of the mixture gave disulfated ste-
roid 11 in 34% yield. The 1H NMR spectrum of 11 showed a multi-
plet at 5.17 ppm (H-5) and two olefinic carbons at 139.4 (C-5) and
120.6 ppm (C-6), characteristic of a D5 steroid [17]. This was con-
firmed by correlation of the C-19 methyl signal at 18.40 ppm with
the multiplet at 5.17 ppm in the HMBC spectrum. In order to avoid
the elimination of the axial hydroxyl group at C-6, we developed a
new strategy based on sulfation of the diol 3 [11], reduction of the
carbonyl group, and further sulfation of the hydroxyl at C-6 in
milder conditions (Scheme 1). In this way, reduction of the disulf-
ated ketone 1 with NaBH4/MeOH, sulfation of the C-6 (b) hydroxyl
of 12 with 5 equiv. of trimethylamine-sulfur trioxide complex for
1 h at 60 °C at a microwave reactor and transformation via ion ex-
change into the sodium salts afforded trisulfated triol 13 in 89%
yield from 1. The 1H NMR spectrum of 13 showed a broad singlet
at 4.06 ppm for H-6b, as confirmed by NOESY correlations.
3.1.4. Evaluation of the acetylcholinesterase inhibitory activity
The acetylcholinesterase inhibitory activity of compounds
6–13, 17 and 18 was evaluated and compared to that of analogs 1
and 3 (Table 1) previously synthesized by our group [10–11]. The
compounds were found to be less active than the reference
compound eserine. From the data shown in Table 1, cis disulfated
compounds 7 and 9 were less active than isomer 1, suggesting the
importance of the sulfate configurations at C-2 and C-3 on the
inhibitory activity. As reported previously [11], steroid 1 binds
reversibly to the enzyme-substrate complex, yielding an inactive
complex. This affinity is favored by hydrogen bonding interactions,
whichinvolve both sulfatesat ring A. Presumably, a change in sulfate
configurations at C-2 and C-3, as in isomers 7 and 9, decreases their
affinity to the enzyme-substrate complex. Interestingly, 2b,3
-trisulfated steroid 18 was the most active compound with an
IC50 value of 15.48 comparable to that of compound
(14.59 M). On the other hand, compound 13 with a sulfate group
at C-6 (b) showed no inhibitory activity (IC50 > 200 M) as well as
a,
6a
lM
1
l
l

V. Richmond et al. / Steroids 78 (2013) 1141–1147
1147
Table 2
[4] Dvir H, Silman I, Harel M, Rosenberry TL, Sussman JL. Acetylcholinesterase:
from 3D structure to function. Chem Biol Interact 2010;187:10–22.
[5] Peng D-Y, Sun Q, Zhu X-L, Lin H-Y, Chen Q, et al. Design, synthesis and
bioevaluation of benzamides: novel AChE inhibitors with multi-functions on
BChE, Ab aggregations and b-secretase. Bioorg Med Chem 2012;20:6739–50.
[6] Enz A, Amstutz R, Boddeke H, Gmelin G, Malonowski J. Brain selective
inhibition of AChE: a novel approach to therapy for Alzheimer’s disease. Prog
Brain Res 1993;98:431–5.
Summary of the in vitro antibutyrylcholinesterase activities.
Compound
IC50
(
l
M)
Ratio IC50 BChE/AChE
1
18
Eserine
>200
176.10 1.29
0.0143 0.0013
>13.7
11.4
1.27
[7] Langjae R, Bussarawit S, Yuenyongsawad S, Ingkaninan K, Plubrukarn A.
Acetylcholinesterase-inhibiting steroidal alkaloid from the sponge Corticium
sp. Steroids 2007;72:682–5.
[8] Porcel J, Montalban X. Anticholinesterasics in the treatment of cognitive
impairment in multiple sclerosis. J Neurol Sci 2006;245:177–81.
[9] Vela Gurovic MS, Castro MJ, Richmond V, Faraoni MB, Maier MS, Murray AP.
Triterpenoids with acetylcholinesterase inhibition from Chuquiraga erinacea D.
Don. Subsp. erinacea (Asteraceae). Planta Medica 2010;76:607–10.
[10] Garrido Santos GA, Murray AP, Pujol CA, Damonte EB, Maier MS. Synthesis and
disulfated analogs 11 and 12 and alcohols 6, 8, 10 and 17. These re-
sults indicate that the presence of trans diaxial sulfate groups at C-
2 and C-3 together with a carbonyl or sulphate group at C-6 (
crease the inhibitory activity.
a) in-
antiviral activity of sulfated and acetylated derivatives of 2b,3a-dihydroxy-5a-
cholestane. Steroids 2003;68:125–32.
[11] Richmond V, Garrido Santos G, Murray AP, Maier MS. Synthesis and
In order to determine the AChE/BChE selectivity of the most ac-
tive compounds 1 and 18, their butyrylcholinesterase activity was
evaluated. As shown in Table 2, the butyrylcholinesterase activity
of 1 and 18 was one magnitude lower than that against AChE,
revealing a selective inhibitor profile. This result is interesting be-
cause BChE has the ability of delaying the onset and decreasing the
rate of Ab fibril formation in vitro, a central event in the pathogen-
esis of AD [19,20].
acetylcholinesterase inhibitory activity of 2b,3a-disulfoxy-5a-cholestan-6-
one. Steroids 2011;76:1160–5.
[12] Becker H, Soler MA, Sharpless KB. Selective asymmetric dihydroxilation of
polyenes. Tetrahedron 1995;51:1345–76.
[13] Ellman GL, Courtney KD, Andres Jr V, Featherstone RM. A new and rapid
colorimetric determination of acetylcholinesterase activity. Biochem
Pharmacol 1961;7:88–95.
[14] Ramirez JA, Gros EG, Galagovsky LR. Effects on bioactivity due to C-5
heteroatom substituents on synthetic 28-homobrassinosteroid analogs.
Tetrahedron 2000;56:6171–80.
[15] Jeong KS, Sjö P, Sharpless KB. Asymmetric dihydroxylation of enynes.
Tetrahedron Lett 1992;33:3833–6.
Acknowledgments
[16] Makarieva TN, Shubina LK, Kalinovsky AI, Stonik VA, Elyakov GB. Steroids in
Porifera. II. Steroid derivatives from two sponges of the family
Halichondriidae. Sokotrasterol sulfate, a marine steroid with a new pattern
of side chain alkylation. Steroids 1983;42:267–81.
[17] Goad LJ, Akihisa T. Analysis of sterols. Blackie; 1997.
[18] Murphy M, Larrivée B, Pollet I, Craig KS, Williams DE, et al. Identification of
We thank the University of Buenos Aires and the National Re-
search Council of Argentina (CONICET) for financial support of this
work. V.R. thanks CONICET for a Doctoral fellowship. A.P.M. and
M.S.M. are Research Members of CONICET.
sokotrasterol sulfate as
2006;99:257–65.
[19] Podoly E, Bruck T, Diamant S, Melamed-Book N, Weiss A, et al. Human
recombinant butyrylcholinesterase purified from the milk of transgenic goats
interacts with beta-amyloid fibrils and suppresses their formation in vitro.
Neurodegener Dis 2008;5:232–6.
[20] Diamant S, Podoly E, Friedler A, Ligumsky H, Livnah O, et al.
Butyrylcholinesterase attenuates amyloid fibril formation in vitro. Proc Natl
Acad Sci 2006;103:8628–33.
a
novel proangiogenic steroid. Circ Res
References
[1] Goedert M, Spillantini MG.
2006;314:777–81.
[2] Parle M, Dhingra D, Kulkarni SK. Neuromodulators of learning and memory.
Asia Pac J Pharmacol 2004;16:89–99.
[3] Rosenberry TL. Acetylcholinesterase. Adv Enzymol Relat Areas Mol Biol
1975;43:103–218.
A century of Alzheimer’s Disease. Science