Synthesis of?oxysterols and?nitrogenous sterols with antileishmanial and?trypanocidal activities

Article abstract of DOI:10.1016/j.ejmech.2006.03.033

Two sterol families have been synthesized: the first one is nitrogenous sterols containing amino, N-hydroxyimino or cyano group and the second one is oxysterols such as ketosterol and hydroxysterols. These compounds were then evaluated in vitro against Leishmania donovani promastigotes and Trypanosoma brucei brucei trypomastigotes. The most active compounds against L.?donovani promastigotes were 7β-aminomethylcholesterol and 7α,β-aminocholesterol (IC₅₀ in a range from 1 to 3?μM, pentamidine: 2.8?μM). These compounds were active on intramacrophage amastigotes with IC₅₀ of 1.3?μM. Such an activity justifies further in vivo antileishmanial evaluation. Against T. b. brucei, (24R,S)-24-hydroxy-24-methylcholesterol (MEC, 12.5?μM) was the most active compound from these series.

Full text of DOI:10.1016/j.ejmech.2006.03.033

European Journal of Medicinal Chemistry 41 (2006) 1109–1116
http://france.elsevier.com/direct/ejmech
Original article
Synthesis of oxysterols and nitrogenous sterols
with antileishmanial and trypanocidal activities
a
b
b
c
Marc-Antoine Bazin , Philippe M. Loiseau , Christian Bories , Yves Letourneux ,
a
a,
*
Sylvain Rault , Laïla El Kihel
a
Centre d’Etudes et de Recherche sur le Médicament de Normandie, UFR des Sciences Pharmaceutiques, 5, rue Vaubénard, 14032 Caen cedex, France
b
Faculté de Pharmacie, Chimiothérapie Antiparasitaire, Université de Paris-XI,
UMR 8076 CNRS, rue Jean-Baptiste-Clément, F-92290 Châtenay-Malabry, France
Université d’Aix-Marseille III, UMR Inra, 1111, avenue Escadrille Normandie-Niemen, 13397 Marseille cedex 20, France
c
Received in revised form 21 March 2006; accepted 23 March 2006
Available online 01 September 2006
Abstract
Two sterol families have been synthesized: the first one is nitrogenous sterols containing amino, N-hydroxyimino or cyano group and the
second one is oxysterols such as ketosterol and hydroxysterols. These compounds were then evaluated in vitro against Leishmania donovani
promastigotes and Trypanosoma brucei brucei trypomastigotes. The most active compounds against L. donovani promastigotes were 7β-
aminomethylcholesterol and 7α,β-aminocholesterol (IC50 in a range from 1 to 3 μM, pentamidine: 2.8 μM). These compounds were active on
intramacrophage amastigotes with IC50 of 1.3 μM. Such an activity justifies further in vivo antileishmanial evaluation. Against T. b. brucei, (24R,
S)-24-hydroxy-24-methylcholesterol (MEC, 12.5 μM) was the most active compound from these series.
©
2006 Published by Elsevier Masson SAS.
Keywords: Antileishmanial; Trypanocidal; Nitrogenous sterols; Oxysterols
1
. Introduction
antileishmanial agent in use. Its activity results from the speci-
fic target of AmB at the level of sterols, mainly ergosterol,
which is found in the membrane of Leishmania genus and
fungi [5]. However, sometimes the high level of toxicity for-
bids its clinical uses. Largely liposomal AmB developed as
Ambisome® gave interesting results by reducing the renal toxi-
city [6]. Other new approaches have been proposed including
the use of other parenteral and non-parenteral agents such as
the aminoglycoside, aminosidine (topical application) or oral
agents. Oral administration has the advantage of reducing
socio-economic difficulties that are present in endemic areas
where health facilities are lacking. Since 4 years, hexadecyl-
phosphocholine (HePC, miltefosine), an antineoplastic agent,
has been identified as the first effective oral treatment in visc-
eral infection [7]. This newly developed drug is a real advance
in antileishmanial chemotherapy. However, the possibility to
obtain miltefosine-resistant parasites by drug pressure is an
indicator of a potential appearance of miltefosine-resistant
cases in the field [8]. This situation justifies the research for
new drugs because new therapeutic agents are urgently
Leishmaniases are parasitic diseases due to several species
of unicellular parasite in the genus Leishmania that is endemic
in several parts of the world. They are a complication of AIDS
in both the developing world and industrialized world [1]. The
parasite is transmitted to some mammals and humans by the
bite of an insect vector, the female phlebotome sandfly. Since
about 50 years, the antimonials sodium stibogluconate
®
(
Pentostam , Glaxo Wellcome, UK) and meglumine antimoni-
®
ate (Glucantime , Aventis, France) are the first-line treatment
for leishmaniases. Amphotericin B (AmB) and pentamidine, as
alternatives to antimony are considered to cause serious and
irreversible toxic effects [2]. The antifungal agent AmB, a
polyene macrolide antibiotic [3], has long been recognized as
a powerful antileishmanial drug [4], and it is the most active
*
Corresponding author.
E-mail address: laila.elkihel@unicaen.fr (L. El Kihel).
0223-5234/$ - see front matter © 2006 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2006.03.033

1110
M.-A. Bazin et al. / European Journal of Medicinal Chemistry 41 (2006) 1109–1116
required. One target of interest is sterol biosynthesis. The main
sterol found in mammalian cells is cholesterol, while the patho-
gens which cause Chagas disease and leishmaniasis (Trypano-
soma cruzi and species of Leishmania, respectively), synthe-
size ergosterol and related 24-alkylated sterol [9,10]. These
sterols have differences in their biosynthetic pathway that are
attractive for drug design. The ergosterol is thought to have
two roles within these kinetoplastid parasites; it has a structural
role in the cell membrane and may have a “sparking” or “ hor-
monal” role similar to that seen in yeast. In Leishmania sp. the
case is less certain.
on resistant strains, these aminosterols were more active than
AmB on Candida tropicalis (AmB-resistant). No significant
variation in the cytotoxicity of the two epimers 7α or 7β was
observed against the three resistant strains [26]. On the other
hand, we have synthesized novel polyaminosterols as squala-
mine analogues. These molecules showed similar antifungal
activities as squalamine [27,28].
Considering the analogy in the sterol biosynthesis pathways
between fungi and Leishmania, we decided therefore to synthe-
size oxysterols and nitrogenous sterols, especially designed to
interact with the sterol metabolism. These molecules were then
evaluated, in vitro, against Leishmania donovani and T. brucei
brucei. The choice of T. brucei relies on the fact that this dis-
ease dramatically increases in Africa and new drugs are needed
urgently whereas T. cruzi infections are in diminution in South
America because of the improvement of habitat suppressing
the contact between triatomine insect vector and humans
Leishmania parasites have a strict requirement for specific
endogenous sterols for survival and growth and they cannot
use the abundant supply of cholesterol present in their mamma-
lian hosts [11]. Therefore, the inhibitory biosynthesis of Leish-
mania sterols was proposed as a source of potential targets for
therapy [12]. It has been shown that various antifungal agents
as sterols biosynthesis inhibitors have been described to have
clinically useful antileishmanial properties [13–15]. For exam-
ple, inhibitors of 14α-demethylase, sterol 24-methyltransferase,
(Scheme 1).
2. Results and discussion
8
7
14
Δ -Δ -sterol isomerase and Δ -sterol reductase have been
shown as having anti-parasitic activity [16,17]. Thus, a large
number of inhibitors of enzymes from the sterol pathway
have been studied for their in vitro and in vivo antileishmanial
properties such as azoles and azasterols [18–20].
2.1. Chemistry
Compounds 1a, 1b, 7, 8a and 8b were prepared according
Concerning Trypanosomatidae, the bloodstream forms of
Trypanosoma brucei brucei differs from other trypanosomes
as it contains predominantly cholesterol and apparently sup-
presses de novo synthesis of C-28-sterols; T. brucei assimilates
host cholesterol from serum lipoproteins to meet its sterol
requirement [21].
The rationale of the present study is based on the fact that
modified sterols cause a depletion of normal sterols and an
accumulation of abnormal amounts of sterol precursors with
cytostatic or cytotoxic consequences [22].
to our procedure described in literature [29].
Dicyanomethylcholesterols 5a and 5b were prepared as
shown in Scheme 2: cholesteryl-3β-acetate 2 was brominated
by N-bromosuccinimide, which gave an epimeric mixture of
monobromide compounds 3 (7α/7β, 7:3 ratio). 7-Bromocholes-
teryl-3β-acetate was prepared by Confalone et al. as intermedi-
ate to prepare the 7-dehydrocholesterol using 5,5-
dimethylhydantoin. Epimeric mixture 2 was not described by
analysis spectra but 7α/7β bromides ratio was determined via
7-phenylsulfide after separation by chromatography on silica
gel column [30]. In our case, epimers bromides were purified
by flash chromatography. The structure and 7α/7β bromides
In the frame of our work, we have focused on a study of
aminosterols [23–26] since we had shown previously that 7α,β-
aminocholesterol (77% α epimer and 23% β epimer) inhibits
1
ratio were established by H NMR spectra. Due to the relative
8
7
14
Δ -Δ -sterol isomerase and Δ -sterol reductase of fungi as
morpholine inhibitors did. In addition, this epimeric mixture
is fungicidal and active against Saccharomyces cerevisiae
resistant strains. Then, 7α and 7β-aminocholesterol were then
selectively synthesized. According to in vitro bioassay studies
instability of the bromide product, the crude product was used
to next step without further purification. The crude bromide
mixture 3 was substituted by malononitrile in the presence of
sodium hydride in THF. The nitrile mixture (4a and 4b) was
easily separated by chromatography. Each epimer 4a and 4b
Scheme 1. Structure of oxysterols and nitrogenous sterols.

M.-A. Bazin et al. / European Journal of Medicinal Chemistry 41 (2006) 1109–1116
1111
Scheme 2. Synthesis of 5a and 5b.
was saponified by potassium hydroxide in methanol to give 5a
and 5b.
showed a mixture of 24-methylenecholesterol, cholesterol and
sitosterol (93:4:3 ratio). (24R,S)-24-hydroxy-24-methylcholes-
terol 11 was prepared from 24-methylenecholesterol (rare
sterol) by hydroxymercuration. P. variabilis lipidic extract
was used as the starting material which was treated with a mer-
curic acetate excess in Brown’s conditions (THF/water, 5:5)
7
N-hydroxyiminocholesterol 6 was obtained by saponifica-
tion of 7N-hydroxyiminocholesteryl-3β-acetate which was
synthesized according to previously described procedure [26].
2
2-Ketocholesterol 9 was prepared from 3β-acetoxy-23,24-bis-
nor-5-cholenic acid according to the published procedure [31].
6(R,S),26-dihydroxycholesterol 10 was prepared from
followed by hydrodemercuration with NaBH /NaOH. After
4
extraction, it was markedly separated by elution (cyclohex-
ane/ethyl acetate, 5:5) on silica gel column. Unreacted sterol
1
diosgenin as the starting material, allowing access to sterols
containing substituents at the 16 and 26 positions using the
established literature method (Scheme 3) [34].
(cholesterol) was eluted first, followed by the hydroxysterol
11 (Scheme 4). Analysis was based on comparison by gas
chromatography of the initial sterols mixture with unreacted
sterol. Structures of the resulting hydroxylated compounds
Compounds 11 and 12 were obtained from a marine inver-
tebrate, Palythoa variabilis (genus Cnidaria). Lipidic extract of
this Cnidaria was analyzed by Diop et al. [33] CPG analysis
1
13
were established by NMR ( H, C and DEPT), mass spectro-
metry and IR.
The 24-methylenecholesterol 12 was isolated from
P. variabilis lipidic extract by hydroxymercuration–desoxymer-
curation. The lipidic extract was treated with a mercuric acetate
excess in THF/water (5:5). The organomercurial compound
was easily separated by flash chromatography on silica gel col-
Scheme 3. Synthesis of compound 10 from diosgenin.
Scheme 4. Synthesis of (24R,S)-24-hydroxy-24-methylcholesterol 11 and isolation of 24-methylenecholesterol 12.

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M.-A. Bazin et al. / European Journal of Medicinal Chemistry 41 (2006) 1109–1116
umn and the reactive alkene was regenerated by treatment in a
biphasic system (ethyl acetate/1 M HCl, 2:1) (Scheme 4).
A high-yielding directed chemoselective hydroxymercura-
tion was achieved on methylenic sterol. These conditions
leave intact the ring double bonds of steroids. No isomerization
of double bond was thus observed in these experimental con-
ditions.
supply of cholesterol from the external medium for its own
sterol synthesis, the activity observed against promastigotes
suggests that oxysterols and nitrogenous sterols easily pene-
trate the parasite provoking the growth inhibition and cell
death.
In our work, two sterol families have been studied: the first
is nitrogenous sterols containing amino, N-hydroxyimino or
cyano group and the second is oxysterols such as ketosterol
and hydroxysterols (Tables 1 and 2).
2
.2. Biology
The most active compounds against Leishmania promasti-
gotes were aminosterols 7, 8a and 8b with IC50 values in a
range from 1.2 to 3.6 μM. 7N-hydroxyiminosterol 6 showed
a comparable activity such as aminosterols (IC50 = 3.7 μM).
Cyanosterols 1a, 1b, 5a and 5b showed a low activity
(IC50 = 26.3–73.2 μM). Aminosterols 7, 8a and 8b have been
These series exhibited a strong activity against Leishmania
whereas the activity against Trypanosoma was slight. The
activities observed on the promastigote form of two
L. donovani strains (LV9 and DD8) were similar. In addition,
a similar activity was observed against a L. donovani DD8
AmB-R clone. This clone is resistant to AmB, a drug having
sterols as the main membrane target [32]. The membranes of
the L. donovani DD8 AmB-R clones lack of 24-methylated
sterols such as ergosterol. This change in the membrane com-
position had no effect on the oxy- and nitrogenous sterols antil-
eishmanial properties. Although Leishmania sp. cannot use the
8
7
evaluated as antifungal agents and inhibit Δ -Δ -sterol isomer-
1
4
ase and Δ -sterol reductase as morpholine inhibitors [23].
Morpholine derivatives contain a nitrogen protonated in biolo-
gical medium, which mimics the carbocationic high energy
8
7
intermediates (HEI) involved in the Δ -Δ -sterol isomerase and
1
4
8
7
Δ -reductase. The Δ -Δ -isomerase reaction is conducted with
Table 1
In vitro antileishmanial and trypanocidal activities of oxysterols and nitrogenous sterols
Compounds
L. donovani
LV9 promastigotes
IC50 (μM ± S.D.)
73.2 ± 6.8
76.3 ± 5.3
62.6 ± 4.1
26.3 ± 2.8
3.7 ± 0.4
1.2 ± 0.2
3.6 ± 0.5
1.4 ± 0.2
4.4 ± 0.4
L. donovani
DD8 WT promastigotes
IC50 (μM ± S.D.)
42.2 ± 5.6
47.3 ± 5.9
> 100
36.8 ± 3.9
3.5 ± 0.4
3.2 ± 0.3
3.6 ± 0.4
1.8 ± 0.2
4.2 ± 0.3
4.7 ± 0.3
L. donovani
DD8 AmB-R promastigotes
IC50 (μM ± S.D.)
35.8 ± 4.1
40.7 ± 3.6
75.7 ± 8.2
27.8 ± 3.2
2.9 ± 0.3
2.2 ± 0.3
3.2 ± 0.4
1.2 ± 0.1
1.6 ± 0.1
T. brucei
MEC (μM)
1
1
5
5
6
7
8
8
9
1
a
b
a
b
100
100
> 100
100
50
100
100
100
100
25
a
b
0
4.3 ± 0.5
2.4 ± 0.2
1
1
4.2 ± 0.2
4.2 ± 0.3
4.5 ± 0.5
4.2 ± 0.4
3.0 ± 0.2
3.1 ± 0.3
12.5
50
12
AmB
0.2 ± 0.1
0.1 ± 0.1
2.2 ± 0.3
/
Pentamidine
2.8 ± 0.3
7.1 ± 0.8
8.6 ± 0.9
2.1 ± 0.3
Results are the mean of three independent experiments ± S.D. Results are expressed in IC50 after a 72 h incubation period for L. donovani promastigote lines
L. donovani LV9, L. donovani DD8 WT and L. donovani DD8 AmB-R). Results are expressed in MEC for T. brucei brucei GVR 35 trypomastigote forms.
(
Table 2
In vitro antileishmanial activity of compounds 7, 8a and 8b against intramacrophage amastigotes of L. donovani LV9
Compounds
Concentration
Mean number and
S.D. of amastigotes/ amastigotes/100
infected macrophage macrophage nuclei
Mean number of
Macrophage
infection (%)
Reduction in number IC50 (μM)
of amastigotes/100
nuclei (%)
–
1
(
μg ml
)
(μM)
7
1.56
3.88
1.94
0.97
0.49
6.00
3.00
6.00
3.00
1.50
0.75
/
/
/
/
/
1.31 (1.20–1.57)
(r = 0.982)
0
0
0
.78
.39
.20
5.24 ± 3.16
8.82 ± 4.80
13.20 ± 7.40
/
12.50 ± 7.20
/
1.25 ± 0.50
6.80 ± 2.61
9.51 ± 5.73
12.50 ± 5.12
208
405
515
/
485
/
40
46
52
/
52
/
16
27
49
53
59
20
0
/
4
8
a
2.50
.25
2.50
ND
1
8
b
/
1.37 (1.17–1.61)
(r = 0.987)
1
0
0
/
.25
.63
.31
20
96
64
8
184
465
660
Untreated control
0
/
Compound 7: toxic on macrophages at 3.88 μM. Compounds 8a and 8b: toxic on macrophages at 6 μM. IC50 of AmB, as reference compound was 1.05
0.87–1.25) r = 0.986.
(

M.-A. Bazin et al. / European Journal of Medicinal Chemistry 41 (2006) 1109–1116
13
1113
1
the initial addition of a proton of C-9 giving a stabilized carbo-
nium ion at C-8 [35]. Amine function at C-7 protonated as
ammonium in physiological media, is specially a better
mimic. Moreover, the basic nitrogen is necessary of protona-
tion and stronger interaction with the enzyme. However,
Δ -Δ -sterol isomerase and Δ -sterol reductase have not yet
been described in Leishmania. Oxysterols 9, 10, 11 and 12
have comparable activities (IC50 = 4.2–4.4 μM). No difference
activity has been shown between these hydroxysterols.
H NMR and C NMR spectra were recorded using CDCl3,
respectively, at 400 MHz (Jeol Lambda 400 spectrometer) and
at 100 MHz. Chemical shifts are reported relative to TMS; J
1
3
1
values are given in Hz. C NMR spectra are H-decoupled.
Compounds 1a, 1b, 7, 8a and 8b were prepared according
to our literature procedure [29].
8
7
14
16(R,S),26-dihydroxycholesterol 10 was obtained according
to the literature procedure starting from diosgenin [4].
Among the three compounds (7, 8a and 8b) tried on the
intramacrophage amastigotes of L. donovani LV9, two of
them (7 and 8b) exhibited an IC50 value of 1.3 μM. Com-
pounds 7, 8a and 8b were responsible for a change in macro-
phage shape at 3.88, 6 and 6 μM, respectively, suggesting a
slight toxicity for macrophages at these concentrations. Despite
this low therapeutic index (1.3/3.88 and 1.3/6) such an activity
similar to those of AmB justifies further in vivo evaluation of
these compounds on the L. donovani/Balb/c mouse model.
Against the bloodstream forms of T. brucei, the most active
compound was compound 11 with a MEC value of 12.5 μM.
Compounds 10, 6 and 12 exhibited MEC values in a range
from 25 to 50 μM. All other compounds, except inactive com-
pound 5a, were active at 100 μM. There is no positive correla-
tion between antileishmanial and trypanocidal properties. Such
results are not encouraging enough to perform further in vivo
evaluation on the T. brucei/mouse model.
4
.1.1. 7α,β-Bromocholesteryl 3β-acetate 3
Cholesteryl-3β-acetate 2 (6 g, 13.9 mmol) was dissolved in
heptane (50 ml) at 50 °C. N-bromosuccinimide (3 g,
6.8 mmol) was added and the solution was irradiated with
1
UV lamp for 15 min (yellow solution). The solution was
cooled to 0 °C, filtered and evaporated. The sample crude pro-
duct (flash chromatography on alumina (cyclohexane/ethyl
acetate, 9:1)) was purified for characterization by H NMR
spectra. The crude product was used to next step without pur-
ification.
1
−1
IR (KBr) υ (cm ): 2946–2868 (C–H alkane), 1732 (C=O
1
ester), 738 (C–Br). H NMR (CDCl , 400 MHz, 25 °C), δ:
3
0
2
.70 (s, 3 H, 18-Me), 0.87 (d, J = 6.5 Hz, 6 H, 26-Me and
7-Me), 0.93 (d, J = 6.5 Hz, 3 H,21-Me), 1.06 (s, 3 H, 19-
Me), 2.03 (s, 3 H, CH COO–), 4.60 (m, 1 H, 3-H), 4.66 (dd,
J7α-8 = 8.0 Hz, J7α-6 = 2.1 Hz, 0.3 H, 7α-H of β epimer), 4.69
3
(
dd, J7β-8 = 5.0 Hz, J7β-6 = 5.0 Hz, 0.7 H, 7β-H of α epimer),
.62 (d, J6-7α = 2.1 Hz, 0.3 H, 6-H of β epimer), 5.80 (d,
J6-7β = 5.2 Hz, 0.7 H, 6-H of α epimer).
5
3
. Conclusion
These series of 12 oxysterols and nitrogenous sterols were
4.1.2. 7α-Dicyanomethylcholesteryl 3β-acetate 4a and 7β-
successfully prepared. The aim of this study was to design
more active and selective compounds. Aminosterols exhibited
a strong antileishmanial activity both against promastigote and
intramacrophagic amastigotes of L. donovani. Such interesting
in vitro results prompt us to carry out in vivo evaluation
against L. donovani.
dicyanomethylcholesteryl 3β-acetate 4b
A solution of malononitrile (1.018 g, 13.9 mmol) in dry
THF (5 ml) was added to a solution of sodium hydride
(0.6 g, 15.4 mmol) in dry THF (40 ml). The solution was stir-
red for 30 min at room temperature under argon atmosphere.
The mixture was then refluxed for 1 h, cooled to 0 °C and 3
(6.5 g as crude product) dissolved in THF (7 ml) was added
4
. Experimental
dropwise. The mixture was stirred for 48 h at room tempera-
ture. The solution was diluted with water (30 ml) and extracted
with dichloromethane (3 × 60 ml). The organic layer was
4
.1. Chemistry
washed with 1 M HCl (10 ml), 5% NaHCO (10 ml), water
3
(
2 × 10 ml), and dried over anhydrous sodium sulfate. The
All solvents were distilled and dried prior to use. Reagents
solution was evaporated under reduced pressure and the crude
product was purified by chromatography on silica gel column
and materials were obtained from commercial suppliers and
were used without further purification. The reactions were
monitored by TLC on Kieselgel-G (Merck Si 254 F) layers
0.25 mm thick). The spots were detected by upon spraying
with sulfuric acid/ethanol (2:8) and heating. Column chroma-
(cyclohexane/dichloromethane, 3:7) to afford compounds 4a (α
epimer, 2.24 g, 34%) and 4b (β epimer, 0.96 g, 14%) as yellow
solids.
(
tography was carried out using silica gel 60 (0.063–0.2 mm)
(
Merck). Melting points were determined on a Kofler block
4.1.3. 7α-Dicyanomethylcholesteryl-3β-acetate 4a
[α] = –30° (C = 1 in CHCl ). IR (KBr) υ (cm ): 2949–
D 3
2870 (C–H alkane), 2215 (C≡N), 1736 (C=O ester). H NMR
−
1
and are uncorrected. IR spectra were recorded on a Perkin–
Elmer 1600 FT-IR spectrometer. Specific rotation was mea-
sured in chloroform with a Perkin–Elmer 343 polarimeter.
Mass spectra were recorded in positive mode on a Finnigan
MAT 95 S spectrometer using electrospray ionization and EI
mass spectra were recorded on a Jeol-GCmate (GC–MS sys-
tem) spectrometer with ionization energy from 30 to 40 eV.
1
(400 MHz, CDCl , 25 °C), δ: 0.71 (s, 3 H, 18-Me), 0.86 (dd,
3
J = 6.5 Hz, J = 2.0 Hz, 6 H, 26-Me and 27-Me), 0.92 (d,
J = 6.5 Hz, 3 H, 21-Me), 1.08 (s, 3 H, 19-Me), 2.05 (s, 3 H,
CH COO–), 2.46 (m, 1H, 7-H), 3.92 (d, J
= 2.7 Hz, 1
= 4.8 Hz,
6-7β
3
Hmalon.-7β
H, –CH(CN) ), 4.72 (m, 1 H, 3-H), 5.65 (dd, J
2

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M.-A. Bazin et al. / European Journal of Medicinal Chemistry 41 (2006) 1109–1116
1
3
J6-4 = 2 Hz, 1 H, 6-H). C NMR (100 MHz, CDCl , 25 °C), δ:
21.3, 22.5, 22.8, 23.8, 26.5, 26.8, 28.0, 28.3, 31.3, 35.5, 35.6,
36.0, 36.1, 36.7, 39.4, 39.5, 42.0, 43.6, 43.7, 49.9, 55.1, 56.4,
70.9, 111.8, 112.7, 117.7, 148.9. FABMS: m/z (%) = ESIMS:
3
1
2
4
2.0, 18.7, 19.4, 21.2, 21.3, 22.5, 22.7, 23.8, 26.4, 26.9, 27.5,
8.0, 28.3, 35.4, 35.6, 36.0, 36.1, 36.4, 37.4, 39.4, 39.4, 43.6,
3.6, 49.9, 55.1, 56.3, 72.8, 111.8, 112.6, 118.5, 147.8, 170.4.
+
m/z = 473.4 [M + Na] .
+
ESIMS: m/z = 515.4 [M + Na] .
4.1.7. N-Hydroxyiminocholesterol 6
4
.1.4. 7β-Dicyanomethylcholesteryl-3β-acetate 4b
7N-(Hydroxy)iminocholesteryl 3β-acetate was obtained to
according procedure [26].
−1
[
α] = +30° (C = 1 in CHCl )IR (KBr) υ (cm ): 2942–2868
D
3
1
(
(
C–H alkane), 2254 (C≡N), 1728 (C=O ester). H NMR
400 MHz, CDCl , 25 °C), δ: 0.72 (s, 3 H, 18-Me), 0.86 (dd,
This oxime (1.2 g, 2.63 mmol) and potassium hydroxide
(0.44 g, 7.89 mmol) were stirred in ethanol (20 ml) for 10 h.
The solution was neutralized (1 M HCl), concentrated and
diluted with water. The mixture was extracted with dichloro-
methane (3 × 20 ml). The organic layer was washed with
water, dried over anhydrous sodium sulfate and evaporated.
The residue 6 was crystallized from acetone (0.582 g, 80%).
3
J = 6.5 Hz, J = 2.0 Hz, 6 H, 26-Me and 27-Me), 0.92 (d,
J = 6.5 Hz, 3 H, 21-Me), 1.09 (s, 3 H, 19-Me), 2.05 (s, 3 H,
CH COO–), 2.43 (m, 1H, 7-H), 4.08 (d, J
H, –CH(CN) ), 4.62 (m, 1 H, 3-H), 5.43 (dd, J
J6-4 = 2.0 Hz, 6-H). C NMR (100 MHz, CDCl , 25 °C), δ:
1
2
4
= 3.2 Hz, 1
= 2.2 Hz,
6-7α
3
Hmalon.-7α
2
1
3
3
−1
2.2, 18.7, 19.5, 21.3, 21.3, 22.5, 22.8, 23.8, 26.5, 26.9, 27.6,
8.0, 28.3, 35.5, 35.6, 36.1, 36.2, 36.5, 37.9, 39.4, 39.5, 43.6,
IR (KBr) υ (cm ): 3452 and 3282 (O–H oxime and alcohol),
1
2944–2870 (C–H alkane), 1646 (C=N oxime). H NMR
3.7, 49.9, 55.3, 56.4, 72.9, 111.9, 112.7, 118.6, 148.0, 170.5.
(400 MHz, CDCl , 25 °C), δ: 0.70 (s, 3 H, 18-Me), 0.86 (dd,
3
+
MS (30 eV, EI): m/z (%) = 432 (13) [M –AcOH], 367 (100)
J = 6.6 Hz, J = 1.5 Hz, 6 H, 26-Me and 27-Me), 0.92 (d,
J = 6.5 Hz, 3 H, 21-Me), 1.13 (s, 3 H, 19-Me), 3.64 (m, 1 H,
+
[
M – (AcOH + CH(CN) )], 145 (66), 109 (28), 81 (80).
2
3
-H), 6.54 (d, J6-4 = 2.0 Hz, 1 H, 6-H), 6.77 (br s, 1 H, N–OH).
1
3
C NMR (100 MHz, CDCl , 25 °C), δ: 12.1, 18.0, 18.9, 20.8,
3
4
.1.5. 7α-Dicyanomethylcholesterol 5a
2
3
1
2.6, 22.8, 23.8, 27.2, 28.0, 28.3, 31.3, 35.6, 36.2, 36.6, 38.0,
8.4, 38.6, 39.5, 42.2, 42.8, 49.7, 50.2, 54.7, 71.1, 112.7,
Compound 4a (1.1 g, 2.23 mmol) was added to a solution
of potassium hydroxide (0.38 g, 6.69 mmol) in methanol
20 ml) and the mixture was stirred for 24 h at room tempera-
+
53.5, 158.2. MS (30 eV, EI): m/z (%) = 415 (36) [M ], 400
(
+
+
(100) [M – Me], 398 (53) [M – OH], 381 (14), 186 (19).
ture. The solution was acidified (pH 6), diluted with water and
extracted with dichloromethane (3 × 20 ml). The organic layer
+
ESIMS: m/z = 438.4 [M + Na] .
was washed with 5% NaHCO , water and dried over anhy-
3
drous sodium sulfate. The solution was evaporated and the
crude product was purified by chromatography (cyclohexane/
ethyl acetate, 8:2) to afford 0.5 g (50%) of nitrile 5a as a white
solid. M.p. 163 °C. [α] = –50° (C = 0.1 in CHCl ). IR (KBr) υ
4.1.8. (24R,S)-24-hydroxy-24-methylcholesterol 11 and 24-
methylenecholesterol 12
Lipidic extract: P. variabilis was crushed (800 g) and
refluxed in a methanol/dichloromethane mixture (ratio 1:2)
for 48 h. The mixture was filtered and the solution was washed
with water, dried under anhydrous sodium sulfate and evapo-
rated to give 5.54 g of extract. The crude product (5.54 g) was
then treated with potassium hydroxide (3 g) in ethanol (50 ml)
and refluxed for 2 h. The solution was acidified (1 M HCl) and
extracted with dichloromethane. The organic layer was washed
with water, dried over anhydrous sodium sulfate and evapo-
rated to give 1.6 g of saponified lipidic mixture. GC analytical
(operating conditions: column temperature: 240 °C; detector
temperature: 320 °C; injector temperature: 300 °C; capillary
column (BPX-35)) showed the mixture of 24-
methylenecholesterol and cholesterol (94.5:4.5 ratio). Relative
retention time/cholesterol (RRT): cholesterol: 1 and 24-
methylenecholesterol: 1.42. No sitosterol was detected.
D
3
−1
(
(
cm ): 3292 (O–H alcohol), 2932–2869 (C–H alkane), 2253
1
C≡N). H NMR (400 MHz, CDCl , 25 °C), δ: 0.70 (s, 3 H,
3
1
8-Me), 0.86 (dd, J = 6.5 Hz, J = 2.0 Hz, 6 H, 26-Me and 27-
Me), 0.93 (d, J = 6.5 Hz, 3 H, 21-Me), 1.04 (s, 3 H, 19-Me),
.39 (m, 1 H, 7β-H), 3.69 (m, 1 H, 3-H), 3.91 (d,
JHmalon.-7β = 2.7 Hz, H, –CH(CN) ), 5.63 (dd,
2
1
2
1
3
J6-7β = 5.0 Hz, J6-4 = 1.7 Hz, 1 H, 6-H). C NMR (100 MHz,
CDCl , 25 °C), δ: 11.7, 18.7, 19.1, 19.2, 20.8, 21.3, 22.5, 23.6,
3
2
4
1
4.3, 24.6, 26.8, 27.9, 31.1, 34.6, 35.5, 36.8, 37.2, 38.8, 39.7,
2.3, 42.4, 42.9, 49.9, 55.5, 56.4, 71.3, 111.8, 113.0, 117.6,
49.0. ESIMS: m/z = 473.4 [M + Na] .
+
4
.1.6. 7β-Dicyanomethylcholesterol 5b
Nitrile 4b (0.5 g, 1.02 mmol) was saponified in the same
manner as 4a to afford 5b (0.35 g, 80%). M.p. 174 °C.
−
1
4
.1.9. (24R,S)-24-hydroxy-24-methylcholesterol 11
[
(
α] = +50° (C = 0.1 in CHCl ). IR (KBr) υ (cm ): 3420
D 3
O–H alcohol), 2950–2868 (C–H alkane), 2254 (C≡N). H
1
Mercuric acetate (2.99 g) in water (15 ml) was added to a
NMR (400 MHz, CDCl , 25 °C), δ: 0.72 (s, 3 H, 18-Me),
stirred solution of lipidic crude extract (0.6 g) in tetrahydro-
furan (15 ml) at room temperature and stirred for 12 h. The
solution was treated with 3 M aqueous sodium hydroxide
(10 ml) and with 0.5 M aqueous sodium borohydride (10 ml)
and stirred for 30 min at room temperature. The solution was
filtered and the solvent was concentrated and extracted with
3
0
0
.86 (dd, J = 6.5 Hz, J = 2.0 Hz, 6 H, 26-Me and 27-Me),
.92 (d, J = 6.3 Hz, 3 H, 21-Me), 1.08 (s, 3 H, 19-Me), 2.35
(
m, 1H, 7-H), 3.56 (m, 1 H, 3-H), 4.09 (d, JHmalon.-7α = 2.9 Hz,
1
6
H, –CH(CN) ), 5.40 (dd, J
-H). C NMR (100 MHz, CDCl , 25 °C), δ: 12.0, 18.7, 19.5,
= 2.2 Hz, J6-4 = 2.2 Hz, 1 H,
6-7α
2
13
3

M.-A. Bazin et al. / European Journal of Medicinal Chemistry 41 (2006) 1109–1116
1115
chloroform. The two compounds were separated by column
chromatography (eluent: cyclohexane/ethyl acetate, 9:1) to
give 0.55 g of (24R,S)-24-hydroxy-24-methylcholesterol 11
as epimeric mixture and 0.019 g of cholesterol. IR (KBr) υ
medium enriched with 10% heat-inactivated fetal calf serum
(hi-FCS) and 50 μg ml gentamycin at 27 °C in a dark envir-
onment.
–
1
−1
1
(
cm ): 3367 (O–H alcohol), 2964–2866 (C–H alkane). H
4.2.2. In vitro evaluation on L. donovani promastigote forms
NMR (400 MHz, CDCl , 25 °C), δ: 0.68 and 0.69 (2 × s,
3
The antileishmanial screening was performed in flat-
2
× 3 H, 18-Me), 0.89 and 0.90 (2 × d, J = 6.9 Hz and
bottomed 96-well plastic tissue-culture trays maintained at
7 °C in an atmosphere of 95% air/5% CO . Promastigote
J = 7.0 Hz, 2 × 3 H, 21-Me), 0.92 and 0.93 (2 × d, J = 6.9 Hz
and J = 6.5 Hz, 2 × 3 H, 26-Me and 27-Me), 1.00 and 1.02
2
2
forms from a logarithmic phase culture were suspended to
(
2 × s, 2 × 3 H, 19-Me), 1.07 and 1.09 (2 × s, 2 × 3 H, 28-
6
1
3
yield 10 cells per ml after hemocytometer counting. Each
Me), 3.52 (m, 2 H, 3-H), 5.36 (s, 2 H, 6-H). C NMR
100 MHz, CDCl , 25 °C), δ : 11.8, 16.9, 16.9, 17.5, 17.5,
well was filled with 100 μl of the parasite suspension, and
plates were incubated at 27 °C for 1 h before drug addition.
The compounds to be tested were dissolved in DMSO and
then added to each well to obtain the final concentration of
100 μM and further concentrations were twice diluted. At up
to 2% (v/v), DMSO had no effect on parasite growth. Each
concentration was screened in triplicate. The viability of pro-
mastigotes was checked using the tetrazolium-dye (MTT) col-
orimetric method. The MTT cell proliferation assay is a colori-
metric assay system, which measures the reduction of a
tetrazolium component (MTT) into an insoluble formazan pro-
duct by the mitochondria of viable cells. After incubation of
the cells with the MTT reagent, a detergent solution was
added to lyse cells and solubilize the colored crystals. The sam-
ples were read using an ELISA plate reader at a wavelength of
(
3
1
3
7
3
8.8, 19.4, 21.1, 23.3, 24.3, 28.2, 29.2, 29.2, 31.6, 31.9,
6.1, 36.5, 36.6, 37.2, 39.7, 42.3, 50.1, 55.9, 56.7, 71.8,
+
4.8, 121.6, 140.7. MS (30 eV, EI): m/z (%) = 416 (12) [M ],
+
98 (28) [M – H O], 373 (31), 314 (100), 271 (39). ESIMS:
2
+
m/z = 439.5 [M + Na] .
4
.1.10. 2.4. Methylenecholesterol 12
Mercuric acetate (1.5 g) in water (10 ml) was added to a
stirred solution of lipidic crude extract (0.5 g) in tetrahydro-
furan (10 ml) at room temperature and stirred for 12 h. The
solution was filtered, diluted with water (30 ml) and extracted
with dichloromethane (3 × 15 ml). The crude product was pur-
ified by flash chromatography (cyclohexane/ethyl acetate, 8:2
and ethyl acetate/acetic acid, 9.4:0.6). The organomercurial in
ethyl acetate (20 ml) was treated by 1 M HCl (10 ml) and
stirred for 2 h at room temperature. The solution was diluted
with water and extracted with dichloromethane. The organic
layer was washed with brine, dried over anhydrous sodium
sulfate and evaporated. The residue was purified by chromato-
graphy on silica gel column (cyclohexane/ethyl acetate, 8:2)
afforded product 12 (0.43 g, 91%) as a white solid. M.p.
570 nm. The amount of color produced was directly propor-
tional to the number of viable cells. The results are expressed
as the concentrations inhibiting parasite growth by 50% (IC )
5
0
after a 3-day incubation period. AmB and pentamidine were
used as antileishmanial reference compounds.
4.2.3. In vitro evaluation on intramacrophage amastigotes
−
1
1
(
41 °C. IR (KBr) υ (cm ): 3435 (O–H alcohol), 2928–2854
Concerning the amastigote in vitro model, peritoneal macro-
1
C–H alkane). H NMR (400 MHz, CDCl , 25 °C), δ: 0.68 (s,
phages were harvested from female CD1 mice (Charles River,
Cléon, France) 3 days after an intraperitoneal injection of
1.5 ml of sodium thioglycolate (Biomérieux) and were dis-
pensed into eight-well chamber slides (LabTek Ltd.) at a con-
3
3
1
1
H, 18-Me), 0.94 (d, J = 6.6 Hz, 3 H, 21-Me), 1.01 (s, 3 H,
9-Me), 1.02 (d, J = 6.5 Hz, 6 H, 26-Me and 27-Me), 3.52 (m,
H, 3-H), 4.65 and 4.71 (d, 2H, J = 1.28, =CH ), 5.34 (d,
2
1
3
4
J6-7β = 5.1 Hz, 1 H, 6-H). C NMR (100 MHz, CDCl3,
centration of 5 × 10 per well (400 μl per well) in RPMI 1640
2
3
5
5 °C), δ: 11.8, 18.7, 19.4, 21.8, 22.0, 22.7, 24.3, 28.2, 29.7,
1.0, 31.6, 31.9, 33.8, 34.7, 35.7, 36.5, 37.2, 39.8, 42.2, 42.3,
0.1, 56.0, 56.7, 71.8, 105.9, 121.7, 140.7, 156.8. MS (30 eV,
medium supplemented with 10% hi-FCS, 25 mM HEPES, and
2 mM L-glutamine (Life Technologies, Cergy-Pontoise,
France). Four hours after the macrophages were plated, they
were washed in order to eliminate fibroblasts. After a 24 h-
incubation period, the macrophages were infected with pro-
mastigote forms of L. donovani LV9 in a stationary phase at
a ratio of 10 parasites per macrophage, to obtain 87% of
infected macrophages and 10 ± 3 amastigotes per macrophage.
At 18 h after the promastigotes had entered macrophages, the
free promastigotes were eliminated and intramacrophagic
amastigotes were treated at various concentrations of the com-
pounds. Pentamidine was used as reference compound. The
culture medium was renewed 48 h later and a new culture med-
ium containing the drug was added. The experiment was
stopped at day 5, and the percentage of infected macrophages
was evaluated microscopically after Giemsa staining. The 50%
inhibitory concentrations (IC ) were determined by linear
+
+
EI): m/z (%) = 398.5 (3) [M ], 314.4 (28) [M –
CHC(CH )CH(CH ) ], 256.3 (48), 129.1 (71), 97.1 (79), 73.9
2
3 2
(
100). Anal. Calcd for C H O: C 84.36; H 11.63. Found: C
28 46
8
4.39; H 11.67.
4
4
.2. Antileishmanial evaluation
.2.1. Parasites
Three strains of L. donovani were used in this study:
L. donovani (MHOM/ET/67/HU3) called LV9 and two strains
of L. donovani (MHOM/IN/80/DD8) promastigotes called
wild-type (WT) and AmB-resistant line (AmB-R). Promasti-
gotes were cultured in HEPES (25 mM)-buffered RPMI 1640
5
0

1116
M.-A. Bazin et al. / European Journal of Medicinal Chemistry 41 (2006) 1109–1116
regression analysis, and expressed in μM ± S.D. Each experi-
ment was performed in triplicate.
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