Oxysterols: Synthesis and anti-leishmanial activities

Article abstract of DOI:10.1016/j.steroids.2015.12.020

Oxygenated sterols (2-16) were synthesized by skeletal rearrangement of steroidal allylic alcohols. All the derivatives were screened for their anti-leishmanial activities. Compounds 3, 11 and 12 showed potent activities. Compound 12 was found least toxic and induced highest nitric oxide (NO) at 48 h. Least toxicity of compound 12 on splenocytes validated its best anti-amastigote effect and induction of NO.

Full text of DOI:10.1016/j.steroids.2015.12.020

Steroids 107 (2016) 65–73
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Oxysterols: Synthesis and anti-leishmanial activities
a
a
b
b
b
Pranab Ghosh a, , Ashim Ghosh , Amitava Mandal , Sirin Salma Sultana , Somaditya Dey , Chiranjib Pal
⇑
a Natural Products and Polymer Chemistry Laboratory, Department of Chemistry, University of North Bengal, Raja Rammohanpur, Darjeeling 734 013, India
b Cellular Immunology and Experimental Therapeutics Laboratory, Department of Zoology, West Bengal State University, Barasat, North 24 PGS, West Bengal 700 126, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 April 2015
Received in revised form 7 December 2015
Accepted 25 December 2015
Available online 29 December 2015
Oxygenated sterols (2–16) were synthesized by skeletal rearrangement of steroidal allylic alcohols. All
the derivatives were screened for their anti-leishmanial activities. Compounds 3, 11 and 12 showed
potent activities. Compound 12 was found least toxic and induced highest nitric oxide (NO) at 48 h.
Least toxicity of compound 12 on splenocytes validated its best anti-amastigote effect and induction of
NO.
Ó 2015 Elsevier Inc. All rights reserved.
Keywords:
Oxygenated sterols
Anti-leishmanial activities
Cytotoxicity
A-nor-cholest-6-hydroxy-5,7-olide
1. Introduction
with important foci of infection in Central and South America,
Southern Europe, North and East Africa, the Middle East and the
Sterols are essential for life in mammals (as well as in lower
eukaryotes such as yeast and fungi) and have a central role in
numerous physiological processes. Oxysterols including both
autoxidation products and sterol metabolites have many impor-
tant biological activities mostly related to the physiological control
Indian subcontinent. In recent times the main foci of VL are in
Sudan and India [13].
Today some antimony containing drugs are generally used to
treat Leishmania. The toxicity of the available drugs and the emer-
gence of strains that are not responsive to drug therapy make the
discovery of novel therapeutic agents imperative [14]. Recently
miltefosine (hexadecylphosphocholine or HPC), originally devel-
oped as an anticancer agent, has been introduced as an effective
anti-leishmanial drug. However, miltefosine-associated gastroin-
testinal toxicity and teratogenicity have already been identified
during clinical trials in India. The long half-life of the drug might
also encourage the emergence of resistant lines, as evidenced from
selected miltefosine resistant line of Leishmania donovani and
Leishmania tropica in vitro, is also another matter of concern [15].
Moreover, cases indicating the relapse of the disease, even after
10 months of a full course of treatment with miltefosine [16]
prompted researchers to search for novel molecules active against
the Leishmania infection. Therefore, development of new
chemotherapeutics with lower toxicity and higher efficiency is
the high demand in contemporary medicinal and pharmaceutical
sciences. In the present study synthesis of some oxygenated
cholesterol derivatives 1–16 along with their anti-leishmanial
activities were evaluated and are being reported here. On prolifer-
ation of HSCs-T6, compound 3 showed the prominent inhibitory
of cholesterol biosynthesis [1]. 7a-Hydroxy-cholest-4-ene-3-one is
an intermediate in the conversion of cholesterol into bile acids [2].
Some of the oxysterol derivatives might be used as markers to
physiological efficiencies [2] and display many biological activities
in normal and diseased states. Derivatives of cholesterol
oxygenated in the 7, 20, 22 or 25 positions depress the activity of
the regulatory enzyme in the sterol synthetic pathway,
3-hydroxy-3-methyl glutaryl-coenzyme (HMG CoA) reductase
within 6 h [1,3]. Specific naturally occurring oxysterols have potent
osteoinductive and anti-adipogenic properties [4,5]. Parhami and
co-workers reported that some oxysterols analogs induced the
osteogenic effects and inhibit the adipogenic differentiation of
bone marrow stromal cells (MSC) through activation of Hedgehog
(Hh) signaling [6]. Researchers have also reported the cytotoxicity
[7–9], anti HIV [10] and anti-asthma [11] properties of various oxy-
genated sterol derivatives.
Leishmaniasis constitutes a group of human diseases caused by
obligate intracellular protozoan parasites of genus Leishmania. It is
the second most common parasitic disease in the modern world,
behind malaria [12]. Leishmaniasis has a worldwide distribution
activity and the LC50 value was 90.5 lg/mL [17]. Compound 12
was previously prepared from the 7-keto compound by using
NaBH4/CeCl3ÁH2O and it showed no significant antimicrobial
activity [18].
⇑
Corresponding author.
E-mail address: pizy12@yahoo.com (P. Ghosh).
http://dx.doi.org/10.1016/j.steroids.2015.12.020
0039-128X/Ó 2015 Elsevier Inc. All rights reserved.

66
P. Ghosh et al. / Steroids 107 (2016) 65–73
2.3.1. Cholest-3(4)epoxy-6-one 7
2. Experimental
White solid; 48% yield; melting point (mp): 120–121 °C; 400.32
[M+]; IR: 2939, 2866, 1714, 1654, 1465, 1379, 880, 669 cmÀ1; UV:
inactive; 1H NMR: dH 0.71 (s, 3H, H3-18), 1.12 (s, 3H, H3-19), 2.42
(m, 2H, H2-2), 4.07 (d, 1H, J = 12 Hz, H-4), 3.83 (d, 1H, J = 12 Hz,
H-3); 13C NMR: dC 212.3, 56.2, 53.7, 46.7, 44.7, 42.5, 39.9, 39.5,
38.5, 38.1, 36.1, 35.7, 35.6, 35.3, 31.7, 28.9, 28.2, 28.0, 24.2, 23.8,
22.8, 22.5, 21.4, 18.6, 12.0; Elemental analysis: C27H44O2 requires
C, 80.94%; H, 11.07%; O, 7.99%; found C, 80.96%; H, 11.04%, O,
7.95%.
Melting points were determined by the open capillary method
and are uncorrected. The NMR spectra were recorded in CDCl3
solutions at ambient temperature on a Bruker Avance 300 MHz
NMR spectrometer using 5 mm BBO (i.e. broadband observe)
probe. The chemical shifts d are given in ppm related to tetram-
ethylsilane (TMS). The coupling constants (J) are reported in Hz.
IR spectra were recorded in Shimadzu 8300 FT-IR spectrophotome-
ter in KBr discs. All solvents were purified according to reported
procedures and reagents were used as commercially available.
The mass spectra were taken in 4800 (ABSciex) MALDI-TOF/TOF
Tandem Mass Spectrometer. Elemental Analyses were done in
Vario EL-III.
2.3.2. 4-Chloro-cholest-3-ol-6-one 8
White solid; 32% yield; melting point (mp): 132–133 °C; 436.65
[M+]; IR: 3396, 2921, 1713, 1463, 1455, 1377, 1305, 1169, 973, 723,
561 cmÀ1; UV: inactive; 1H NMR: dH 0.71 (s, 3H, H3-18), 1.12 (s, 3H,
H3-19), 2.68 (dd, 2H, J = 3.8 and 15.8 Hz, H2-2); Elemental analysis:
2.1. Jone’s Oxidation of Cholesterol 1
C
27H45O2Cl requires C, 74.19%; H, 10.38%; O, 7.32%; Cl, 8.11%;
Jone’s Oxidation of Cholesterol was carried out following the
found C, 74.23%; H, 10.41%, O, 7.29%; Cl, 8.12%.
previous reports [19] and Cholest-4(5)en-3-one
2 (47%) and
Cholest-4(5)en-3,6-dione (40%) were obtained. However,
compound 3 was prepared earlier from the 5a,6b-diols by using
N-bromosuccinimide [20].
3
2.3.3. Treatment of H2O2-p-TsOH on Cholest-4(5)en-3,6-diol 6
The reaction was carried out following the same method as
described above by taking 402 mg (1 mmol) of 6 in 50 mL methy-
lene chloride. After purification over a column of silica gel (60–120
mesh, 100 g, 25 cm) it yielded two compounds. One was found
identical with cholest-3(4) epoxy-6-one, 7 (eluent 1% EA in PE)
and another as the nor lactone, 9 (eluent 2% EA in PE).
2.2. Sodium borohydride reduction of Cholest-4(5)en-3,6-dione 3
Cholest-4(5)en-3,6-dione (650 mg, 1.63 mmol) was dissolved in
a mixture of dioxane and methanol (15 mL each) was added with
cooling (0–5 °C)
a slurry of sodium borohydride (20.45 mg,
2.3.4. A-nor-cholest-6-hydroxy-5,7-olide 9
0.54 mmol) prepared in an ammonium chloride-ammonium
hydroxide buffer (pH = 8, 3 mL) and the mixture was stirred at
room temperature for 3 h. Most of the solvents were removed by
distillation, cooled and acidified with 1 M HCl and then extracted
with ether (3 Â 50 ml). The ethereal layer was washed with water
till neutral and dried by anhydrous sodium sulfate and purified
over a column of silica gel [60–120 mesh, 100 g, 25 cm, eluent 1%
ethyl acetate (EA) in pet. ether (PE)].
White solid; 45% yield; melting point (mp): 144–145 °C; IR:
3442, 2949, 2867, 1767 (c-lactone), 1735, 1461, 1440, 1375,
1252, 969 cmÀ1; UV: inactive; 1H NMR: dH 0.71 (s, 3H, H3-18),
1.12 (s, 3H, H3-19), 4.946 (m, 1H, AOH), 2.32 (q, 2H, J = 14.9 Hz,
H2-6), 2.034 (m, 2H, H2-3). Elemental analysis: C27H44O3 requires
C, 77.84%; H, 10.64%; O, 11.52%; found C, 77.80%; H, 10.61%, O,
11.49%.
2.4. Acetylation of cholesterol 1
2.2.1. Cholest-4(5)en-3-ol-6-one 5
White solid; 35% yield; melting point (mp): 142–143 °C; 400.34
[M+]; IR: 3386, 2841, 2669, 1716, 1652, 1508, 1462, 1377, 1168,
850, 723, 669 cmÀ1; UV: 232 nm (3320); 1H NMR: dH 0.71 (s, 3H,
H3-18), 1.12 (s, 3H, H3-19), 2.40 (m, 2H, H2-2); 3.21 (s, 1H, AOH
at C3), 4.31 (d, 1H, J = 9.3 Hz, H-3), 6.17 (s, 1H, H-4); 13C NMR: dC
200.1, 172.7, 119.6, 68.4, 56, 55.5, 53.7, 42.4, 41.2, 39.4, 39, 36.1,
36, 35.7, 34.1, 33.7, 28.1, 27.9, 24.1, 23.8, 22.8, 22.5, 21.0, 18.6,
18.2, 11.9. Elemental analysis: C27H44O2 requires C, 80.94%; H,
11.07%; O, 7.99%; found C, 80.98%; H, 11.01%, O, 7.92%.
Acetylation was carried out following the previous reports [22].
2.5. Oxidation of cholesteryl acetate with sodium dichromate
This reaction was performed following the literature to obtain
7-Keto cholesteryl acetate 10 [22].
2.5.1. 7-Keto cholesteryl acetate 10
White solid (85% yield, eluent 1% EA in PE); IR: 2945, 2868,
1676, 1616, 1460, 1375, 1248, 1228, 867 cmÀ1; UV: 242 nm; 1H
NMR: dH 0.68 (s, 3H, H3-18), 1.21 (s, 3H, H3-19), 0.88 (d, 3H,
J = 6.6 Hz, H3-21), 2.06 (s, 3H, H3-OAc), 4.21 (m, 1H, H-3), 5.71 (s,
1H, H-6); 13C NMR: dC 202, 170.3, 163.8, 126.7, 72.2, 54.7, 49.9,
49.7, 45.4, 43.1, 39.4, 38.6, 38.3, 37.7, 36.1, 35.9, 35.7, 28.5, 28,
27.3, 26.3, 23.8, 22.8, 22.5, 21.2, 21.1, 18.8, 17.2, 11.9.
2.2.2. Cholest-4(5)en-3,6-diol 6
The observed spectroscopic analysis agreed well with the liter-
ature data [21].
2.3. Treatment of H2O2-p-TsOH on Cholest-4(5)en-3-ol-6-one 5
To a solution of Cholest-4(5)en-3-ol-6-one (200 mg, 0.49 mmol)
in methylene chloride (50 mL) was added 40 mL of oxidizing solu-
tion prepared from 3 mL 30% H2O2 in 80 mL of t-BuOH containing
3.0 g of p-toluene sulfonic acid at room temperature. The solution
was stirred slowly for 72 h was then poured onto water. The mix-
ture was extracted with methylene chloride (3 Â 50 ml), washed
with water, aqueous sodium bicarbonate solution and then again
with water. The organic layer was dried off, evaporated and purifi-
cation by column chromatography (silica gel 60–120 mesh, 50 g,
12 cm) yielded two compounds (eluent 1% EA in PE and 2% EA in
PE).
2.6. Sodium borohydride reduction of 7-Keto cholesteryl acetate 10
Compound 10 (700 mg, 1.58 mmol) was reduced by sodium
borohydride following the method as described above.
2.6.1. 3-Acetoxy cholest-5(6)en-7-ol 11 (30%) and Cholest-5(6)en-3,7-
diol 12 (37%)
The analytical data agreed well with the literature report
[23,24].

P. Ghosh et al. / Steroids 107 (2016) 65–73
67
2.7. Treatment of H2O2-p-TsOH on 3-acetoxy cholest-4(5)en-7-ol 11
2.8.1. A-nor-3-hydroxy-3-tbutoxy-cholest-5(6)en-7-one 16
White solid; 47% yield; eluent 3% EA in PE; melting point (mp):
121–123 °C; IR: 3424, 2940, 2867, 1735, 1672, 1461, 1375, 1238,
1148, 1033, 668 cmÀ1; UV: inactive; 1H NMR: dH 0.68 (s, 3H,
H3-18), 1.21 (s, 3H, H3-19), 0.88 (d, 3H, J = 6.6 Hz, H3-21), 2.05 (s,
9H), 4.72 (m, AOH at C3), 5.22 (s, 1H, H-6); Elemental analysis:
Compound 11 (250 mg, 0.56 mmol) was treated with H2O2-p-
TsOH in methylene chloride following the method as described
above.
C
31H50O3 requires C, 79.10%; H, 10.71%; O, 10.20%; found C,
2.7.1. Cholest-7-keto-3(4),5(6)-diene 13
79.15%; H, 10.76%, O, 10.26%.
White solid; 20% yield; eluent PE; melting point (mp):
118–119 °C (Lit. 109–119) 382.45 [M+]; IR: 2945, 2868, 1674,
1616, 1463, 1377, 1331, 1265, 1229, 1191, 1020 cmÀ1. [25].
2.9. Anti-proliferative effect on parasitic protozoa L. donovani and
Leishmania major
2.7.2. Cholest-5(6)epoxy-3,7-diol 14
The cytotoxicity assay was performed on three different species
of Leishmania in three independent experiments as per the guide-
lines of biosafety committee of West Bengal State University. L.
donovani AG83 (MHOM/IN/83/AG83) was originally obtained from
Indian Institute of Chemical Biology, Kolkata, India [26]. L. donovani
LV9 (MHOM/ET/67/HU3/LV9) was obtained from Dr. Bhaskar Saha,
NCCS, Pune, India as gift. L. major LV39 (MRHO/Sv/59/P strain) was
obtained from Dr. Fabienne Tacchini-Cottier, Director, WHO-IRTC,
University of Lausanne, Switzerland as gift [27]. The parasites were
maintained in animal facility as per the guidelines of institutional
animal ethics committee of West Bengal State University.
Promastigote morphs of Leishmania sp. were transformed from
intracellular amastigotes, acquired from splenic aspirates of
infected BALB/c mice in complete M 199 medium (Invitrogen) sup-
plemented with 1% penicillin-streptomycin (Invitrogen) and 10%
FCS (GIBCO) at requisite temperature, 22 °C for L. donovani (AG83
and LV9) and at 26 °C for L. major LV39. To estimate the percentage
of inhibition the derivative, the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide (MTT) micro method was used, as
described previously [28]. Briefly, late-log-phase promastigotes
White solid; 25% yield; (eluent 1% EA in PE; melting point (mp):
114–116 °C; 418.56 [M+]; IR: 3421, 2947, 2869, 1670, 1465, 1379,
1332, 1274, 1170, 1055, 893, 665 cmÀ1; UV: inactive; 1H NMR: dH
0.68 (s, 3H, H3-18), 1.21 (s, 3H, H3-19), 0.88 (d, 3H, J = 6.6 Hz,
H3-21), 3.54 (m, 1H, H-3), 3.87 (m, 1H, H-7), 5.98 (m, 1H, H-6);
2.74 (dd, 2H, J = 9.9 and 15.6 Hz, H2-2). Elemental analysis:
C27H46O3 requires C, 77.46%; H, 11.07%; O, 11.47%; found C,
77.45%; H, 11.11%, O, 11.53%.
2.7.3. Cholest-3(5)epoxy-6,7-diol 15
White solid; 32% yield; eluent 2% EA in PE; melting point (mp):
138–139 °C; 418.48 [M+]; IR: 3423, 2935, 2868, 1718, 1652, 1460,
1380, 1261, 1170, 1053, 887, 792 cmÀ1; UV: inactive; 1H NMR: dH
0.68 (s, 3H, H3-18), 1.21 (s, 3H, H3-19), 0.88 (d, 3H, J = 6.6 Hz,
H3-21), 3.54 (m, 1H, H-6), 3.87 (m, 1H, H-7),4.15 (m, 1H, H-3);
2.74 (dd, 2H, J = 9.9 and 15.6 Hz, H2-2), Elemental analysis:
C27H45O3 requires C, 77.65%; H, 10.86%; O, 11.47%; found C,
77.59%; H, 10.92%, O, 11.53%.
were seeded in a 96-well flat-bottom plate (200 lL per well; BD
2.8. Treatment of H2O2-p-TsOH on Cholest-5(6)en-3,7-diol 12
Falcon) in complete M 199 medium in presence or absence of the
derivative. To examine the effect of all sixteen compounds (deriva-
tive 1 to 16), the cultures were additionally supplemented without
(DMSO control) or with increasing concentrations of the derivative
(0.5 lg/mL, 1 lg/mL, 2.5 lg/mL and 5 lg/mL) soluble in DMSO
(0.2% v/v). Equal volume of DMSO only was added in control exper-
Compound 12 (402 mg, 1 mmol) was treated with H2O2-p-TsOH
in methylene chloride at room temperature following the method
as described above. After purification over a column of silica gel
(60–120 mesh, 100 g, 25 cm) it yielded three compounds. First
two compounds were found identical with 13 and 14 respectively.
The third compound was identified as 16.
iments. After 72 h of incubation in requisite temperature, MTT
(10 mg/mL, 10
lL per well) was added to each well and the plates
1. Py/(CH3COO)2O
2. Na2Cr2O7/CH3COOH
benzene/reflux, 70 oC
Jone's
HO
Oxidation
r.t
O
AcO
O
3
O
10
O
2
1
NaBH4/MeOH
Ice cold
NaBH4/MeOH
Ice cold
AcO
OH
HO
OH
HO
HO
5
11
12
6
O
OH
H2O2/p-TsOH
t-BuOH, DCM
H2O2/p-TsOH
t-BuOH, DCM
H2O2/p-TsOH
t-BuOH, DCM
H2O2/p-TsOH
t-BuOH, DCM
r.t
r.t
r.t
r.t
O
13
14
HO
O
O
C
O
7
HO
O
OH
HO
Cl
8
H
O
13
O
C
O
H
7
O
14
OH
16
O
9
O
OH
OH
15
Scheme 1. Steroidal allylic rearrangement to different oxysterol derivatives.

68
P. Ghosh et al. / Steroids 107 (2016) 65–73
H+
-H2O
HO
H
OH
HO
H2O / p-TsOH
OH
6
6b
O
6a
H2O2
OH
OH
OH
O
6c
OH
OH
HO
H+, semipinacol- pinacolone type
H2O2
Oxidative
OH
lactonization
OH
O
OH
O
O
H
6d
9
Scheme 2. Mechanism for the formation of 9 from 6.
were incubated for an additional 4 h at 37 °C. The enzyme reaction
was then stopped by addition of acidic isopropanol (0.4 mL 10(N)
HCl in 100 mL isopropanol, 100 lL per well), and the absorbance
electronic pulse with a height (amplitude) proportional to the total
fluorescence emission from the cell. Thereafter, such fluorescence
data are considered as a measurement of the cellular DNA content.
For flow cytometry analysis of DNA content, exponentially grown
L. donovani promastigote cells (2 Â 106) were incubated without
was measured at 595 nm. Degree of cytotoxicity of conventional
antileishmanial drug (reference drug), sodium antimony gluconate
(SAG), was also measured on L. donovani AG83 [28] at the increas-
ing concentration. Percentage of inhibition was measured in
respect to the proliferation of Leishmania promastigotes of DMSO
control group [28]. Statistical analyses for all experiments were
performed by Student’s t test with the program Sigma Plot using
alpha adjustment.
or with IC50 concentrations of compound 3 (1
lg/mL), compound
12 (5 g/mL) and compound 11 (2.5
l
lg/mL) in complete M199
media for 48 h. Cells were then harvested and washed three times
with 1Â PBS, fixed in 45% ethanol (diluted in 1Â PBS), treated with
500 lg/mL RNAse A and then suspended in 0.5 M sodium citrate
containing 69 lM PI. These samples were analyzed through flow
cytometry (FACS Verse, Becton & Dickinson, USA) after keeping
them in the dark for 45 min [29].
2.10. Analysis of cell cycle progression
The nuclear DNA content of a cell can be quantitatively mea-
sured by flow cytometry. Initially, a fluorescent dye that binds sto-
2.11. DNA fragmentation assay by agarose gel electrophoresis
ichiometrically to the DNA is added to
a
suspension of
To determine the fragments of DNA generated during cell death,
total cellular DNA was isolated by a previously described [30] and
analyzed by agarose gel electrophoresis. Briefly, pellets of 107 pro-
mastigotes were treated with sarkosyl detergent lysis buffer
(50 mM Tris, 10 mM EDTA, 0.5% w/v sodium-N-lauryl sarcosine,
permeabilized single cells or nuclei. The principle is that the
stained material has incorporated an amount of dye proportional
to the amount of DNA. The stained material is then measured in
the flow cytometer and the emitted fluorescent signal yields an
H
AcO
O
AcO
O H
AcO
OH
11
H
-H
-H
-OAc
O
AcO
O H
13
Scheme 3. Proposed mechanism for the formation of 13.

P. Ghosh et al. / Steroids 107 (2016) 65–73
69
Fig. 1. Determination of 50% inhibitory concentration of active compounds against Leishmania promastigotes. Leishmania promastigotes were incubated without (control) or
with graded concentrations (0.5 g/mL to 5 g/mL) of compounds 3 (d), 11 (.) and 12 (s) in complete M-199 medium for 48 h. Cells were washed and incubated with MTT
l
l
(10 mg/mL) and percentage of viable Leishmania donovani AG83 (A), Leishmania donovani LV9 (B) and Leishmania major LV39 (C) were estimated as described in materials and
methods. Values represent the mean SD of three independent experiments in triplicate.
pH 7.5) and proteinase K (15.6 mg mLÀ1), vortexed and allowed to
Table 1
digest overnight at 50 °C. RNase A (0.3 mg mlÀ1) treatment was
Anti-proliferative effect of the compounds 3, 11 and 12 against Leishmania
given for 1 h at 37 °C. The lysates were then extracted with phe-
nol/chloroform/isoamyl alcohol (25:24:1) and centrifuged at
16,000g for 5 min. The upper phase was treated with 3 M sodium
acetate and 100% ethanol for overnight at A20 °C. The sample
was centrifuged at 16,000g for 10 min, the supernatant removed
and 0.5 ml of 70% ethanol added. DNA was solubilized in Tris/EDTA
(10 mM/1 mM) buffer and quantitated spectrophotometrically at
260/280 nm. Total DNA was mixed with tracking dye and loaded
on 1% agarose gel containing ethidium bromide. Gel was run for
2.5 h at 50 V.
promastigotes and reference drug sodium antimony gluconate (SAG).
Compound tested
Concentration
g/mL)
% of inhibition vs. DMSO
control
P
(
l
value
Leishmania donovani AG83 (MHOM/IN/83/AG83)
Sodium Antimony
Gluconate
5
1.375
2.275
5.45
9.8275
10.0275
NS
NS
NS
NS
NS
10
15
20
25
Leishmania donovani AG83 (MHOM/IN/83/AG83)
Compound 3
Compound 12
Compound 11
0.5
1
2.5
5
0.5
1
2.5
5
31.6
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
49.61
70.54
78.44
16.06
25.07
34.74
48.64
22.34
37.0
2.12. Determination of anti-proliferative activity against intracellular
amastigotes
To check the anti-proliferative effect on amastigotes, macro-
phages were collected from thioglycolate elicited (1.5 mL of 4%
for 5 days) peritoneal exudates of male BALB/c mice as per the
guidelines of Institutional Animal Ethics Committee, West Bengal
State University. Macrophages were allowed to adhere in 8 cham-
0.5
1
2.5
5
47.57
61.67
bered slides in complete RPMI 1640 (supplemented with 1%
L-glu-
Leishmania donovani LV9 (MHOM/ET/67/HU3/LV9)
tamine, 1% penicillin-streptomycin, 50 M 2-ME, 1% essential
l
Compound 3
Compound 12
Compound 11
0.5
1
2.5
5
0.5
1
2.5
5
0.5
1
2.5
5
13.05
30.5
49.5
87.0
15.5
57
81
89.15
2.5
<0.016
<0.001
<0.001
<0.001
<0.006
<0.001
<0.001
<0.001
<0.221
<0.006
<0.001
<0.001
amino acids and 10% FCS). Macrophages were infected with L.
donovani AG83 promastigotes (1:10) for 12 h, after washing, fur-
ther incubated for another 60 h without treatment. After treatment
with indicated doses of compound 3, compound 11 and compound
12 the cultures were terminated after 48 h, fixed with methanol,
stained with Giemsa stain and counted under a phase contrast
microscope. Extent of infection was expressed as the number of
amastigotes per hundred macrophages as described earlier [30].
16.0
37.0
82.5
2.13. Estimation of nitric oxide
Leishmania major LV39 (MRHO/Sv/59/P)
Compound 3
Compound 12
Compound 11
0.5
1
2.5
5
0.5
1
2.5
5
0.5
1
2.5
5
19.76
25.31
29.25
35.0
<0.001
<0.001
<0.001
<0.001
<0.006
<0.001
<0.001
<0.001
<0.333
<0.001
<0.001
<0.001
Uninfected and Leishmania-infected peritoneal macrophages
(1 Â 106 cells/ml) were treated with indicated doses of compound
3, compound 11 and compound 12 for 48 h. The culture super-
natants were collected. The nitrite concentration was measured
by Griess reagent (Sigma-Aldrich), as described earlier [18,31].
15.5
21.85
35.15
37.5
2.14. Splenocyte proliferation for toxicity assay
7.0
22.55
28.95
34.65
Toxicity of compounds towards host cells was assessed on
splenocytes in vitro using the MTT assay as previously described

70
P. Ghosh et al. / Steroids 107 (2016) 65–73
[32]. Briefly, 1 Â 105 splenocytes per well were cultured in 96-well
allylic alcohols (Scheme 1) by the Corey’s method [33]. Cholesterol
does not possess any allylic substitution. To apply Corey’s method
first allylic substitutions were made in cholesterol skeleton
through different approaches to 2, 3 and 10. Jone’s oxidation of
cholesterol gave two conjugated ketones 2 and 3, 3 being the major
product. Compound 3 on reduction (NaBH4) under cold produced a
partially reduced product 5 and another fully reduced product, 6
(Vide infra). The structures of these compounds were established
by spectral data. Both the synthesized allylic alcohols were then
treated with H2O2-p-TsOH in DCM at room temperature for 72 h
following Corey’s method.
plates in 100 lL complete RPMI 1640 without phenol red and stim-
ulated with phytohemagglutinin (5
with or without graded concentrations of compound 3, compound
11 and compound 12 (0.5 g/mL, 1 g/mL, 2.5 g/mL and 5 g/
mL). After 48 h of incubation, 20 L of MTT (5 mg/mL in PBS,
lg/mL). Cells were incubated
l
l
l
l
l
Sigma) were added to each well and the cells were incubated for
4 h. The resulting formazan product was dissolved with acid-iso-
propanol-SDS and the absorbance at a wavelength of 595 nm
(A490) was read by iMark Microplate Reader (Biorad, USA). Statis-
tical analyses for all experiments were performed by Student’s t-
test with the program Sigma Plot using alpha adjustment [32].
Purification of the reaction mixture of 5 yielded two compounds,
a 6-ketoepoxide, 7 and the chlorinated one 8. Such kind of chlorine
incorporation by oxidative skeletal rearrangement was reported
earlier from our laboratory [34,35]. Compound 6 on the other hand
3. Results and discussion
gave 7 and 9, a c-lactone as the final rearranged products.
3.1. Chemistry
In an attempt, oxidation of cholesteryl acetate with sodium
dichromate gave 7-keto cholesteryl acetate, 10 as the final product.
Mild borohydride reduction on 10 yields two compounds 11 and
In the present study various oxygenated cholesterol derivatives
were synthesized by oxidative skeletal rearrangement of steroidal
Fig. 2. Compounds 3, 12 and 11 disrupted the cell cycle progression in promastigotes. 2.5 Â 106 cells/mL exponential phase Leishmania donovani AG83 promastigotes were
incubated for 6 h (A), 12 h (B) and 24 h (C) in complete M199 medium in the presence or absence of IC50 concentration of compound 3, 12 and 11 on promastigotes at 22 °C;
washed in 1Â PBS and treated with RNase A and PI to analyze the cell cycle progression as described in materials & methods. Values represent the percentage of positive cells
(representative of three independent experiments).

P. Ghosh et al. / Steroids 107 (2016) 65–73
71
12. Compounds 11 and 12 were then separately treated with
H2O2-p-TsOH in DCM at r.t for 72 h. Compound 11 yields 13, 14
and 15 as the rearranged products whereas 13, 14 and 16 were
produced by 12 (Scheme 1). All the new compounds were charac-
terized with the help of spectroscopic analysis (UV, IR, NMR and
mass).
3.1.1. Proposed mechanisms
Mechanism proposed here (Scheme 2) is an acid catalyzed car-
bocationic rearrangement by the involvement of ring A and B of the
system. Acid catalyzed dehydration of 6 yielded the intermediate
diene 6a and subsequently 6b. Reaction of 6b in presence of
H2O2 followed by epoxide ring opening gives intermediate diol
6c. Acid catalyzed rearrangement of 6c and finally oxidative lac-
tonization of the rearranged product 6d yielded the lactone 9. Such
kind of rearrangements in case of friedelan skeleton involving C/D
rings was also observed [33–35].
Formation of 13 can be explained by the initial oxidation of
allylic hydroxyl group to conjugated ketone [34]. The conjugated
ketone then undergoes rearrangement via protonation of the car-
bonyl group followed by elimination of AcOH to form more stable
hetero annular dienone 13. Such kind of rearrangement for the
steroidal system is already reported in literature [34,35].
Formation of 13 from 12 is also expected in presence of acid
through the expulsion of H2O instead of AcOH as depicted in
Scheme 3.
3.2. Anti-leishmanial activity
3.2.1. Anti-proliferative effect on Leishmania promastigotes and
estimation of IC50
Fig. 3. DNA fragmentation profile in agarose gel from untreated and treated
promastigotes with compound 3, 12 and 11.
Anti-proliferative effects of all the oxygenated derivatives were
evaluated on L. donovani, causative agent of visceral leishmaniasis
Fig. 4. Anti-survival activities of Compounds 3, 12 and 11 on intracellular amastigotes and induction of nitric oxide. (A) Resting peritoneal macrophages were infected with
Leishmania donovani (1:10) and treated without or with compound 3, compound 12 and compound 11 (0.125 g/mL). The Giemsa stained cell-micrographs were observed
under Carl Zeiss microscope and the anti-leishmanial activity was expressed as the number of amastigotes/100 macrophages. Values represent the mean SD of three
l
independent experiments in triplicate [Pa < 0.01; Pb < 0.005; Pc < 0.008]. (B) Nitric oxide was measured by Griess reagent and expressed in
lM, as described in materials and
methods. Compound 12 was found to induce highest NO at 48 h. Values represent the mean SD of three independent experiments in triplicate. Pa < 0.02; Pb < 0.003;
Pc < 0.002 vs. DMSO control.

72
P. Ghosh et al. / Steroids 107 (2016) 65–73
and L. major, causative agent of cutaneous leishmaniasis. We found
that compounds 3, 11 and 12 inhibited the proliferation of L. dono-
vani AG83, L. donovani LV9 and L. major LV39 promastigotes
in vitro. The 50% inhibitory concentration of 3, 11 and 12 has been
estimated against L. donovani AG83 promastigotes as 1
2.5 g/mL and 5 g/mL respectively. However, the highest dose
(5 g/mL) of the conventional drug SAG inhibited the promastigote
lg/mL,
l
l
l
growth only by 1.37% in respect to DMSO control. Significantly, it
has been found that 3, 11 and 12 could also inhibit the proliferation
of L. donovani LV9 in comparison to DMSO control. However, the
effects of the compounds were found costly against L. major
LV39. Our data clearly indicated that the three compounds have
a noteworthy anti-proliferative effect on L. donovani AG83 and
LV9 strain (Fig. 1 and Table 1).
3.2.2. Compounds 3, 11 and 12 disrupted the cell cycle progression in
L. donovani promastigotes
Cell-cycle analysis complemented the cytotoxicity of the com-
pounds obtained from MTT assay. It demonstrated that at 48 h.
Fig. 5. Splenocyte proliferation assay in presence of compound 3, compound 12 and
compound 11. MTT survival assay of splenocytes treated by graded concentration of
compound 3, 11 and 12 (0.5–5 lg/mL). Splenocyte proliferation index was
of culture, IC50 concentration of compounds 3, 11 and 12 (1
lg/
mL, 2.5 g/mL and 5 g/mL respectively) caused L. donovani pro-
l
l
mastigotes to remain as resting G0/G1 (M2) cells and inhibited
their entry into the S phase (M3). The percentage of dead cells
(M1) increased during this incubation period and growth arrest
was also visible. At 12 h the test compounds started blockage the
entry of L. donovani promastigotes in to S phase from G0/G1, how-
ever, compound 3 at 24 h completely blocked the entry dose
dependently (Fig. 2). Our results suggested that compound 3 pref-
erentially disrupted the cell cycle phases in L. donovani promastig-
otes followed by death in vitro.
Tests for another hallmark of apoptosis, the internucleosomal
degradation of genomic DNA, showed that the experimental group
had nucleosome-sized DNA fragments, giving a DNA-ladder-like
pattern identified by agarose gel electrophoresis of DNA from trea-
ted cells (Fig. 3).
determined relative to untreated control cells. Values represent mean SD of three
independent experiments in triplicate, Pa < 0.001 vs. DMSO control.
Table 2
Cytotoxicity assay on murine splenocytes.
Compound tested
Splenocytes
% of inhibition vs. control
P value
Compound 3
0.5
1
2.5
5
16.93
21.56
38.73
42.5
<0.333
<0.001
<0.001
<0.001
Compound 11
Compound 12
0.5
1
2.5
5
3.16
8.7
12.83
25.33
<0.333
<0.333
<0.333
<0.001
0.5
1
2.5
5
10.33
23.53
31.91
40.9
<0.333
<0.001
<0.001
<0.001
3.2.3. Compound 3, 11 and 12 significantly inhibited the intracellular
amastigotes
Compound 3, 11 and 12 were evaluated for its ability for
inhibiting the intracellular L. donovani amastigotes in peritoneal
macrophages. Significantly, 0.125 lg/mL compound 3, compound
splenocytes endorsed its best anti-amastigote effect and induction
of nitric oxide.
11 and compound 12 were found to inhibit the replication of intra-
cellular amastigotes in macrophages by 31.6% (P < 0.01), 40%
(P < 0.008), 50% (P < 0.005) respectively (Fig. 4A). Thus compound
12 was found most effective against the L. donovani amastigotes,
clinically important morphs of this parasite in mammalian host.
4. Conclusion
In a nutshell, the present paper described the synthesis of dif-
ferent oxysterol derivatives by skeletal rearrangement of steroidal
allylic alcohol. Compounds 3, 11 and 12 showed good activities
against different Leishmania species. Compound 12 was found least
toxic and induced highest NO at 48 h. Least toxicity of compound
12 on splenocytes authorized its best anti-amastigote effect and
induction of nitric oxide. The study will definitely help to develop
potent, least toxic steroidal anti-leishmanial drug.
3.2.4. Compound 3, 11 and 12 significantly induced nitric oxide in
macrophages
Since nitric oxide (NO) is one of the key anti-leishmanial mole-
cules [32], we were interested to check the induction of NO by the
three compounds in Leishmania-infected and uninfected macro-
phages (Fig. 4B). Compound 3, 11 and 12 were found efficient in
production of NO in infected-macrophages among which com-
pound 12 was found to induce the highest NO in infected
macrophages. The result can be correlated with the highest anti-
amastigote competence of this fraction.
Funding
Financial support from UGC was cordially acknowledged by A.
M. and P.G. C.P. has been supported by Department of
Biotechnology, Government of India (Project ref: BT/217/NE/
TBP/2011, dated 15.12. 2011).
3.2.5. Compound 3, 11 and 12 were found considerably nontoxic to
murine splenocytes
In order to test the safety of compound 3, 11 and 12 on mam-
malian cells, murine splenocytes were treated with these com-
pounds (0.5
checked by MTT assay. Compounds were found considerably non-
toxic to phytohemagglutinin (5 g/mL) stimulated BALB/c spleno-
cytes at 96 h (Fig. 5 and Table 2). Least toxic compound 12 towards
l
g/mL to 5
l
g/mL), and the viability of cells was
Acknowledgements
l
The authors are indebted to Vice Chancellor, North Bengal
University and Vice Chancellor, West Bengal State University for

P. Ghosh et al. / Steroids 107 (2016) 65–73
73
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