Synthesis and antimicrobial activity of novel oxysterols from lanosterol

Article abstract of DOI:10.1016/j.tet.2013.10.090

Chemically diverse oxysterols and their synthetic manipulations were carried out from variety of Δ⁸⁽⁹⁾-lanosterol derivatives and evaluated for their in vitro antimicrobial activities. Most of the synthesized oxysterols exhibited significant antifungal activity against the tested strains.

Full text of DOI:10.1016/j.tet.2013.10.090

Tetrahedron 69 (2013) 11155e11163
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and antimicrobial activity of novel oxysterols from
lanosterol
,
b
,
a
a
a
Bapurao B. Shingate a , Braja G. Hazra , Deepak B. Salunke , Vandana S. Pore ,
*
Fazal Shirazi c, Mukund V. Deshpande c
a Division of Organic Chemistry, National Chemical Laboratory, Pune 411 008, India
b Department of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431 004, India
c Biochemical Sciences Division, National Chemical Laboratory, Pune 411 008, India
a r t i c l e i n f o
a b s t r a c t
Chemically diverse oxysterols and their synthetic manipulations were carried out from variety of D8(9)
lanosterol derivatives and evaluated for their in vitro antimicrobial activities. Most of the synthesized
oxysterols exhibited significant antifungal activity against the tested strains.
Ó 2013 Elsevier Ltd. All rights reserved.
-
Article history:
Received 13 August 2013
Received in revised form 25 October 2013
Accepted 29 October 2013
Available online 1 November 2013
Keywords:
Allylic oxidation
Oxygenated steroids
Dihydroxylation
Antifungal activity
Antibacterial activity
1. Introduction
low density lipoprotein (LDL) also leads to the formation of
oxysterols, which actually contributes to the cytotoxicity of ox-
Major focuses for the 21st century are the sterol intermediates
in cholesterol synthesis and their metabolites. Oxygenated de-
rivatives of cholesterol are potent inhibitors of sterol bio-
synthesis in mammalian cells, which are of major interest.1 Most
of the oxysterols are autoxidation products2 but some of them
are formed in vivo by enzymatic means and their formation can
also result from the oxidation of cholesterol by free radicals,
generated by xenobiotics or by ionizing radiation.2b In the
ygenated LDL.5
Oxysterols exhibit marked biological activities including in-
hibition of endogenous cholesterol biosynthesis, antitumour and
immunomodulating activities.6 Oxysterols with multiple oxo-
functionalities in ring A and B were found to be selectively toxic
to cancer cells.7 There are several reports8 on the isolation,
bioevaluation and synthesis of oxygenated steroids having
anti-proliferative, anti-HIV and anti-asthma properties.9
body, oxysterols, such as, 7
ketocholesterol, 25-hydroxycholesterol, 5
and 5 ,6 -dihydroxycholestanol are derived from dietary sour-
ces. 22(R)-Hydroxycholesterol, 24(S)-hydroxycholesterol, 20(S)-
hydroxycholesterol and 27-hydroxycholesterol are formed en-
zymatically and present in the liver and blood circulation3 and
carries oxygenated functions in the steroidal side chain. Oxy-
sterols are also found in foam cells, lipid-laden macrophages that
accumulate in atherosclerotic lesions.4 Moreover, oxidation of
a
- and 7
b
-hydroxycholesterols, 7-
24,25-Epoxylanosterol is a potent inhibitor of 3-hydroxy-3-
b
,6b-epoxycholesterol
methylglutaryl CoA reductase (HMG-CoA), which is a key regula-
tory enzyme in the biosynthesis of sterols in humans.10 Oxygenated
cholesterol derivatives were reported to exhibit high activities
against Candida albicans, against the standard drugs amphotericin B
a
b
and miconazole at a concentration of 1.5
rivatives of lanosterol (Fig. 1) have been shown to act as inhibitors
m
g/mL.11 Side chain de-
of D
24(25) sterol methyl transferase, which is an essential enzyme in
the sterol biosynthesis pathway of protozoa, fungi and plants.12
These molecules presumably alter the viscosity of the lipid core
of the plasma membrane of fungal cells by interfering in the er-
gosterol biosynthesis.13
* Corresponding author. Tel.: þ91 240 2403311x13; fax: þ91 240 2403113; e-mail
addresses: bapushingate@gmail.com (B.B. Shingate), brajahazra@gmail.com
(B.G. Hazra).
In continuation of our work14,15 on biologically active sterol
derivatives, herein we would like to report, synthetic
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.10.090

11156
B.B. Shingate et al. / Tetrahedron 69 (2013) 11155e11163
2. Results and discussion
The synthesis of various oxygenated derivatives has been
attempted from the commercially available lanosterol 1 (a mixture
of D24 and C-24 dihydrolanosterol in 3:2 ratio). Lanosterol 1 on
catalytic hydrogenation with PdeC in ethyl acetate, resulted into
the formation of pure 24,25-dihydrolanosterol 2 in excellent yield
(Scheme 1). Acetylation of 2 with acetic anhydride and catalytic
amount of 4-N,N-dimethylaminopyridine (DMAP) afforded 3b-
Fig. 1. Lanosterol.
acetoxy-24,25-dihydrolanosteryl acetate 3 in 96% yield. Morisaki
and co-workers described,16 allylic oxidation of 24,25-
dihydrolanosterol acetate 3 to 7,11-dioxo-compound 4 by CrO3 in
acetic acid with low yield. Rodewald and Jagodzinski reported,17
manipulations of synthesized oxysterols and their promising anti-
microbial properties, since no biological activities of such de-
rivatives were described to till date, even if numerous similar
natural products possessing interesting biological activities. In the
present study, we have synthesized and evaluated a library of ox-
ygenated sterols in the ring B, C and side chain at C-24, C-25 po-
sitions and their functional group modifications.
conversion of the 24,25-dihydrolanosterol acetate
3
with
RuO2eNaIO4 to 3b-acetoxylanost-8-ene-7,11-dione 4 in 15% yield.
Very recently, we have reported15 the allylic oxidation of 3 with
RuCl3$3H2O and tBuOOH (tert-butyl hydroperoxide) in cyclohexane
at 20 ꢀC, resulted into the formation of 3
b-acetoxylanost-8-en-7,11-
Scheme 1. Reagents and conditions: (a) 10% PdeC, ethyl acetate, H2, 45 psi, 6 h, 98%; (b) Ac2O, pyridine, DMAP, 30 ꢀC, 2 h, 96%; (c) RuCl3$3H2O, tBuOOH, cyclohexane, 20 ꢀC, 2 h, 4
(89%) and 5 (8%); (d) NH2OH$HCl, pyridineeEtOH, reflux, 12 h, 6 (98%), 9 (95%); (e) KOH, MeOHeTHFeH2O (1:1:0.1), 30 ꢀC, 2 h, 7 (98%), 11 (94%); (f) Zn, acetic acid, reflux, 2 h, 98%;
(g) DIBAL-H, THF, reflux, 4 h, 83%.

B.B. Shingate et al. / Tetrahedron 69 (2013) 11155e11163
11157
dione 4 and 3
one 5 in 89% and 8% yields, respectively (Scheme 1).
b
-acetoxy-7
b
-tert-butyl hydroperoxy-lanost-8-en-11-
The reaction of unsaturated sterols or tetracyclic tri-terpenes
with mercuric acetate followed by reduction with sodium boro-
hydride/sodium hydroxide in water is highly selective for the side
chain double bonds, i.e., 24-methylene or 24(25). Compound 12
on treatment with Hg(OAc)2 in THFeH2O, gave the organo-
mercuric compound in situ, which was reduced with NaBH4 to
Oxygenated groups are known to be essential for antimicrobial
activity in natural occurring sterols,7e9 herein, other functionalities
have been introduced to get diverse and amplified synthetic library.
Compound 4 on treatment with NH2OH$HCl, resulted into the
formation of selective C-7 oxime derivative 6 in 98% yield (Scheme
1). The selective formation of C-7 oxime may be due to the steric
hindrance at C-11 position. This has been confirmed by spectro-
scopic data. In the 13C NMR spectrum of compound 6, the signals
afford 3
(Scheme 3). Rodewald and Jagodzinski described,19 conversion of
-acetoxylanost-8-en-25-ol 16 with RuO2eNaIO4 to 3 -acetox-
b-acetoxylanost-8-en-25-ol 16 along with compound 3
3
b
b
ylanost-8-ene-7,11-dione-25-ol 17in 17% yield. We have carried
appeared at d 200.9 and 158.1 ppm, were assigned to C-11 carbonyl
out15 allylic oxidation of compound 16 with ruthenium chloride
and C-7 oxime groups, respectively. According to the 1H NMR data
of compound 6, the signals at 3.19 and 3.27 for hydrogen at C-6
position, suggest the formation of C-7-oxime. Hydrolysis of acetate
functionality in compound 6 with KOH, afforded compound 7 in
98% yield.
and tert-butyl hydroperoxide, resulted into the formation of 3b-
acetoxylanost-8-en-7,11-di-oxo-25-ol 17 in 93% yield (Scheme 3).
Hydrolyzed product 18, has been obtained from compound 17 in
97% yield.
Epoxidation of 12 obtained from commercially available lano-
sterol with m-chloroperbenzoic acid afforded epimeric 24,25-
epoxylanosterol acetate 19 in good yield.20 Allylic oxidation of 19
with RuCl3 and tert-butyl hydroperoxide resulted into the forma-
tion of15 7,11-oxo derivative 20 in 78% yield (Scheme 4).
Reduction of D8(9) double bond in 3
b-acetoxylanost-8-en-7,11-
dione 4 with zinc in acetic acid resulted,17 the compound 8 in
98% yield (Scheme 1). According to the IR spectrum of compound 8,
two carbonyls are present at 1714 and 1703 cmꢁ1 and 13C NMR
spectrum shows signals at
d
209.4 and 208.8 ppm for 7,11-oxo
Dihydroxylation of olefins proven to be very useful for the
synthesis of biologically active steroids having a vicinal diol in the
side chain, such as brassinosteroids, potent plant growth regula-
tors, ecdysteroid, an insect molting hormones, as well as active
metabolites of vitamin D.21 Use of ruthenium tetroxide (RuO4) as an
oxidant for organic functional group transformations has been well
recognized.22,23 Treatment of 12 with aqueous solution of
RuCl3$3H2O and an excess of NaIO4 afforded the epimeric mixture
of 24,25-diol 21 in 91% yield (Scheme 5). This is the first report of
RuO4 mediated dihydroxylation of lanosteryl acetate. Selective
acetylation of C-24 hydroxy group in 21 with Ac2O, afforded 3,24-
diacetoxy compound 22 in 89% yield. Allylic oxidation of 22 with
ruthenium chloride and tert-butyl hydroperoxide resulted15 into
the formation of 7,11-oxo derivative 23 in 87% yield. Hydrolysis of
acetate functionality in 21 and 23 with KOH, led to the formation of
compounds 24 and 25 in 96% and 98% yield, respectively.
functionality. Compound 8 on treatment with NH2OH$HCl, resul-
ted into the formation of selective C-7 oxime derivative 9 in 95%
yield. This has been confirmed by 13C NMR spectrum, in which the
signals appeared at d 210.5 and 159.3 ppm, assigned to C-11 and C-
7, respectively. DIBAL-H reduction of 9, stereoselectively afforded
compound 10 in 83% yield. Under this condition, the selective re-
duction of C-11 keto-functionality and acetate to hydroxy groups;
but the reduction of oxime to amine does not takes place. It may be
due to the steric hindrance of C-14 axial methyl group. Acetate
functionality in 8 was hydrolyzed to give 3
compound 11 in 94% yield.
b-hydroxy-7,11-oxo
Lanosterol 1 on acetylation with acetic anhydride and catalytic
amount of DMAP afforded lanosterol acetate 12 in 96% yield
(Scheme 2). Compound 12 on treatment18 with BH3$Me2S in THF at
0
ꢀC, followed by in situ oxidation of organoborane by pyridinium
fluorochromate (PFC) gave 3
high yield (96%, yield based on presence of lanosteryl acetate along
with dihydrolanosteryl acetate 3). 3 -Acetoxylanost-8-en-24-one
13, a key intermediate in the sterol biosynthesis of C-24 alkylated
b
-acetoxylanost-8-en-24-one 13 in
To study the biological activity of oxygenated cholesterol 29,
following the procedure of Brunel and co-workers,11 we have also
synthesized the same compound 29 by RuCl3etBuOOH mediated15
allylic oxidation of 27, followed by alkaline hydrolysis of 28. Cho-
lesterol 26 on acetylation, followed by allylic oxidation of choles-
teryl acetate 27 with RuCl3eTBHP in cyclohexane resulted into
the formation 3b-acetoxy-cholest-5-en-7-one 28 in 74% yield
(Scheme 6). Hydrolysis of acetate functionality in compound 28
with KOH, afforded 7-oxo cholesterol 29 in 95% yield.
b
metabolites and potential regulators.18
3b-Acetoxylanost-8-en-24-
one 13 on allylic oxidation15 with ruthenium chloride and tert-
butyl hydroperoxide resulted into the formation of 7,11,24-tri-oxo
lanosterol derivative 14 in 89% yield. Hydrolysis of acetate func-
tionality in compound 14 with KOH afforded 15 in 91% yield.
Scheme 2. Reagents and conditions: (a) Ac2O, pyridine, DMAP, 30 ꢀC, 2 h, 96%; (b) (i) BH3$Me2S, THF, 0 ꢀC, 2 h; (ii) PFC, CH2Cl2, molecular sieves 4 ꢀA, reflux, 4 h, 90%; (c)
t
RuCl3$3H2O, BuOOH, cyclohexane, 20 ꢀC, 6 h, 89%; (d) KOH, MeOHeTHFeH2O (1:1:0.1), 30 ꢀC, 2 h, 91%.

11158
B.B. Shingate et al. / Tetrahedron 69 (2013) 11155e11163
Scheme 3. Reagents and conditions: (a) Hg(OAc)2, THFeH2O, NaBH4, NaOH, 91%; (b) RuCl3$3H2O, tBuOOH, cyclohexane, 20 ꢀC, 6 h, 93%; (c) KOH, MeOHeTHFeH2O (1:1:0.1), 30 ꢀC,
2 h, 97%.
t
Scheme 4. Reagents and conditions: (a) m-CPBA, CHCl3, 4 h, 86%; (b) RuCl3$3H2O, BuOOH, cyclohexane, 20 ꢀC, 6 h, 78%.
Scheme 5. Reagents and conditions: (a) RuCl3$3H2O, NaIO4, ethyl acetateeacetoneewater, 0 ꢀC, 10 min, 89%; (b) Ac2O, pyridine, DMAP, 30 ꢀC, 2 h, 91%; (c) RuCl3$3H2O, tBuOOH,
cyclohexane, 20 ꢀC, 6 h, 87%; (d) KOH, MeOHeTHFeH2O (1:1:0.1), 30 ꢀC, 2 h, 24 (96%) and 25 (98%).
t
Scheme 6. Reagents and conditions: (a) Ac2O, pyridine, DMAP, 30 ꢀC, 2 h, 98%; (b) RuCl3$3H2O, BuOOH, cyclohexane, 20 ꢀC, 6 h, 74%; (c) KOH, MeOHeTHFeH2O (1:1:0.1), 30 ꢀC,
2 h, 95%.

B.B. Shingate et al. / Tetrahedron 69 (2013) 11155e11163
11159
2.1. Bioevaluation
and 25. In particular, compounds 7, 14, 16, 17, 21e23 and 29 have
shown higher activity than reference drug fluconazole with 8 g/
mL as an MIC value. Among the all, compounds 15 and 18 have
shown promising activity with 2 g/mL as an MIC value against C.
albicans with reference to standard drug fluconazole and compa-
rable with amphotericin B. It is due to the presence of hydroxyl
group at C-3 along with carbonyl at C-24 and additional hydroxyl
group at C-25 position in compounds 15 and 18, respectively. It can
be seen that, the compounds 2e4, 6e25 and 29, showed very good
m
In vitro antifungal and antibacterial activity of the structurally
promising steroidal derivatives against eight fungal strains and one
strain of Gram-positive bacteria and one strain of Gram-negative
bacteria was investigated using micro-dilution method in com-
parison to the reference drugs amphotericin B, fluconazole, tetra-
cycline and erythromycin. The results are shown in Table 1. The
results clearly revealed that, all tested compounds in the present
study were found to have significant antifungal and moderate an-
tibacterial activity against the used strains.
m
inhibitory activity with 2e8
neoformans1. In particular, compounds 4 and 10 possess excellent
inhibitory effect with 2 g/mL as an MIC value. C. neoformans2 was
adversely affected by the compounds 4, 10, 16, 23 and 25 with MIC
value of 4e8 g/mL higher than the reference drugs amphotericin B
mg/mL as an MIC value against C.
m
Table 1
In vitro antifungal activity of compounds 1e25 and 29
m
Compound
Minimum inhibitory concentration (MIC)a
Fungal strains
(mg/mL)
and fluconazole. In this case, only two compounds 23 and 25 show
comparable activity against amphotericin B and higher than flu-
conazole. Compounds 4, 8e10, 19, 22, 25 and 29 also possess sig-
nificant fungicidal effect against C. neoformans3 with MIC value of
CA
16
CN1
CN2
CN3
CN4
BP
YL
32
FO
64
32
16
2
16
>64
16
4
1
16
4
8
2
>64
4
4
4
4
2
4
4
4
8
8
8
8
4
4
8
8
4
4
4
8
8
NT
NT
NT
8
NT
32
16
16
32
8
>64
NT
32
16
16
8
>64
32
16
>64
NT
>64
4
NT
NT
NT
4
NT
16
16
4
NT
NT
NT
2
NT
>64
64
16
>64
64
8
NT
16
>64
32
16
16
32
>64
4
32
16
16
16
32
32
8
4e8
significant antifungal activity against C. neoformans4 with MIC
value of 4e8 g/mL. Oxygenated derivatives 7, 10, 11, 14, 15, 17, 18,
mg/mL. Similarly, compounds 4, 11, 20 and 22 have observed
2
3
4
5
6
7
64
16
16
32
64
8
32
32
8
8
8
64
16
16
16
8
m
20 and 21 exhibits better antifungal activity against B. poitrasii than
reference drugs amphotericin B and fluconazole.
The activity of the compounds 4e6,11, 20 and 22e24 was higher
to that of amphotericin B and fluconazole against Y. lipolytica with
8
9
10
11
12
13
14
16
32
32
32
64
16
8
32
16
4
4
8
8
>64
16
16
32
8
MIC value of 8
showed superior antifungal activity with MIC value of 2e4
and compounds 9 and 14 with MIC value of 8 g/mL.
m
g/mL. In case of F. oxysporum, compounds 4 and 8
16
NT
16
64
32
16
16
16
8
32
NT
8
16
16
8
8
mg/mL
64
32
8
8
32
4
8
32
8
16
>64
16
32
16
16
16
16
8
m
15
2
>64
16
16
16
16
16
64
64
>64
16
32
>64
16
8
2.3. Antibacterial activity of compounds 1e25 and 29
16
8
17
8
In case of antibacterial activity, almost all the compounds do not
show activity against Gram-positive Escherichia coli and Gram-
negative Staphylococcus aureus strains (Table 2). Furthermore,
only compound 18 has shown higher antibacterial activity than
standard drug tetracycline and erythromycin against E. coli having
18
2
19
20
21
>64
16
8
NT
8
8
32
8
8
22
8
32
32
16
16
16
16
32
23
8
16
16
32
>64
2
MIC value of 4
mg/mL. However, compounds 10 and 17 showed
24
25
29
16
>64
8
32
4
32
4
8
higher or comparable antibacterial activity against S. aureus to the
reference drug tetracycline. However, compounds 2e4, 6, 9, 12, 16,
18, 22 and 23 showed comparable activity against S. aureus to the
reference drug erythromycin.
16
64
16
64
8
16
8
Ampho. B
Fluconazole
2
32
16
32
8
16
CA, Candida albicans (NCL1); CN1, Cryptococcus neoformans (NCL2); CN2, Cryptococ-
cus neoformans (NCIM-3542; ATCC No. 34664); CN3, Cryptococcus neoformans (NCIM-
3541; ATCC No. 32045); CN4, Cryptococcus neoformans (NCIM-33378); BP, Benjami-
niella poitrasii (NCL3); YL, Yarrowia lipolytica (NCL4); FO, Fusarium oxysporum (NCL5).
NT: Not tested.
3. Conclusions
Chemically diverse oxysterols were prepared from a variety of
D
8(9)-lanosterol derivatives and evaluated for their in vitro anti-
Negative control, DMSO and THF (50:50), No inhibition.
a
MIC (Minimum inhibitory concentration) was determined as 90% inhibition of
microbial activities against eight human/plant pathogenic as well
as nonpathogenic fungi along with Gram-positive and Gram-
negative bacterial strains. The influence of oxo-functionalities
(such as oxo-, hydroxyl-, oxime-, epoxy- and acetate) in rings B, C
and side chain of lanosterol, the antimicrobial activity was sys-
tematically studied. Most of the compounds exhibit significant
antifungal and moderate antibacterial activity against all the tested
strains. As reported by Brunel and co-workers,11 the compound 29
is highly active against the fungal strains, but in the present in-
vestigation, there are several oxysterols synthesized from lano-
sterol are more active than compound 29.
growth with respect to the growth control.
The synthesized structurally promising oxysterols 1e25 and 29
were tested in vitro for antifungal and antibacterial activity. The
antifungal activity was tested using NCL isolate fungal strains C.
albicans, Cryptococcus neoformans (human pathogen), Benjaminiella
poitrasii, Yarrowia lipolytica (saprophytes) and Fusarium oxysporum
(plant pathogen). Most of the pathogen fungi viz C. albicans are
dimorphic in nature. However, their use as model faces a number of
problems of slow growth rate and difficulties in getting synchro-
nous growth.24 Therefore nonpathogenic dimorphic fungus B. poi-
trasii was used as a model, which exhibits a rapid and simple one-
step process of yeastemycelium transition in response to temper-
ature and/or glucose change.25
4. Experimental section
4.1. General methods
2.2. Antifungal activity of compounds 1e25 and 29
All melting points were determined on Yanco Micro melting
point apparatus and are uncorrected. Optical rotations were ob-
tained on Bellingham and Stanly ADP-220 Polarimeter. Yields refer
to chromatographically and spectroscopically (1H and 13C NMR)
All the tested oxygenated lanosterol derivatives have shown
significant activity against C. albicans, except compounds 2, 6, 12, 19

11160
B.B. Shingate et al. / Tetrahedron 69 (2013) 11155e11163
Table 2
In vitro antibacterial activity of compounds 1e25 and 29
dried and concentrated to dryness. The residue was purified by
flash column chromatography (ethyl acetateepetroleum ether
Minimum inhibitory concentration (MIC)a
Bacterial strains
(mg/mL)
3:97) to give 3b-acetoxyl-7b-tert-butylhydroperoxy-lanost-8-ene-
Compound
11-one 5 (0.091 g, 8%) as a colourless solid. Mp: 133e135 ꢀC
(Methanol). IR (Nujol, cmꢁ1): 1741 (OCOCH3), 1676 (C]O). 1H NMR
EC
SA
(CDCl3, 200 MHz)
OCOCH3), 1.23 (s, 9H, OOCMe3), 1.12 (3H), 0.86e0.95 (18H), 0.63 (s,
3H, 18-H3). 13C NMR (CDCl3, 75 MHz)
¼199.7 (C), 170.6 (C), 156.1
d
¼4.69 (m, 1H, 6-H), 4.58 (s, 1H, 3-H), 2.07 (s, 3H,
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
8
>64
32
32
32
64
32
>64
64
32
8
64
32
64
64
>64
32
16
32
64
64
64
32
32
>64
64
64
16
32
32
64
16
64
64
16
32
16
64
>64
16
16
64
32
32
32
4
>64
64
>64
32
32
>64
64
32
8
d
(C),144.5 (C), 79.1 (C), 79.8 (CH), 75.7 (CH), 50.4 (CH), 50.1 (CH), 48.1
(C), 47.8 (C), 40.7 (CH2), 40.3 (C), 39.5 (CH2), 38.0 (C), 36.8 (CH2),
36.3 (CH), 36.3 (CH2), 33.5 (CH2), 32.9 (CH2), 28.1 (CH2), 28.0 (CH2),
27.5 (CH), 27.0 (3ꢃ CH3), 25.2 (CH3), 24.1 (CH2), 24.0 (CH2), 22.8
(CH3), 22.5 (CH3), 21.2 (CH3), 19.4 (CH3), 18.7 (CH3), 17.7 (CH3), 16.5
(CH3). MS (LCeMS) m/z: 573.3 (Mþ1). Anal. Calcd for C36H60O5: C,
75.48; H, 10.56. Found: C, 75.59; H, 10.47. Further elution with same
solvent system afforded
3b-acetoxylanost-8-ene-7,11-dione 4
(0.886 g, 89%) as a pale yellowish solid. Mp: 159e161 ꢀC (Metha-
nol). IR (Nujol, cmꢁ1): 1741 (OCOCH3), 1716, 1676 (C]O). 1H NMR
(CDCl3, 200 MHz)
d
¼4.52 (m, 1H, 3-H), 2.65 (dd, J¼5 and 14 Hz, 2H,
6-H2), 2.46 (s, 2H, 12-H2), 2.06 (s, 3H, OCOCH3), 1.33 (s, 3H, 19-H3),
1.17 (s, 3H, 14-H3), 0.95 (s, 3H, 4-H3), 0.90 (s, 3H, 4-H3), 0.89 (d, 6H),
0.85 (s, 6H), 0.81 (s, 3H, 18-H3). 13C NMR (CDCl3, 75 MHz)
d¼202.0
(C), 201.5 (C), 170.4 (C), 151.6 (C), 150.5 (C), 79.2 (CH), 51.6 (CH2),
50.1 (CH), 49.0 (CH), 48.9 (C), 47.4 (C), 39.3 (CH2), 36.1 (2ꢃ CH2),
36.1 (CH), 33.7 (CH2), 32.1 (CH2), 27.9 (CH), 27.7 (CH3), 27.3 (CH2),
25.8 (CH3), 23.9 (2ꢃ CH2), 22.74 (CH3), 22.5 (CH3), 21.1 (CH3), 18.5
(CH3), 17.5 (CH3), 16.7 (CH3), 16.5 (CH3). MS (LCeMS) m/z: 499.3
(Mþ1). Anal. Calcd for C32H50O4: C, 77.06; H, 10.10. Found: C, 76.89;
H, 9.89.
25
29
Tetracycline
Erythromycin >64
EC, Escherichia coli (NCIM No. 2574); SA, Staphylococcus aureus (NCIM No.
2122).
4.1.2. 3b-Acetoxylanost-8-ene-7-oxime-11-one (6). To the solution
Negative control, DMSO and THF (50:50), No inhibition.
of compound 4 (0.249 g, 0.5 mmol) in ethanol (10 mL) and pyridine
(2 mL) was added hydroxylamine hydrochloride (NH2OH$HCl)
(0.14 g, 2 mmol) and was refluxed for 12 h. Solvent was removed
under reduced pressure. Crushed ice was added and extracted with
ethyl acetate (2ꢃ50 mL). The organic extracts were washed with
brine (2ꢃ25 mL), dried over anhydrous Na2SO4, evaporated at re-
duced pressure to afford crude compound 6. Flash chromatographic
purification over silica gel using ethyl acetateepetroleum ether
a
MIC (Minimum inhibitory concentration) was determined as 90% in-
hibition of growth with respect to the growth control.
homogeneous materials, unless otherwise stated. Reactions were
monitored by thin-layer chromatography (TLC) using TLC alumin-
ium sheets, silica gel 60-F254 precoated, Merck, Germany and lo-
cating the spots using UV light as the visualizing agent or spraying
with ethanolic phosphomolybdic acid (PMA) solution followed by
heating. Flash column chromatography was carried out with silica
gel (300e400 mesh). Preparative thin-layer chromatography sep-
arations were carried out on 0.25 mm E Merck silica gel plates (60-
(7:93) as an eluent gave pure 3b-acetoxylanost-8-ene-7-oxime-11-
one 6 (0.251 g, 98%) as a colourless solid. Mp: 147e148 ꢀC (Meth-
anol). IR (Nujol, cmꢁ1): 3367 (eOH), 1731 (OCOCH3), 1710 (C]O),
F
254). 1H and 13C NMR spectra were recorded on Bruker AC-200
(200 MHz) at 200.13 and 50.32 and on a Bruker MSL-300
(300 MHz) at 300.13 and 75.47 or on Bruker DRX-500
(500 MHz) spectrophotometer at 500.13 and 125.78, respectively.
Chemical shifts are given in values relative to TMS (tetrame-
1643 (eC]N). 1H NMR (CDCl3, 200 MHz)
d
¼4.52 (m, 1H, 3-H), 2.06
(s, 3H, OCOCH3), 1.23 (s, 3H,19-H3),1.18 (s, 3H), 0.84e0.97 (18H). 13
NMR (CDCl3, 125 MHz)
¼200.9 (C), 171.1 (C), 158.1 (C), 152.4 (C),
C
a
d
144.9 (C), 80.1 (CH), 51.5 (CH2), 51.0 (C), 49.3 (CH), 48.7 (CH), 48.2
(C), 39.4 (CH2), 38.5 (C), 37.9 (C), 36.2 (CH), 36.2 (CH2), 34.6 (CH2),
34.1 (CH2), 27.9 (2ꢃ CH3), 27.4 (CH2), 26.4 (CH3), 24.1 (CH2), 23.9
(CH2), 22.7 (CH3), 22.5 (CH3), 21.2 (CH3), 19.5 (CH2), 18.6 (CH3), 18.1
(CH3), 16.8 (CH3), 16.3 (CH3). MS (LCeMS) m/z: 514.3 (Mþ1). Anal.
Calcd for C32H51NO4: C, 74.81; H, 10.01; N, 2.73. Found: C, 74.72; H,
10.13; N, 2.56.
d
thylsilane) as internal standard. IR spectra were recorded on Shi-
madzu 8400 series FTIR instrument and values are reported in
cmꢁ1 unit. Specific rotations ([
a]D) are reported in deg/dm and the
concentration (c) is given in g/100 ml in the specific solvent. Mass
spectra were recorded by either LCeMS or MS-TOF API QSTAR
PULSAR spectrophotometer, samples introduced by infusion
method using Electrospray Ionization Technique. Elemental anal-
yses were performed by CHNS-O EA 1108-Elemental analyzer,
Carloerba Instrument (Italy) or Elementor Vario EL (Germany) and
were within ꢂ0.4% of calculated values.
4.1.3. 3
b-Hydroxylanost-8-ene-7-oxime-11-one (7). To a stirred so-
lution of acetate compound
6
(0.102 g, 0.20 mmol) in
MeOHeTHFeH2O (1:1:0.1) (5 mL) was added aqueous solution of
KOH (0.70 g,1.1 mmol). The reaction mixture was stirred at 30 ꢀC for
2 h. It was quenched with water (5 mL), methanol and THF was
removed under reduced pressure. The residue was extracted with
ethyl acetate (2ꢃ100 mL). The combined organic extracts were
washed with brine (2ꢃ25 mL), dried over anhydrous Na2SO4,
4.1.1. 3
b
-Acetoxylanost-8-ene-7,11-dione (4) and
3b
-acetoxy-7
b
-
-
tert-butylhydroperoxy-lanost-8-ene-11-one (5). To a solution of 3
b
acetoxylanost-8-ene 3 (0.936 g, 2 mmol) and RuCl3$3H2O (6 mg,
0.023 mmol, 1.4 mol %) in cyclohexane (20 mL) was added 70% tert-
butyl hydroperoxide (TBHP, 6.0 mL, 7 mmol) via syringe pump at
20 ꢀC for 1 h. The resulting mixture was stirred at 20 ꢀC for 5 h. The
mixture was treated with saturated NaHCO3 solution and then
extracted with EtOAc. The organic layer was washed with brine,
concentrated under reduced pressure yielded 3
b-hydroxylanost-8-
ene-7-oxime-11-one (0.092 g, 98%) as
7
a colourless solid.
Mp: 236e238 ꢀC (Methanol). IR (Nujol, cmꢁ1): 3380 (eOH), 1643
(eC]N). 1H NMR (CDCl3, 200 MHz)
19-H3), 1.18 (s, 6H, 14-H3), 1.08 (s, 3H), 0.84e0.91 (12H). 13C NMR
d¼3.30 (m, 1H, 3-H), 1.24 (s, 3H,

B.B. Shingate et al. / Tetrahedron 69 (2013) 11155e11163
11161
(CDCl3þCD3OD, 50 MHz)
d
¼201.4 (C), 157.4 (C), 153.3 (C), 144.3 (C),
Yield: 94%. Mp: 191e193 ꢀC (Methanol). IR (Nujol, cmꢁ1): 3469
(eOH), 1697 (C]O). 1H NMR (CDCl3, 200 MHz)
¼3.24 (m, 1H, 3-H),
1.28 (s, 3H, 19-H3), 1.20 (s, 6H, 14-H3), 0.96 (s, 3H), 0.84e0.88 (12H),
0.71 (s, 3H, 18-H3). 13C NMR (CDCl3, 50 MHz)
¼209.6 (C), 209.1 (C),
77.8 (CH), 51.5 (CH2), 51.0 (CH), 49.2 (CH), 48.5 (CH), 48.0 (C), 39.2
(CH2), 38.9 (C), 38.5 (C), 36.1 (CH), 36.0 (CH2), 34.5 (CH2), 34.3 (CH2),
27.8 (CH3), 27.7 (CH), 27.3 (CH2), 26.1 (CH3), 23.8 (CH2), 22.6 (CH3),
22.3 (CH3), 19.4 (CH2), 18.4 (CH3), 17.9 (CH3), 16.6 (CH3), 15.1 (CH3).
MS (LCeMS) m/z: 472.3 (Mþ1). Anal. Calcd for C30H49NO3: C, 76.39;
H, 10.47; N, 2.97. Found: C, 76.18; H, 10.24; N, 2.86.
d
d
78.2 (CH), 60.5 (CH), 52.9 (CH), 52.5 (CH2), 52.4 (CH), 48.9 (C), 48.6
(CH), 46.3 (C), 39.3 (CH2), 39.3 (C), 39.3 (CH2), 36.8 (C), 36.2 (CH2),
36.0 (CH2), 35.8 (CH), 33.0 (CH2), 28.6 (CH2), 27.9 (CH3), 27.7 (CH),
27.2 (CH2), 23.9 (CH2), 22.8 (CH3), 22.5 (CH3), 18.4 (CH3), 17.5 (CH3),
16.1 (CH3), 14.9 (CH3), 13.6 (CH3). MS (LCeMS) m/z: 459.3 (Mþ1).
Anal. Calcd for C30H50O3: C, 78.55; H, 10.99. Found: C, 78.37; H,
10.69.
4.1.4. 3b-Acetoxylanost-7,11-dione (8). To the reflux solution of 3b-
acetoxylanost-8-ene-7,11-dione 4 (0.200 g, 0.4 mmol) in a glacial
acetic acid (15 mL), zinc dust (1 g) was added portion wise during
1 h and the heating was continued for 1 h. Zinc was filtered and
washed with hot acetic acid. The filtrate and washings were com-
bined and were added to the ice. The total precipitate so obtained
was filtered off, washed with water and dried and on crystallization
from methanol yielded pure compound 8 (0.196 g, 98%). Mp:
218e220 ꢀC (Methanol). IR (Nujol, cmꢁ1): 1741 (OCOCH3), 1714,
4.1.8. 3b-Acetoxylanost-8-en-24-one (13). Lanosteryl acetate 12
(6.0 g, 7.8 mmol) was dissolved in THF (30 mL) and cooled to 0 ꢀC in
an ice bath. While maintaining a nitrogen atmosphere, 5 mL
(5 mmol) of 1 M BH3$Me2S solution was added over 10 min. The
reaction was stirred for 1 h at 0 ꢀC under nitrogen. Ice was cau-
tiously added to decompose the excess hydride, H2O was added and
the reaction was extracted with diethyl ether. The organic extracts
were dried over anhydrous Na2SO4 and evaporated at reduced
pressure. The residue was dried in a vacuum desiccator over P2O5
for 2 h and dissolved in dichloromethane (50 mL), pyridinium
fluorochromate (8 g) and molecular sieves (0.050 g, type 4 ꢀA) were
added, and were refluxed for 3 h. The solvent was removed under
reduced pressure and the residue was subjected to column chro-
1703 (C]O). 1H NMR (CDCl3, 200 MHz)
d
¼4.52 (m, 1H, 3-H), 2.65
(dd, J¼5 and 14 Hz, 2H, 6-H2), 2.04 (s, 3H, OCOCH3), 1.27 (s, 3H, 19-
H3),1.18 (s, 3H,14-H3), 0.82e0.89 (15H), 0.69 (s, 3H,18-H3). 13C NMR
(CDCl3, 50 MHz)
d
¼209.4 (C), 208.8 (C), 170.6 (C), 79.8 (CH), 60.2
(CH), 52.9 (CH), 52.3 (CH2), 52.2 (CH), 48.9 (CH), 48.5 (CH), 46.3 (C),
39.3 (CH2), 39.0 (CH2), 38.0 (C), 36.6 (C), 36.1 (CH2), 35.7 (CH) 35.5
(CH2), 32.9 (CH2), 28.5 (CH2), 27.8 (CH), 27.5 (CH3), 23.8 (CH2), 23.5
(CH2), 22.7 (CH3), 22.4 (CH3), 21.1 (CH3), 18.3 (CH3), 17.4 (CH3), 16.0
(CH3), 15.9 (CH3), 13.6 (CH3). MS (LCeMS) m/z: 501.3 (Mþ1). Anal.
Calcd for C32H52O4: C, 76.75; H, 10.47. Found: C, 76.49; H, 10.41.
matography (ethyl acetateepetroleum ether 8:92) resulted 3b-
acetoxylanost-8-en-24-one 13. Yield: 86%. Mp: 135e137 ꢀC. IR
(Nujol, cmꢁ1): 1735 (eOCOCH3), 1710 (C]O). 1H NMR (CDCl3,
4.1.5. 3
b
-Acetoxylanost-7-oxime-11-one (9). Compound 9 was pre-
200 MHz)
d
¼4.52 (m, 1H, 3-H), 2.62 (m, 1H, 25-H), 2.05 (s, 3H,
pared from 8 using the same procedure for synthesis of 6 from 4.
OCOCH3), 1.10 (d, J¼6 Hz, 6H, 26 and 27-H3), 1.0 (s, 3H), 0.68 (s, 3H,
Yield: 98%. Mp: 209e211 ꢀC (Methanol). IR (Nujol, cmꢁ1): 1739
18-H3).
(OCOCH3),1703 (C]O). 1H NMR (CDCl3, 200 MHz)
d
¼4.47 (m,1H, 3-
H), 2.04 (s, 3H, OCOCH3), 1.28 (s, 3H, 19-H3), 1.16 (s, 3H, 14-H3),
4.1.9. 3b-Acetoxylanost-8-en-7,11,24-trione (14). Compound 14 was
0.82e0.91 (15H), 0.72 (s, 3H, 18-H3). 13C NMR (CDCl3, 50 MHz)
prepared from 13 using the same procedure for synthesis of 4 from
3. Yield: 89%. Mp: 135e137 ꢀC (Methanol). IR (Nujol, cmꢁ1): 1733
(OCOCH3), 1718, 1714, 1676 (C]O). 1H NMR (CDCl3, 200 MHz)
d
¼210.5 (C), 170.9 (C), 159.3 (C), 80.3 (CH), 60.9 (CH), 52.6 (CH2),
51.8 (CH), 49.3 (C), 49.0 (CH), 47.1 (C), 45.2 (CH), 39.4 (CH2), 38.3 (C),
36.9 (C), 36.2 (CH2), 35.9 (CH), 35.9 (CH2), 33.7 (CH2), 28.6 (CH2),
27.9 (CH), 27.9 (CH3), 23.9 (CH2), 23.7 (CH2), 22.8 (CH3), 22.5 (CH3),
21.3 (CH3), 20.3 (CH2), 18.4 (CH3), 17.4 (CH3), 16.1 (2ꢃ CH3), 13.7
(CH3). MS (LCeMS) m/z: 515.3 (Mþ1). Anal. Calcd for C32H53NO4: C,
74.52; H, 10.36; N, 2.72. Found: C, 74.37; H, 10.29; N, 2.61.
d
¼4.51 (m, 1H, 3-H), 2.05 (s, 3H, OCOCH3), 1.32 (s, 3H, 19-H3), 1.17 (s,
3H), 1.10 (s, 6H), 1.07 (s, 3H), 0.95 (s, 3H), 0.89 (6H), 0.79 (s, 3H, 18-
H3). 13C NMR (CDCl3, 50 MHz)
d¼214.8 (C), 202.0 (C), 201.6 (C),170.6
(C), 151.5 (C), 150.4 (C), 79.1 (CH), 51.4 (CH2), 50.0 (CH), 48.9 (CH),
48.9 (C), 47.3 (C), 40.7 (CH), 39.4 (C), 37.6 (C), 37.1 (CH2), 36.1 (CH2),
35.6 (CH), 33.6 (CH2), 32.0 (CH2), 29.6 (CH2), 27.7 (CH3), 27.1 (CH2),
25.7 (CH2), 23.8 (CH2), 20.2 (CH3), 18.2 (3ꢃ CH3), 17.4 (CH3), 16.7
(CH3), 16.4 (CH3). MS (LCeMS) m/z: 513.3 (Mþ1). Anal. Calcd for
C32H48O5: C, 74.96; H, 9.44. Found: C, 74.87; H, 9.47.
4.1.6. 3b,11b-Dihydroxylanost-7-oxime (10). DIBAL-H (0.75 mL of
1.67 M toluene solution, 1.25 mmol) was added dropwise to a so-
lution of compound 9 (0.129 g, 0.25 mmol) in dry tetrahydrofuran
(5 mL) at 0 ꢀC. The mixture was refluxed for 4 h. The solution was
diluted with ethyl acetate and treated with cold saturated ammo-
nium chloride. It was extracted with ethyl acetate and washed with
brine and dried over anhydrous sodium sulfate. The solvent was
evaporated under reduced pressure afforded crude compound. The
crude product was purified by column chromatography on silica gel
4.1.10. 3b-Hydroxylanost-8-en-7,11,24-trione (15). Compound 15
was prepared from 14 using the same procedure for synthesis of 7
from 6. Yield: 91%. Mp: 85e86 ꢀC (Methanol). IR (Nujol, cmꢁ1):
3432 (eOH), 1714 (C]O), 1697 (C]O). 1H NMR (CDCl3, 200 MHz)
d
¼3.27 (m, 1H, 3-H), 1.30 (s, 3H, 19-H3), 1.26 (s, 3H), 1.18 (s, 3H), 1.11
(s, 3H), 1.08 (s, 3H), 1.02 (s, 3H), 0.88 (6H), 0.80 (s, 3H, 18-H3). 13
NMR (CDCl3, 50 MHz)
(ethyl acetateepetroleum ether 2:8) to give 3
b
,11
b
-dihydrox-
C
ylanost-7-oxime 10 (0.098 g, 83%) as a colourless solid. Mp:
d¼215.1 (C), 202.3 (C), 202.2 (C), 151.7 (C),
97e99 ꢀC. IR (Nujol, cmꢁ1): 3407 (eOH). 1H NMR (CDCl3, 200 MHz)
150.6 (C), 77.6 (CH), 51.6 (CH2), 50.1 (CH), 49.1 (CH), 48.9 (C), 47.3
(C), 40.9 (CH), 39.7 (C), 38.8 (C), 37.3 (CH2), 36.4 (CH2), 35.7 (CH),
34.0 (CH2), 32.1 (CH2), 29.7 (CH2), 27.8 (CH3), 27.5 (CH2), 27.2 (CH2),
23.8 (CH3), 18.3 (2ꢃ CH3), 18.2 (CH3), 17.5 (CH3), 16.8 (CH3), 15.4
(CH3). MS (LCeMS) m/z: 471.3 (Mþ1). Anal. Calcd for C30H46O4: C,
76.55; H, 9.85. Found: C, 76.39; H, 9.89.
d
¼4.32 (d, J¼2 Hz, 1H, 11-H), 3.27 (m, 1H, 3-H), 1.08 (3H), 1.07 (3H),
0.82e0.91 (15H), 0.72 (s, 3H, 18-H3). 13C NMR (CDCl3, 50 MHz)
d
¼161.3 (C), 79.1 (CH), 68.7 (CH), 51.4 (CH), 51.0 (CH), 50.6 (CH), 50.1
(C), 45.7 (C), 43.0 (CH2), 40.3 (CH), 39.5 (C), 39.4 (CH2), 36.4 (CH2),
36.2 (C), 35.9 (CH), 35.9 (CH2), 35.1 (CH2), 29.7 (CH2), 27.9 (CH3),
27.8 (CH2), 27.5 (CH), 27.2 (CH2), 24.1 (CH2), 22.8 (CH3), 22.6 (CH2),
22.5 (CH3), 18.8 (CH3), 18.6 (CH3), 18.4 (CH3), 15.6 (CH3), 14.4 (CH3).
MS (LCeMS) m/z: 476.4 (Mþ1). Anal. Calcd for C30H53NO3: C, 75.74;
H, 11.23; N, 2.94. Found: C, 75.53; H, 11.03; N, 2.69.
4.1.11. 3b-Acetoxylanost-8-en-7,11-oxo-25-ol (17). Compound 17
was prepared from 16 using the same procedure for synthesis of 4
from 3. Yield: 93%. Mp: 199e200 ꢀC (Methanol) (lit.19 196e197 ꢀC).
IR (Nujol, cmꢁ1): 3490 (eOH),1710 (OCOCH3), 1683, 1660 (C]O). 1H
4.1.7. 3
b
-Hydroxylanost-7,11-dione (11). Compound 11 was pre-
NMR (CDCl3, 200 MHz)
1.33 (s, 3H, 19-H3), 1.21 (s, 9H), 0.94 (d, 3H), 0.90 (s, 6H), 0.80 (s, 3H,
d
¼4.5 (m, 1H, 3-H), 2.06 (s, 3H, OCOCH3),
pared from 8 using the same procedure for synthesis of 7 from 6.

11162
B.B. Shingate et al. / Tetrahedron 69 (2013) 11155e11163
18-H3). 13C NMR (CDCl3, 125 MHz)
d
¼202.3 (C), 201.8 (C), 170.7 (C),
0.88 (9H), 0.70 (s, 3H, 18-H3). MS (LCeMS) m/z: 545.3 (Mþ1). Anal.
151.7 (C), 150.6 (C), 79.2 (CH), 70.9 (C), 51.6 (CH2), 50.1 (CH), 49.0
(CH), 49.0 (C), 47.4 (C), 44.2 (CH2), 39.5 (C), 37.7 (C), 36.4 (CH2), 36.2
(CH2), 36.1 (CH), 33.7 (CH2), 32.1 (CH2), 29.3 (CH3), 29.2 (CH3), 27.8
(CH3), 27.3 (CH2), 25.9 (CH3), 23.9 (CH2), 21.2 (CH3), 21.0 (CH2), 18.5
(CH3), 17.5 (CH3), 16.7 (CH3), 16.5 (CH3). MS (LCeMS) m/z: 515.3
(Mþ1). Anal. Calcd for C32H50O5: C, 74.67; H, 9.79. Found: C, 74.58;
H, 9.63.
Calcd for C34H56O5: C, 74.96; H, 10.36. Found: C, 74.69; H, 10.18.
4.1.16. 24(R,S)-3b,24-Diacetoxylanost-8-en-7,11-oxo-25-ol
(23). Compound 23 was prepared from 22 using the same pro-
cedure for synthesis of 4 from 3. Yield: 87%. Mp: 145e147 ꢀC
(Methanol). IR (Nujol, cmꢁ1): 3442 (eOH), 1735 (OCOCH3), 1718,
1676 (C]O). 1H NMR (CDCl3, 200 MHz)
d
¼4.7 (m, 1H, 24-H), 4.54
(m, 1H, 3-H, 24-H), 2.11 (s, 3H, OCOCH3), 2.06 (s, 3H, OCOCH3), 1.32
(s, 3H,19-H3),1.20 (s, 9H), 1.16 (s, 3H), 0.95 (s, 3H), 0.90 (6H), 0.79 (s,
3H). MS (LCeMS) m/z: 573.3 (Mþ1). Anal. Calcd for C34H52O7: C,
71.30; H, 9.15. Found: C, 71.51; H, 9.23.
4.1.12. 3b-Hydroxylanost-8-en-7,11-oxo-25-ol (18). Compound 18
was prepared from 17 using the same procedure for synthesis of 7
from 6. Yield: 97%. Mp: 183e185 ꢀC (Methanol). IR (Nujol, cmꢁ1):
3550 (eOH), 3454 (eOH), 1668 (C]O). 1H NMR (CDCl3, 200 MHz)
d
¼3.26 (m, 1H, 3-H), 1.30 (s, 3H, 19-H3), 1.21 (s, 6H), 1.17 (s, 3H), 1.01
4.1.17. 24(R,S)-3
(24). Compound 24 was prepared from 23 using the same
procedure for synthesis of from 6. Yield: 96%. Mp:
153e156 ꢀC (Methanol). IR (Nujol, cmꢁ1): 3541 (eOH), 3404 (eOH),
1674 (C]O). 1H NMR (CDCl3, 200 MHz)
1.31 (s, 3H, 19-H3), 1.26 (s, 3H), 1.22 (s, 3H), 1.17 (s, 6H), 1.02 (s, 3H),
0.88 (d, 6H), 0.81 (s, 3H). MS (LCeMS) m/z: 489.3 (Mþ1). Anal. Calcd
for C30H48O5: C, 73.73; H, 9.90. Found: C, 73.58; H, 9.74.
b,24,25-Trihydroxylanost-8-en-7,11-dione
(s, 3H), 0.90 (s, 6H), 0.80 (s, 3H, 18-H3). 13C NMR (CDCl3, 50 MHz)
d¼202.5 (C), 202.3 (C), 151.8 (C), 150.6 (C), 77.5 (CH), 71.0 (C), 51.6
7
(CH2), 50.1 (CH), 49.1 (CH), 48.9 (C), 47.3 (C), 44.2 (CH2), 39.7 (C),
38.8 (C), 36.4 (2ꢃ CH2), 36.1 (CH), 34.0 (CH2), 32.1 (CH2), 29.3 (CH3),
29.1 (CH3), 27.8 (CH3), 27.5 (CH2), 27.3 (CH2), 20.9 (CH2), 18.5 (CH3),
17.5 (CH3), 16.8 (CH3), 15.4 (CH3). MS (LCeMS) m/z: 473.3 (Mþ1).
Anal. Calcd for C30H48O4: C, 76.23; H, 10.24. Found: C, 76.18; H,
10.13.
d¼3.32 (m, 2H, 3-H, 24-H),
4.1.18. 24(R,S)-3b,24,25-Trihydroxylanost-8-ene (25). Compound 25
4.1.13. 24(R,S)-3
b
-Acetoxy-24,25-epoxy-lanost-8-en-7,11-one
was prepared from 21 using the same procedure for synthesis of 7
(20). Compound 20 was prepared from 19 using the same pro-
cedure for synthesis of 4 from 3. Yield: 78%. Mp: 101e102 ꢀC
(Methanol). IR (Nujol, cmꢁ1): 1731 (OCOCH3), 1674 (C]O). 1H NMR
from 6. Yield: 98%. Mp: 156e158 ꢀC (Methanol). IR (Nujol, cmꢁ1):
3330 (eOH). 1H NMR (CDCl3, 200 MHz)
d¼3.20e3.38 (m, 2H, 3-H,
24-H), 1.22 (s, 3H, 19-H3), 1.17 (s, 3H), 1.01 (s, 3H), 0.99 (s, 3H), 0.89
(d, 6H), 0.82 (s, 3H), 0.70 (s, 3H, 18-H3). MS (LCeMS) m/z: 461.3
(Mþ1). Anal. Calcd for C30H52O3: C, 78.21; H, 11.38. Found: C, 78.09;
H, 11.21.
(CDCl3, 200 MHz)
d¼4.5 (m, 1H, 3-H), 2.06 (s, 3H, OCOCH3), 1.33 (s,
3H,19-H3),1.31 (s, 3H), 1.27 (s, 3H), 1.17 (s, 3H), 0.90e0.94 (9H), 0.81
(s, 3H, 18-H3). MS (LCeMS) m/z: 513.3 (Mþ1). Anal. Calcd for
C
32H48O5: C, 74.96; H, 9.44. Found: C, 75.18; H, 9.53.
4.2. Antimicrobial activity: materials and methods
4.1.14. 24(R,S)-3b-Acetoxylanost-8-en-24,25-diol (21). A solution of
RuO4 was prepared by adding a solution of NaIO4 (639 mg,
3.0 mmol) in H2O (2.4 mL) to RuCl3$3H2O (36.5 mg, 0.14 mmol).
To a stirred solution of 12 (0.466 g, 1.0 mmol) in ethyl acetate
(14 mL), acetone (6.0 mL), acetonitrile (6.0 mL) at 4 ꢀC was added
RuO4 solution at once and the reaction mixture was stirred at
5e6 ꢀC for a further period of 5 min. To the mixture was added
20% Na2S2O5 solution (10 mL), and stirred for 5 min. All the sol-
vents were evaporated and the residue was extracted with ethyl
acetate (3ꢃ50 mL). The combined ethyl acetate extracts were
washed with H2O (2ꢃ50 mL) and brine (2ꢃ50 mL), and dried
(Na2SO4). Removal of the solvent afforded the crude product,
which was purified by column chromatography on silica gel (ethyl
C. albicans, C. neoformans (human pathogen), B. poitrasii and Y.
lipolytica (non pathogen) strains were maintained on YPG (yeast
extract, 0.3%, peptone, 0.5%, and glucose, 1%) agar slants. F. oxy-
sporum (plant pathogen) strain was maintained on PDA (potato,
20% dextrose, 2%) agar slants at 28 ꢀC. E. coli (NCIM No. 2574) and S.
aureus (NCIM No. 2122) were maintained on NA (beef extract, 0.3%,
peptone, 0.5%, sodium chloride, 0.5%) slants. Strains of C. albicans, C.
neoformans, Y. lipolytica were inoculated in YPG broth at 28 ꢀC and
B. poitrasii at 37 ꢀC for 24 h, respectively, F. oxysporum in potato
ꢀ
dextrose at 28 C for 48 h, whereas bacterial strains E. coli and S.
aureus in NA broth for 24 h. Compounds 1e25 and 29 were solu-
bilized in DMSO and THF (50:50), and stock solutions of 1.28 mg/mL
were prepared. Amphotericin B, fluconazole, tetracycline and
erythromycin were also dissolved in DMSO and THF (50:50), and
were used as a positive control.
acetateepetroleum ether 1:9) afforded 3b-acetoxylanost-8-en-
24,25-diol 21. Yield: 89%. Mp: 185e187 ꢀC (Methanol). IR (Nujol,
cmꢁ1): 3388 (eOH), 1720 (OCOCH3). 1H NMR (CDCl3, 500 MHz)
d
¼4.52 (m, 1H, 3-H), 3.31 (m, 1H, 24-H), 1.22 (s, 3H, 19-H3), 1.17 (s,
3H), 1.01 (s, 3H), 0.88 (9H), 0.70 (s, 3H, 18-H3). MS (LCeMS) m/z:
503.3 (Mþ1). Anal. Calcd for C32H54O4: C, 78.21; H, 11.38. Found:
C, 78.09; H, 11.21.
4.3. MIC determination
In vitro antifungal and antibacterial activity of newly synthe-
sized compounds were studied against fungal strains viz., C. albi-
cans, C. neoformans, B. poitrasii, Y. lipolytica, F. oxysporum and
bacterial strains E. coli (NCIM No. 2574), and S. aureus (NCIM No.
2122), respectively, to find out MIC (Minimum Inhibitory Concen-
tration). All the experiments were done in triplicate under similar
experimental conditions. MIC of the synthesized compounds was
determined according to standard broth micro-dilution technique
as per NCCLS guidelines.26 Testing was performed in U bottom 96
well tissue culture plates in YPG, PDA for fungal strains and NA for
bacterial strains. The concentration range of tested compounds and
4.1.15. 24(R,S)-3
b
,24-Diacetoxylanost-8-en-25-ol (22). To the solu-
tion of 3 -acetoxylanost-8-en-
b
x24,25-diol 21 (0.502 g, 1 mmol) in
pyridine (5 mL) were added acetic anhydride (0.5 mL, 5 mmol) and
DMAP (0.012 g, 0.1 mmol). The reaction mixture was stirred at 30 ꢀC
for 1 h. The reaction was quenched with crushed ice and extracted
with ethyl acetate (2ꢃ50 mL). The combined organic extracts were
washed with brine (2ꢃ10 mL), dried over Na2SO4, concentrated
under reduced pressure afforded crude product. Column chro-
matographic purification over silica gel using ethyl aceta-
teepetroleum ether (6:94) as eluent afforded 22 (0.517 g, 95%) as
a colourless solid. Mp: 152e154 ꢀC (Methanol). IR (Nujol, cmꢁ1):
standard was 64e0.5 m
g/mL. The plates were incubated at 28 ꢀC for
all the microorganisms except for B. poitrasii at 37 ꢀC, absorbance at
600 nm was recorded to assess the inhibition of cell growth after
24 h for B. poitrasii and Y. lipolytica, 48 h for C. albicans and F.
3356 (eOH),1735 (OCOCH3). 1H NMR (CDCl3, 50 MHz)
24-H), 4.52 (m, 1H, 3-H), 1.22 (s, 3H, 19-H3), 1.17 (s, 3H), 1.01 (s, 3H),
d
¼4.7 (m,1H,

B.B. Shingate et al. / Tetrahedron 69 (2013) 11155e11163
11163
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oxysporum, 72 h for C. neoformans and 24 h for bacterial cultures.
MIC was determined as 90% inhibition of growth with respect to the
growth control was observed.
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Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2013.10.090.
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