Inhibitory effect of oxygenated cholestan-3-ol derivatives on the growth of Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmcl.2013.09.013

A variety of cholestan-3-ol derivatives, which are oxygenated at different positions of the steroid ring system, were prepared and tested for their inhibition of the Mycobacterium tuberculosis H₃₇Rv strain. Several compounds showed significant antitubercular activities with MIC₉₀ values in the range 4-8 μM and low or non-detectable toxicity against mammalian cells.

Full text of DOI:10.1016/j.bmcl.2013.09.013

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6111–6113
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitory effect of oxygenated cholestan-3b-ol derivatives on the
growth of Mycobacterium tuberculosis
Arndt W. Schmidt a, Taylor A. Choi b, Gabriele Theumer a, Scott G. Franzblau b, Hans-Joachim Knölker a,
⇑
a Department of Chemistry, Technische Universität Dresden, Bergstr. 66, 01069 Dresden, Germany
b Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A variety of cholestan-3b-ol derivatives, which are oxygenated at different positions of the steroid ring
system, were prepared and tested for their inhibition of the Mycobacterium tuberculosis H37Rv strain. Sev-
Received 14 August 2013
Revised 2 September 2013
Accepted 5 September 2013
Available online 14 September 2013
eral compounds showed significant antitubercular activities with MIC90 values in the range 4–8
low or non-detectable toxicity against mammalian cells.
lM and
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Anti-TB activity
Cholestanols
Inhibition
Steroids
Tuberculosis
Tuberculosis (TB) remains to represent a major global threat to
the human population. For the year 2011, 8.7 million cases of new
infections and 1.4 million deaths resulting from TB were esti-
mated.1 Mycobacterium tuberculosis (MTB), the pathogen causing
TB, can develop efficient survival strategies and is becoming resis-
tant towards key antibiotics currently in use to treat TB infections.
Especially the global spreading of multidrug-resistant TB (MDR-TB)
and the growing number of cases reported for extensively drug-
resistant TB (XDR-TB) increased this problem dramatically in re-
cent years.1 Thus, it is an important challenge for medicinal chem-
istry to identify new lead structures which eventually may lead to
more efficacious drugs for the treatment of TB. Over the past dec-
ade, an intense research activity, including screening of compound
libraries and bioactivity-guided isolation of natural products, was
directed towards the development of new potential drugs against
TB.1–6 Investigations of diverse natural sources led to a range of
phytosterols and ergosterols which were identified to exhibit sig-
nificant antitubercular (anti-TB) activities.7 Previously, we had re-
ported on the inhibition of M. tuberculosis by carbazole alkaloids
which were obtained by means of total synthesis.8 In another pro-
ject, we studied substituted cholestan-3b-ols for their hormonal
activity on the nematode Caenorhabditis elegans.9–11 Herein, we de-
scribe the anti-TB activity of these synthetic cholestan-3b-ol deriv-
atives which are oxygenated at different positions of the steroidal
framework.
Starting from commercially available 7-ketocholesterol (1), the
7-oxygenated cholestan-3b-ol derivatives 2–6 are readily
prepared (Scheme 1).10 Acetylation of compound
1 afforded
7-ketocholesteryl acetate (2), which on catalytic hydrogenation
gave 7-ketocholestan-3b-yl acetate (3). Cleavage of the ester led to
7-ketocholestan-3b-ol (4). Reduction of compound 3 using lithium
aluminum hydride provided cholestane-3b,7a-diol (5) and choles-
tane-3b,7b-diol (6), which can be separated by chromatography.
H
H
b
H
H
O
H
H
O
RO
RO
H
1
2
R = H
R = Ac
3
R = Ac
a
c
4 R = H
d
H
H
3
+
H
H
H
OH
H
H
OH
HO
HO
H
5
6
Scheme 1. Synthesis of the cholestane-3b,7-diols 5 and 6. Reagents and conditions:
(a) Ac2O, Et3N, cat. DMAP, THF, rt, 20 h (99%); (b) 10% Pd/C, H2, CH2Cl2, rt, 24 h
(85%); (c) KOH, MeOH, reflux, 48 h (100%); (d) LiAlH4, THF, –78 °C to rt, 24 h (57% 5,
9% 6).
⇑
Corresponding author. Fax: +49 351 463 37030.
E-mail address: hans-joachim.knoelker@tu-dresden.de (H.-J. Knölker).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.013

6112
A. W. Schmidt et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6111–6113
Commercial 25-hydroxycholesterol (7) served as starting mate-
H
O
rial for the synthesis of the 7,25-dioxygenated cholestan-3b-ols 11
and 12 (Scheme 2).10 Acetylation of 7 to compound 8 followed by
allylic oxidation using Chandrasekaran’s procedure12 led to 25-hy-
droxy-7-ketocholesteryl acetate (9). Transfer hydrogenation of 9
using ammonium formate as hydrogen source13 provided 25-hy-
droxy-7-ketocholestan-3b-yl acetate (10) in an improved yield of
90%.10 Direct reduction of 10 with lithium aluminum hydride affor-
ded an unseparable mixture of the triols 11 and 12. As the diols 5
and 6 could be separated (see above), the 25-hydroxy group was
protected first. Indeed, after protection of the tertiary hydroxy
group of compound 10 by silylation and reduction of the 7-keto
H
a
O
OH
H
H
OH
H
H
H
H
HO
HO
17
18
b
e
OH
H
H
H
HO
19
group with lithium aluminum hydride, the 7a-hydroxy and 7b-hy-
droxy diastereoisomers could be separated by chromatography.
Cleavage of the trimethylsilyl ethers of the individual compounds
Scheme 4. Synthesis of (25R)-cholest-5-ene-3b,26-diol (19). Reagents and condi-
tions: (a) Zn dust, 19% HCl, EtOH, reflux, 4 h (85%); (b) TBSCl, DBU, THF, rt, 16 h
(85%); (c) MsCl, pyridine, 0 °C to rt, 16 h; (d) LiAlH4, Et2O, reflux, 4 h (89%, two
steps); (e) TBAF, THF, reflux, 20 h (89%).
provided cholestane-3b,7a,25-triol (11) and cholestane-3b,7b,25-
triol (12).
O-Silylation of 6-ketocholestan-3b-ol (13) gave compound 14
(Scheme 3).10 Deprotonation of 14 with lithium diisopropylamide
Table 1
Antituberculosis activities, cytotoxicities, and selectivity indices of the oxygenated
cholestan-3b-ol derivatives 1–19
Ref.a
MIC90
(l
M)
IC50
75
(lM)
SId
b
c
Compound
OH
OH
H
H
b
1
2
3
4
5
6
7
8
—
>128
>128
>128
8.0
>128
>128
>128
>128
>128
>128
>128
24
6.0
3.5
4.6
>128
>128
6.0
>128
0.4
<0.6
—
—
7.8
—
—
—
—
—
—
H
H
H
H
O
10
10
10
10
10
—
10
10
10
10
10
—
10
10
10
—
11
11
—
>128
>128
62
RO
AcO
OH
7
8
9
R = H
R = Ac
a
n.d.
n.d.
>128
n.d.
>128
>128
n.d.
>128
92
H
d
f
c
9
H
H
O
10
11
12
13
14
15
16
17
18
19
INHe
RMPe
AcO
—
H
>5.3
15.3
10.9
8.7
<0.4
—
>21
—
>320
1543
10
38
40
41
OH
OH
H
H
+
n.d.
>128
n.d.
>128
108
H
H
H
H
OH
OH
HO
HO
H
H
11
12
—
0.07
Scheme 2. Synthesis of the cholestane-3b,7,25-triols 11 and 12. Reagents and
conditions: (a) Ac2O, Et3N, cat. DMAP, THF, rt, 20 h (100%); (b) PDC, Celite, tert-
BuOOH, C6H6, 0 °C to rt, 48 h (85%); (c) 10% Pd/C, HCOONH4, MeOH, reflux, 2 h
(90%); (d) TMSCl, pyridine, rt, 1 h; (e) LiAlH4, THF, –78 °C to rt, 16 h, separation of
a
Reference for the synthesis.
Minimum inhibitory concentration (90% growth inhibition) against MTB H37Rv
b
in MABA assay.
c
the 7a
-hydroxy and 7b-hydroxy isomers; (f) 10% HCl, THF, rt, 30 min (63% 11, 16%
Concentration effecting a 50% decrease in tetrazolium dye reduction by Vero
12, over three steps).
cells (African green monkey kidney cells). Both values are means of three replicate
experiments; for experiments that gave a value higher than the maximum con-
centration used, >128 is denoted; n.d. = not determined.
d
Selectivity index: SI = IC50/MIC90
INH = isoniazid and RMP = rifampicin (rifampin) used as positive controls; sol-
.
e
vent used as negative control.
H
H
b
c
H
O
H
H
O
H
F
RO
HO
(LDA) followed by addition of trimethylsilyl chloride afforded the
trimethylsilyl 6,7-enol ether, which on treatment with Selectfluor
H
H
13 R = H
14 R = TMS
15
a
and subsequent desilylation provided 7
3b-ol (15). Reduction of 15 using sodium borohydride stereoselec-
tively afforded 7 -fluorocholestane-3b,6b-diol (16).
a-fluoro-6-ketocholestan-
a
Clemmensen reduction of commercial diosgenin (17) afforded
(25R)-cholest-5-ene-3b,16b,26-triol (18) (Scheme 4).11,14 Silyl pro-
tection of the two hydroxy groups at C3 and C26, followed by
mesylation of the hydroxy group at C16, removal of the mesylate
by reduction with lithium aluminum hydride, and double desilyla-
tion led to (25R)-cholest-5-ene-3b,26-diol (19).
d
H
H
H
F
HO
H
OH
16
The oxygenated cholestan-3b-ols 1–19 described above were
investigated for their potential anti-TB activity and several hits
could be identified (Table 1). The minimum inhibitory concentra-
tions (MIC90 values) against the MTB strain H37Rv were obtained
Scheme 3. Synthesis of 7a-fluorocholestane-3b,6b-diol (16). Reagents and condi-
tions: (a) TMSCl, DMAP, THF, rt, 1 h (96%); (b) LDA, THF, –78 °C, 3 h, then TMSCl and
warming to rt (72%); (c) Selectfluor, DMF, rt, 15 min, then TBAF, THF, rt, 5 min
(71%); (d) NaBH4, MeOH, rt, 2 h (100%).

A. W. Schmidt et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6111–6113
6113
by the microplate alamar blue assay (MABA).2,15 In vitro cytotoxic-
References and notes
ities (IC50 values) were determined using Vero cells.2,16 The results
of our initial screening demonstrate that cholestan-3b-ol deriva-
tives which are oxygenated at specific positions of the steroidal
framework exhibit a significant anti-TB activity. The following con-
clusions could be drawn from our initial screening of oxygenated
cholestan-3b-ols. Among the 7-oxygenated cholestan-3b-ols
shown in Schemes 1 and 2, only 7-ketocholestan-3b-ol (4) and cho-
lestane-3b,7b,25-triol (12) showed anti-TB activity. Comparing
compounds 5 and 6, the orientation of the OH group at C-7 is not
important as both are inactive; whereas in the presence of an OH
group at C-25, the configutaion of OH at C-7 matters as compound
12 shows activity >5-fold than 11. The 6-ketocholestan-3b-ols
13–15 exhibit substantial anti-TB activities with MIC90 values
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cytotoxicity. A significant anti-TB activity (MIC90 = 6 lM) with no
toxicity for the mammalian cell line has been found for (25R)-
cholest-5-ene-3b,16b,26-triol (18). However, for the diol 19, result-
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group, no anti-TB activity has been detected.
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In summary, several oxygenated cholestan-3b-ols show signifi-
cant inhibitory activity against MTB H37Rv with no or relatively
low toxicity against the mammalian cell line (see compounds 4,
13, and 18). Additional structural modification of cholestane deriv-
atives directed towards improvement of their anti-TB potency may
perhaps pave the way to find new lead structures for potential
anti-TB drugs.
Acknowledgement
We are grateful to the ESF EuroMembrane Network (DFG grant
KN 240/13-1) for financial support.