Syntheses and biological activity studies of novel sterol analogs from nitroso diels-alder reactions of ergosterol

Article abstract of DOI:10.1021/ol900997t

A serles of novel sterol analogs was prepared using nitroso Dlels-Alder reactions with ergosterol. Most cycloaddltlon reactions proceeded In an excellent reglo- and stereoselective fashion. Further N-O bond cleavage of cycloadducts generated compounds with biological activity In PC-3 and MCF-7 cancer cell lines

Full text of DOI:10.1021/ol900997t

ORGANIC
LETTERS
2009
Vol. 11, No. 13
2828-2831
Syntheses and Biological Activity
Studies of Novel Sterol Analogs from
Nitroso Diels-Alder Reactions of
Ergosterol
Baiyuan Yang,† Patricia A. Miller,† Ute Mo¨llmann,‡ and Marvin J. Miller*,†
Department of Chemistry and Biochemistry, UniVersity of Notre Dame, Notre Dame,
Indiana 46556, and Leibniz Institute for Natural Products Research and Infection
Biology - Hans Knoell Institute, Beutenbergstrausse 11a, D-07745 Jena, Germany
mmiller1@nd.edu
Received May 9, 2009
ABSTRACT
A series of novel sterol analogs was prepared using nitroso Diels-Alder reactions with ergosterol. Most cycloaddition reactions proceeded
in an excellent regio- and stereoselective fashion. Further N-O bond cleavage of cycloadducts generated compounds with biological activity
in PC-3 and MCF-7 cancer cell lines.
Natural products and their derivatives represent the most
prolific source of molecular diversity in drug discovery.1
Functional-group transformation of natural products con-
stitutes one of the main avenues for generating pharma-
cologically relevant compounds with altered and some-
times improved biological properties. Typical chemical
derivatizations of natural products are often limited to
standard modification of nucleophilic or electrophilic
functional groups. Since many natural products contain
multiple functional groups of the same or similar type,
derivatization selectivity is often problematic. Hence, new
derivatization methods are still needed. Our effort in this
area has involved nitroso Diels-Alder (NDA) reactions
as efficient methods for derivatization and functionaliza-
tion of diene-containing natural products. We and others2
have demonstrated that many complex diene-containing
natural products readily undergo nitroso cycloadditions,
generating 1-amino-4- hydroxy-2-ene heterocycle scaffolds
with high regio- and stereoselectivity. Ergosterol (Figure
1), a natural sterol containing a conjugated diene, was
Figure 1. Ergosterol (1a) and ergosterol acetate (1b).
chosen for further exploration of this method for modular
enhancement of Nature’s diversity (MEND).2a Ergosterol
1a (ergosta-5,7,22-trien-3ꢀ-ol) is biologically relevant as
a precursor (a provitamin) to Vitamin D23 and an integral
component of fungal cell membranes. The presence of
ergosterol in fungal cell membranes coupled with its
† University of Notre Dame.
‡ Hans Knoell Institute.
(1) (a) Butler, M. S. Nat. Prod. Rep. 2005, 22, 162. (b) Newman, D. J.;
Cragg, G. M.; Snader, K. M. J. Nat. Prod. 2003, 66, 1022.
10.1021/ol900997t CCC: $40.75
Published on Web 06/04/2009
 2009 American Chemical Society

Scheme 1
absence in animal cell membranes makes it a useful target
N-adduct 5a, a much more sterically congested isomer
compared to adduct 4a.2d Reaction with 4-methylbenzo-
hydroxamic acid 2b gave a similar result, although
dioxazine 6b was only isolated as a mixture with adduct
5b (entry 2). In contrast, oxidation of 2-methoxybenzo-
hydroxamic acid 2c, phenylacetohydroxamic acid 2d,
L-leucine-derived hydroxamic acid 2e and tert-butyl
N-hydroxycarbamate 2f gave 4c-f as the sole products
in good to excellent yields (entry 3-6). Further K2CO3
mediated deacetylation of 4a-f, 6a gave compounds 7a-f
and 8a in 90% average isolated yields.
for antifungal drugs.4 Several groups have shown that
sterols with a heteroatom substituent at C-24 or C-25 are
effective antifungal compounds.5 Nitroso Diels-Alder
reactions with ergosterol acetate 1b were reported by
Kirby et al.;2d,e however, this method was never exploited
as a tool for derivatization of sterol compounds and the
dienophiles used were often limited to transient aroyl nitroso
species. Herein we report the syntheses of novel C5 and C8
disubstituted sterol analogs using nitroso Diels-Alder reac-
tions of ergosterol, and its acetate, with acyl- and iminoni-
troso agents, and wish to demonstrate the utility of this
methodology in natrual product derivatization by evaluation
of their biological activity.
A nitroso Diels-Alder reaction with ergosterol 1a was
also investigated using nitrosobenzene 8 as a representative
aryl nitroso dienophile (entry 1, Scheme 2). The NDA
1
reaction occurred based on TLC and H NMR analysis;
A series of hydroxamic acids 2a-e was obtained by
reaction of the corresponding methyl esters with hydroxy-
lamine;6 N-hydroxycarbamate 2f was prepared from direct
acylation of hydroxylamine with di-tert-butyl dicarbonate.
Oxidation of compounds 2a-f to acyl nitroso dienophiles
3a-f with tetrabutylammonium periodate in the presence
of ergosterol acetate 1b gave the 5R-N-8R-O-adducts 4a-f
in various yields (Scheme 1, Table 1). The stereoisomeric
however, adduct 11 was not able to be isolated in pure form
by chromatography. Additionally, cycloadditions with vari-
ous iminonitroso agents 10, mainly 2-nitrosopyridine deriva-
tives, were examined (Scheme 2). Those nitroso compounds
were prepared from aminoheterocyclic precursors 9 in a two-
step sequence (N, N-dimethyl sulfilimine intermediate forma-
tion, followed by oxidation using m-CPBA).7 Most of the
(2) (a) Li, F. Z.; Yang, B. Y.; Miller, M. J.; Zajicek, J.; Noll, B. C.;
Mollmann, U.; Dahse, H.-M.; Miller, P. Org. Lett. 2007, 9, 2923. (b)
Krchnak, V.; Waring, K. R.; Noll, B. C.; Moellmann, U.; Dahse, H.-M.;
Miller, M. J. J. Org. Chem. 2008, 73, 4559. (c) Ruan, B. F.; Pong, K.;
Jow, F.; Bowlby, M.; Crozier, R. A.; Liu, D.; Liang, S.; Chen, Y.; Mercado,
M. L.; Feng, X. D.; Bennett, F.; Schack, D. V.; McDonald, L.; Zaleska,
M. M.; Wood, A.; Reinhart, P. H.; Magolda, R. L.; Skotnicki, J.; Pangalos,
M. N.; Koehn, F. E.; Carter, G. T.; Abou-Gharbia, M.; Graziani, E. I. Proc.
Natl. Acad. Sci. U.S.A. 2008, 105, 33. (d) Kirby, G. W.; Mackinnon,
J. W. M. J. Chem. Soc., Perkin Trans. 1 1985, 887. (e) Kirby, G. W.;
Mackinnon, J. W. M. J. Chem. Soc., Chem. Commun. 1977, 1, 23. (f) Kirby,
G. W.; Bentley, K. W.; Horsewood, P.; Singh, S. J. Chem. Soc., Perkin
Trans. 1 1979, 3064. (g) Kirby, G. W.; Sweeny, J. G. Chem. Commun.
1973, 704.
Table 1. Results of Acylnitroso Diels-Alder Reactions
entry
compd
adduct
yield (%)
dioxazine
yield (%)
1
2
3
4
5
6
2a
2b
2c
2d
2e
2f
4a
4b
4c
4d
4e
4f
29
12
74
95
93
93
6a
6b
6c
6d
6e
6f
54
63a
0b
0b
0b
0b
a As a nonseparable mixture of dioxazine 6b and 5R-O-8R-N-adduct
(3) Rajakumar, K.; Greenspan, S. L.; Thomas, S. B.; Holick, M. F. Am. J.
Public. Health 2007, 97, 1746.
5b. b Not detected.
(4) (a) Keith, B. B.; Graham, D. Acta Biochim. Polonica 1995, 42, 465.
(b) Walsh, T. J.; Viviani, M. A.; Arathoon, E.; Chiou, C.; Ghannoum, M.;
Groll, A. H.; Odds, F. C. Med. Mycol. 2000, 38, 335.
(5) (a) Nes, W. D.; Guo, D.; Zhou, W. Arch. Biochem. Biophys. 1997,
342, 68. (b) Ator, M. A.; Schmidt, S. J.; Adams, J. L.; Dolle, J. M.; Kruse,
R. E.; Frey, L. I.; Barone, C. L. J. Med. Chem. 1992, 35, 100. (c) Acuna-
Johnson, A. P.; Oehlschlager, C.; Pierce, A. M.; Pierce, H. D.; Czyzewska,
E. K. Bioorg. Med. Chem. 1997, 5, 821. (d) Beuchet, P.; Dherbomez, M.;
Elkiel, L.; Charles, G.; Letourneux, Y. Bioorg. Med. Chem. Lett. 1999, 9,
1599.
1
configuration of 4a-f was assigned by H NMR studies
based on literature analysis of 4a.2d In the case of
benzohydroxamic acid 2a (entry 1, Table 1), similar to
Kirby’s observation,2d dioxazine 6a was isolated as the
major product in 54% yield, with adduct 4a as the minor
product in 29% yield. Compound 6a was formed as a
result of [3, 3] sigmatropic rearrangement of 5R-O-8R-
(6) Miller, M. J.; Biswas, A.; Krook, M. A. Tetrahedron 1983, 39, 2571.
(7) (a) Taylor, E. C.; Tseng, C. P.; Rampal, J. B. J. Org. Chem. 1982,
47, 552. (b) Sharma, A. K.; Swern, D. Tetrahedron. Lett. 1974, 16, 1503.
Org. Lett., Vol. 11, No. 13, 2009
2829

Scheme 2
Figure 2. X-ray of cycloadduct 11k with ergosterol acetate 1b.
further diversification. Adduct 11a was chosen as model for
optimizing reaction conditions. Reactions with SmI2/THF,8
Zn/CH3COOH, Mo(CO)6,9 etc. were attempted; however, all
of these conditions failed to yield desired product 12a
(Supporting Information). Detailed screenings showed that
Na amalgam10 successfully reduced the N-O bond of 11a
to give 12a in 82% yield (entry 1, Scheme 3). Then, a series
Scheme 3
a 11 failed to be isolated through silica gel chromatography. b 1.2 equiv
of nitroso was used. c Ergosterol acetate 1b was used.
iminonitroso compounds could be isolated and stored in pure
form, except for 10k. The NDA reactions between ergosterol
1a and iminonitroso species 10a-j usually were complete
within 30 min at 0 °C. In each case, the cycloadducts 11a-j
were obtained as single 5RO-8RN-adducts in good to
excellent yields after column chromatography (Scheme 2).
The regio- and stereochemistry of the adducts was assigned
based on our previous report and NMR studies.2a The
instability of nitroso agent 10k required its immediate use,
so it was trapped in situ with ergosterol acetate 1b.
Interestingly, both regioisomers 11k and 11l were obtained
in a 8.5:1 ratio in 95% combined yield (entry 12, Scheme
2). The major adduct 11k has the same stereochemistry as
cycloadducts 11a-j, based on X-ray crystallography (Figure
2). Further deacetylation of 11k generated compound 11m.
Clearly, 2-nitrosopyridines 10, as stabilized forms of imi-
nonitroso reagents, constitute an ideal combination of
reactivity and stability for NDA reactions with ergosterol,
relative to benzenenitroso 8 and acylnitroso moieties 3,
respectively
a
-Cl of R was partially reduced. b -Cl of R was fully reduced.
of ergosterol nitroso adducts was subjected to the optimized
Na(Hg) conditions. While low transformation was found with
acylnitroso adducts 7d and 7f (entries 2-3), moderate yields
were achieved for pyridinylnitroso adducts. Not surprisingly,
these conditions also reduced the -Cl substituent (entries
5-6).
Ergosterol peroxide 1311 is a natural sterol derivative that
possesses a variety of biological properties including im-
munosuppressive and antitumor activity. Simply removing
the N-Boc group of cycloadduct 7f afforded a quick access
We next set out to demonstrate the synthetic utility of
ergosterol nitroso cycloadducts through N-O bond cleavage,
a method for generating 1,4-amino alcohols suitable for
(8) Revuelta, J.; Cicchi, S.; Brandi, A. Tetrahedron. Lett. 2004, 45, 8375.
Org. Lett., Vol. 11, No. 13, 2009
2830

to compound 14 as an isoelectronic analog of ergosterol
peroxide 13 (Scheme 4).
Table 2. Results of Anticancer Screeningsa
% inhibition at 20 µM
IC50 (µM)
entry
compd
PC-3
MCF-7
PC-3 MCF-7
Scheme 4
1
2
3
4
5
6
7
8
1a
7a
7b
7c
7d
7e
7f
14
11a
11b
11c
11d-e, g-h
11i
11j
11k
11m
12
12a
12b
12e
12h
12j
10
64
40
94
76
57
73
48
<10
15
76
<10
22
15
15
15
97
97
97
96
95
89
82
15
-
-
-
14
14
6
14
8
20
>20
-
-
15
-
17
-
-
16
11
12
12
17
>20
-
-
-
-
-
-
-
-
9
6
6
7
10
4
9
Broad antibacterial testing of these novel sterol analogs
using the argar diffusion method revealed no significant
activity against Gram-positive or Gram-negative organ-
isms. However, N-O cleaved analogs had their largest
zone of inhibition against the fungal strain, Sporobolo-
myces salmonicolor, whereas ergosterol, 1a, and adducts
did not (Supporting Information). More interestingly,
N-O reduced compounds 12, 12a-b,e,h,j, along with
ergosterol acylnitroso adducts 7a-f, showed growth
inhibitory activity in MCF-7 (breast cancer) and PC-3
(prostate cancer) tumor cell assays, while parent erogsterol,
1a, and most pyridinylnitroso adducts were relatively
inactive (entries 1, 9, 10, 12, 14-16, Table 2). All N-O
reduced analogs reached the low micromolar range of
inhibition against both PC-3 and MCF-7 cells (entries
17-22). In general, all active compounds showed sig-
nificantly improved inhibitory activity against the MCF-7
cell line. These biological assays clearly suggest that the
nitroso heterocycle changed the biological activity profile
of its parent natural product.
In summary, we have reported that nitroso Diels-Alder
reactions between ergosterol (or ergostrol acetate) and
2-nitrosopyrdines as well as acylnitroso agents proceeded
with excellent regio- and stereoselectivity. A new class
of sterol analogs that show encouraging anticancer activity
was generated through N-O bond cleavage of the
cycloadducts. The method presented herein provides a
novel and efficient way to derivatize diene-containing
natural products. Efforts to determine the exact mechanism
10
11
12
13
14
15
16
17
18
19
20
21
22
29
100
<10
90
<10
29
8
94
20
94
-
6.5
2
3.5
3
100
100
100
100
100
100
100
100
100
100
2.5
2.5
a Trichostatin A was used as the positive control (MCF-7 IC50 ) 16
nM, PC-3 IC50 ) 160 nM).
of anticancer action and further diversification of ergos-
terol adducts are in progress.
Acknowledgment. We acknowledge NIH (GM075885)
for support of this research. We also thank Timothy
Wencewicz (UND) and Uta Wohlfeld (HKI) for performing
antibacterial assays, the NMR facility at Notre Dame, Dr.
Allen Oliver (UND) for X-ray crystallography, Nonka
Sevova (UND) for mass spectroscopic analyses. B.Y.Y
acknowledges a Reilly Graduate Fellowship (2008-2009,
UND). We dedicate this paper to Prof. Jeremiah P. Freeman
on the occasion of his 80th birthday and with deep gratitude
for his 25 years of service to organic chemistry as secretary
of Organic Syntheses.
Supporting Information Available: Full experimental
procedures, characterization data and copies of 1H NMR and
13C NMR spectra, protocols of antibacterial and anticancer
assay. This material is available free of charge via the Internet
at http://pubs.acs.org.
(9) Cicchi, S.; Goti, A.; Brandi, A.; Guarna, A.; De Sarlos, F. D.
Tetrahedron Lett. 1990, 31, 3351.
(10) Keck, G. E.; Fleming, S.; Nickell, D.; Weider, P. Syn. Commun.
1979, 9, 281.
(11) (a) Bok, J. W.; Lermer, L.; Chilton, J.; Klingeman, H. G.; Towers,
G. H. N. Phytochem. 1999, 51, 891. (b) Kim, D. S.; Baek, N. I.; Oh, S. R.;
Jung, K. Y.; Lee, I. S.; Kin, J. K.; Lee, H. K. Arch. Pharm. Res. 1997, 20,
201.
OL900997T
Org. Lett., Vol. 11, No. 13, 2009
2831