Novel endoperoxides: Synthesis and activity against Candida species

Article abstract of DOI:10.1016/j.bmcl.2005.10.101

Fifteen new endoperoxides have been synthesised and tested for activity against pathogenic Candida species. These endoperoxides can be prepared in high yields, in one to three steps, from inexpensive starting materials. Despite chemical and structural sim

Full text of DOI:10.1016/j.bmcl.2005.10.101

Bioorganic & Medicinal Chemistry Letters 16 (2006) 920–922
Novel endoperoxides: Synthesis and activity against
Candida species
Peter Macreadie,a Thomas Avery,b Ben Greatrex,b Dennis Taylorb and Ian Macreadiea,*
aCSIRO, Molecular and Health Technologies, Parkville, 3052 Victoria, Australia
bUniversity of Adelaide, Department of Chemistry, Adelaide 5005, South Australia, Australia
Received 14 October 2005; revised 28 October 2005; accepted 28 October 2005
Available online 15 November 2005
Abstract—Fifteen new endoperoxides have been synthesised and tested for activity against pathogenic Candida species. These end-
operoxides can be prepared in high yields, in one to three steps, from inexpensive starting materials. Despite chemical and structural
similarities, their inhibitory activity against Candida growth varied greatly from one endoperoxide to another, and one species to
another. This study of susceptibility to endoperoxide compounds presented here may lead to the development of potent new
antifungal agents.
Ó 2006 Published by Elsevier Ltd.
Endoperoxides are a remarkable class of organic perox-
ides, having significant chemical and biochemical prop-
erties.1 One endoperoxide of particular importance is
the plant-derived drug, artemisinin (Fig. 1), which has
been shown to be a potent and fast-acting antimalarial
drug.2,3 More recently, the activity of artemisinin and
derivatives against other tropical parasites4 and various
cancer cells5,6 has been demonstrated. The high cost of
Figure 1. Artemisinin.
artemisinin has led to searches for synthetic endoperox-
ides with antimalarial activity.7 We have made similar
pounds with divergent mechanisms of action, appropri-
searches and produced a series of endoperoxides with
ate pharmaceutical properties, with high potency and
weak antimalarial activity.8 In this study, we investigat-
broad spectrum of activity.
ed these compounds for activity against three clinically
important Candida species.
A robust synthetic procedure for the ready construc-
tion of endoperoxides of types 4 and epoxy-endoperox-
ides (5 and 6) has been developed according to the
The occurrence of life-threatening fungal infections is
increasing worldwide.9–12 The most common fungal
generalised scheme shown in Figure 2.8 Key features
include a cycloaddition of a singlet oxygen with the
infections of humans are caused by Candida yeast.13
Of the antifungals currently available to suppress
appropriate 1,3-butadiene (3a–e) to generate the core
Candida growth, none satisfy the medical world com-
of the endoperoxide ring and form compounds of type
pletely.14 Their limitations include weak potency, high
4a–e. Further oxidation with m-CPBA at ambient tem-
cost of development, host toxicity, limited spectrum
of efficacy and deleterious drug–drug interactions.15
In addition, the emergence of drug-resistant isolates
continues to increase.16 Therefore, there is an acute
perature produces the epoxy-endoperoxides 5 and 6.8,17
This latter conversion produces, from a single precur-
sor, both epoxy-endoperoxides 5 and 6, which have
the epoxide oxygen atom either on the opposite face
need for the development of novel antifungal com-
(compound 5) or the same face (compound 6) to the
alkyl or aryl substituents. Consequently, two unique
versions of these new pro-drugs are readily available
for examining structure–activity relationships. Further-
more, our previous studies17,18 reported that exposure
Keywords: Antifungal; Artemisinin; Candida; Endoperoxide.
*
Corresponding author. Tel.: +61 3 9662 7299; fax: +61 3 9662
7266; e-mail: ian.macreadie@csiro.au
0960-894X/$ - see front matter Ó 2006 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2005.10.101

P. Macreadie et al. / Bioorg. Med. Chem. Lett. 16 (2006) 920–922
921
R1
O
(a) R1 = R2 = cyclohexyl
O
OH
(b) R1 = R2 = cyclopentyl
R2
H
(c) R1 = R2 = cyclobutyl
(d) R1 = cyclohexyl R2 = H
(e) R1 = cyclopentyl R2 = H
7a
iii
R1
R1
R2
H
R1
R2
H
R1
R2
H
H
H
H
H
i
ii
H
H
O
O
O
O
+
O
O
O
O
H
R2
H
H
3
5
4
6
43-55%
major isomer
49-68%
minor isomer
27-41%
Figure 2. Scheme for synthesis of novel endoperoxides. Reagents and conditions: (i) O2, Rose Bengal bis(triethylammonium) salt, hm, CH2Cl2,
7 h, 5 °C; (ii) m-CPBA, CH2Cl2, 25 °C; (iii) Co(II)salen or NEt3, CH2Cl2, 25 °C.
of epoxy-endoperoxides (5 and 6) to catalytic amounts
of cobalt(II) salen complexes resulted in a clean free
radical rearrangement resulting in the formation of
the potential downstream product (7). Subsequently,
one of these ring-opened products (7a) was purified
in order to evaluate its activity.
compound 4d displayed high growth inhibitory activity
to C. albicans, C. krusei and C. tropicalis.
The work presented here describes the examination of a
series of related endoperoxide compounds plus a down-
stream derivative for their potential activity as anti-
fungal compounds.
The inhibitory activity of amphotericin B, ketoconazole
and our endoperoxide compounds was assayed against
Candida albicans, Candida krusei and Candida tropicalis
(Table 1).19 Growth of Candida species was inhibited by
all our compounds to greater or lesser degrees. The
activity of individual compounds generally varied with
species. Amphotericin B was the most potent compound
tested, but some of our endoperoxides had activities
comparable with that of ketoconazole. In particular,
For all compounds, the route for construction was ro-
bust, safe, inexpensive and could be performed on a
large scale. Many of the endoperoxides in this series
not only had the endoperoxide linkage but also had a
third oxygen atom (forming the epoxide group), which
is positioned in an environment similar to that of the
ether oxygen atom within artemisinin. In addition, co-
balt catalysis was used to induce ring-opening of the
endoperoxides, providing a synthetic route to the con-
struction of isomeric ring-opened compounds with po-
tential use as pro-drugs. This free radical mechanism
to open the ring is not an unlikely fate of endoperoxides
in the body due to the presence of a relatively weak and
easily cleaved oxygen–oxygen bond. For example, the
prostaglandin endoperoxide exists as a short-lived inter-
mediate in the body.20 The synthesis of ring-opened ana-
logues allowed the possible downstream products to be
tested for activity. For example, it appears that epoxide
7a, a downstream product for epoxy-endoperoxide
5a, has a higher activity against C. krusei and C. tropi-
calis than its parent compound. While the apparent
minimum pharmacophore for biological activity of most
of the endoperoxides is the endoperoxide ring, activity
was slightly increased for compound 7a (ring-open
structure).
Table 1. Growth inhibition of some Candida species by endoperoxide
compounds and reference drugs
Compound
IC50 (lM)
Candida
albicans
Candida
Candida
krusei
tropicalis
Amphotericin B
Ketoconazole
<0.5
0.1–0.4
12–47
0.2–0.4
100–200
100–200
400–800
>1000
250–500
250–500
>1000
Nystatin
4a
5a
100–200
200–400
200–400
25–50
100–200
100–200
250–500
250–500
125–250
250–500
125–250
250–500
250–500
9.5–37
6a
7a
400–800
500–1000
>1000
63–125
125–250
125–250
125–250
125–250
500–1000
250–500
40–80
4b
5b
63–125
6b
4c
125–250
250–500
500–1000
500–1000
75–150
A number of endoperoxide compounds showed inhibi-
tory activity against the pathogenic yeast C. krusei. This
is of notable importance since C. krusei is resistant to
fluconazole and many other antifungal agents.21 It has
been hypothesised that the extensive use of fluconazole
is responsible for the changing epidemiology of fungal
infections, leading to the emergence of drug-resistant
5c
6c
4d
5d
250–500
500–1000
500–1000
250–500
125–250
250–500
250–500
75–150
500–1000
>1000
4e
5e
500–1000
300–600
6e

922
P. Macreadie et al. / Bioorg. Med. Chem. Lett. 16 (2006) 920–922
5. Chen, H. H.; Zhou, H. J.; Fang, X. Pharmacol. Res. 2003,
48, 231.
strains such as C. krusei; however, a causal relationship
has not yet been established and the theory remains con-
troversial.22 Nevertheless, the activity of the endoperox-
ides described here against C. krusei demonstrates their
potential utility as novel antifungals for drug-resistant
non-albicans Candida species.
6. Efferth, T.; Dunstan, H.; Sauerbrey, A.; Miyachi, H.;
Chitambar, C. R. Int. J. Oncol. 2001, 18, 767.
7. Vennerstrom, J. L.; Arbe-Barnes, S.; Brun, R.; Charman,
S. A.; Chiu, F. C.; Chollet, J.; Dong, Y.; Dorn, A.;
Hunziker, D.; Matile, H.; McIntosh, K.; Padmanilayam,
M.; Santo Tomas, J.; Scheurer, C.; Scorneaux, B.; Tang,
Y.; Urwyler, H.; Wittlin, S.; Charman, W. N. Nature 2004,
430, 900.
8. Taylor, D. K.; Avery, T. D.; Greatrex, B. W.; Tiekink, E.
R.; Macreadie, I. G.; Macreadie, P. I.; Humphries, A. D.;
Kalkanidis, M.; Fox, E. N.; Klonis, N.; Tilley, L. J. Med.
Chem. 2004, 47, 1833.
9. Anaissie, E. Clin. Infect. Dis. 1992, 14, S43.
10. Greenwood, D. J. Med. Microbiol. 1998, 47, 751.
11. Kam, L. W.; Lin, J. D. Am. J. Health Syst. Pharm. 2002,
59, 33.
12. Krcmery, V., Jr.; Matejicka, F.; Pichnova, E.; Jurga, L.;
Sulcova, M.; Kunova, A.; West, D. J. Chemother. 1999,
11, 385.
Research aimed at assessing the clinical usefulness of
novel drugs depends critically on in vitro toxicity assays.
Some of the endoperoxides described here have high
haemolytic activity,8 suggesting that potential problems
with haemolysis may result from internal applications.
However, the high haemolytic potential of some endop-
eroxides may not be a major toxic liability when they are
to be used to treat superficial fungal infections. A dis-
tinct pattern implicating the presence or absence of cer-
tain structural features in haemolytic activity has been
reported.8 Modification of these haemolysis-causing
structural features should allow an improvement to the
overall therapeutic profile of these endoperoxides by
reducing their haemolytic activity.
13. McCullough, M. J.; Ross, B. C.; Reade, P. C. Int. J. Oral
Maxillofac. Surg. 1996, 25, 136.
14. Georgopapadakou, N. H. Curr. Opin. Microbiol. 1998, 1,
547.
15. Barrett, D. Biochim. Biophys. Acta 2002, 1587, 224.
16. Kontoyiannis, D. P.; Lewis, R. E. Lancet 2002, 359, 1135.
17. Greatrex, B. W.; Jenkins, N. F.; Taylor, D. K.; Tiekink, E.
R. J. Org. Chem. 2003, 68, 5205.
As part of continuing studies, we propose to further
improve the efficacy of these endoperoxides by adding
substituents onto the cyclic ring systems. In the case of
the endoperoxide artemisinin, addition of an amino side
chain has been shown to improve efficacy,23 presumably
by enhancing drug uptake. If the efficacy of the endop-
eroxides described here can be improved and effective in
vivo, it would provide a much-needed alternative for
treating fungal infections. The endoperoxide com-
pounds described in this work are structurally unrelated
to any other antifungals in clinical use and provide an
account of the utility of endoperoxides as biologically
active agents.
18. Avery, T. D.; Jenkins, N. F.; Kimber, M. C.; Lupton, D.
W.; Taylor, D. K. Chem. Commun. (Camb) 2002, 28.
19. In vitro testing was performed by the microbroth dilution
technique (96-well microplate) with a starting inoculum
of optical density at A595 of 0.2–0.3 in YEPD (1% yeast
extract, 2% peptone, 2% glucose and 1.5% agar). Drugs
and endoperoxide compounds were then added as
twofold serial dilutions down from 1 mM concentrations.
A growth control was included in the same microplate.
The microplate was incubated in a microplate shaker at
35 °C, and the absorbance at 595 nm was measured at
the time of compound addition and 2 h later using a
microplate reader (Labsystem Multiscan Ascent). Each
sample was assayed in triplicate. Absorbance values were
averaged and are plotted against the drug and compound
concentration, and the concentration required to inhibit
50% growth (IC50) was calculated. Candida strains
employed in this study consisted of two clinical isolates,
C. albicans JRW #5 and C. krusei WM 03,204 and the
American Type Culture Collection strain C. tropicalis
750.
Acknowledgments
The study described here was funded by the Brailsford
Robertson Award. The provision by Prof. Wieland
Meyer and Dr. John Warmington of strains of Candida
is gratefully acknowledged. We also thank Dr. Morry
Frenkel and Paul Vaughan for helpful comments in
the preparation of this manuscript.
20. Smith, W. L.; Song, I. Prostaglandins Other Lipid Mediat.
2002, 68-69, 115.
References and notes
21. Samaranayake, Y. H.; Samaranayake, L. P. J. Med.
Microbiol. 1994, 41, 295.
1. Saito, I.; Nittala, S. The Chemistry of Peroxides; Wiley:
New York, 1983.
2. Meshnick, S. R. Int. J. Parasitol. 2002, 32, 1655.
3. OÕNeill, P. M.; Posner, G. H. J. Med. Chem. 2004, 47, 2945.
4. Utzinger, J.; Chollet, J.; Tu, Z.; Xiao, S.; Tanner, M.
Trans. R. Soc. Trop. Med. Hyg. 2002, 96, 318.
22. White, T. C.; Holleman, S.; Dy, F.; Mirels, L. F.; Stevens,
D. A. Antimicrob. Agents Chemother. 2002, 46, 1704.
23. Hindley, S.; Ward, S. A.; Storr, R. C.; Searle, N. L.; Bray,
P. G.; Park, B. K.; Davies, J.; OÕNeill, P. M. J. Med.
Chem. 2002, 45, 1052.