Design of endoperoxides with anti-Candida activity

Article abstract of DOI:10.1016/j.bmc.2006.10.021

Broad antifungal structure-activity relationships governing epoxy-endoperoxides 2 and 3 and their parent endoperoxides 1 are reported. Their inhibitory activity against Candida albicans in conjunction with hemolytic activity and/or growth inhibition of cu

Full text of DOI:10.1016/j.bmc.2006.10.021

Bioorganic & Medicinal Chemistry 15 (2007) 36–42
Design of endoperoxides with anti-Candida activity
a
b
a
a
Thomas D. Avery, Peter I. Macreadie, Ben W. Greatrex, Tony V. Robinson,
a,
b,
*
*
Dennis K. Taylor and Ian G. Macreadie
a
Department of Chemistry, The University of Adelaide, 5005 SA, Australia
CSIRO, Molecular and Health Technologies, Parkville, 3052 Vic., Australia
b
Received 29 June 2006; revised 21 September 2006; accepted 11 October 2006
Available online 13 October 2006
Abstract—Broad antifungal structure–activity relationships governing epoxy-endoperoxides 2 and 3 and their parent endoperoxides
are reported. Their inhibitory activity against Candida albicans in conjunction with hemolytic activity and/or growth inhibition of
1
cultured mammalian cells are reported. This information provided guidance for the further development of endoperoxide and
epoxy-endoperoxides as topical antifungal agents.
ꢀ
2006 Elsevier Ltd. All rights reserved.
1
. Introduction
addressing these issues are needed. One avenue is to ex-
plore novel chemical moieties with action through diver-
gent mechanisms; high potency and broad-spectrum
activity are also of paramount importance.
There is an ever-increasing number of peroxide-contain-
ing natural products being isolated, with nearly all
showing interesting and important biological activities
1
worthy of investigation, Figure 1.
We were inspired, whilst evaluating epoxy-endoperox-
8
ides as antimalarials, to investigate the potential of
9
these compounds as antifungals. Our study highlighted
None has had more interest than leading antimalarial
2
natural product artemisinin: the importance of this
compound and its expensive production has been the
driving force behind an incredible number of analogue
a robust, concise and inexpensive synthesis of endoper-
oxides which in some cases demonstrated better broad-
spectrum antifungal activity than two of the current
drugs on the market. Importantly, some endoperoxide
candidates showed good activity against Candida krusei,
3
syntheses. More recently endoperoxides exhibiting anti-
fungal activity have been isolated from marine sponges
of the species Plakortis, Figure 1, however a general syn-
thesis of endoperoxides of this structure is yet to be
4
5
CH3
reported. Fungal infections in humans due to aging
H
populations and an increased number of medically in-
duced immunodepressed patients, coupled with relative-
ly low activities of current drugs, are becoming an issue
requiring urgent attention. Primarily the source of infec-
tion is Candida yeast strains and current drugs are limit-
ed in their spectrum of efficacy and in many cases display
O
O
H3C
O
(
CH ) Ph
O
2 10
O
H
O
H
O
O
H
CH3
O
Artemisinin: potent commercial
6-epiplakortolide E:
potent cytotoxicity to various
cancer lines.
6
antimalarial drug
host toxicity and adverse drug–drug interactions. Drug
resistance is an emerging trend in virtually all treatments
7
O O
of disease and Candida is no exception. New antifungals
Et
MeO2C
Et
Et
Keywords: Antifungal; Endoperoxide; Candida; Hemolytic activity;
Artemisinin; Growth inhibition.
Plakortis isolate: micromolar active
*
Corresponding authors. Tel.: +61 8 8303 5494; fax: +61 8 8303 4358
D.K.T); Tel.: +61 3 9662 7299; fax: +61 3 9662 7266 (I.G.M); e-mail
addresses: dennis.taylor@adelaide.edu.au; ian.macreadie@csiro.au
antifungal.
(
Figure 1. Bioactive endoperoxide natural products.
0
968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.021

T. D. Avery et al. / Bioorg. Med. Chem. 15 (2007) 36–42
37
a strain resistant to Fluconazole and other commercial
Table 1. Growth inhibition of Candida albicans exposed to endoper-
oxide compounds and commercial drugs
1
0
antifungal agents. Prompted by the promising results
of this limited study, we now present the synthesis and
antifungal activity of a large range of epoxy-endoperox-
ides and their parent endoperoxides.
Compound
IC50 (lM)
Amphotericin B
Ketoconazole
Nystatin
<0.5
250–500
250–500
31–63
1a
2a
1b
2b
3b
31–63
2
. Results and discussion
125–250
125–250
125–250
63–125
125–250
>1000
Endoperoxides 1a–r were synthesized from the appro-
priate 1,3-butadiene through photosensitized cycloaddi-
1c
2c
1d
1
1
tion of singlet oxygen. Epoxidation was achieved in
good yield through treatment of endoperoxides with
1
2
2
1
2
d
e
e
>1000
m-chloroperbenzoic acid, generating isomers 2 and 3.
Endoperoxides 1m and 1n were synthesized from 1k
through DCC coupling with the appropriate carboxylic
63–125
500–1000
125–250
125–250
125–250
63–125
63–125
63–125
250–500
500–1000
250–500
500–1000
500–1000
250–500
>1000
13
14
1f
acid. Tricyclic endoperoxide 4 was synthesized and
evaluated for antifungal activities as a mixture of iso-
mers Scheme 1.
2
3
1
2
f
f
g
g
Generally a mono-substituted alkyl chain or cycloalkyl
grouping at the R position resulted in good antifungal
activity (1a, 1b, 1f, and 1g), with individual series
3g
1h
2h
1
1
2
3
i
i
i
(
2
parent endoperoxide and epoxy-endoperoxides) 1b,
b, 3b, etc., appearing in the same IC50 brackets,
Table 1. Notably cis or trans orientation of the epoxide
1
3
1j
moiety with respect to the R and R groups made no
difference to anti-Candida activity in these series.
2
3
1
2
j
j
>1000
16–31
>1000
k
k
Deviation from the general trend (where epoxides and
parent endoperoxides have similar activity) was ob-
served with endoperoxides containing tethered hydroxyl
1l
2l
3l
125–250
250–500
125–250
125–250
>1000
(
1k and 1o), ester (1l–n) or ether groups (1p and 1q) at
3
1
2
3
1
2
3
1
2
1
m
the R position, with the epoxides generally showing
much poorer activity than the parent endoperoxides,
compare compounds 1k and 2k, for example. Addition-
ally it was noted that within each series the cis epoxides
showed equal to or better activity than the correspond-
m
m
n
250–500
250–500
>1000
n
n
>1000
250–500
>1000
o
o
p
250–500
125–250
63–125
16–31
O
O
a
O
O
O O
R1
R3
R1
R3
+
R1
R3
2p
3
p
R2
R2
O
R2
a: R1 = Ad, R2 = R = H
O
1q
2q
3q
1r
2r
4
3
250–500
63–125
31–63
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2a-r
3b,f,g,i,j,l-n,p,q
1
2
3
b: R = CH2Ad, R = R = H
c: R1 = H, R2 = Ad, R3 = H
d: R1 = Ad, R2 = H, R3 = Ad
500–1000
16–31
e: R = H, R2 = cC6H11, R = H
f: R = CH2cC6H11, R2 = R3 = H
1
3
1
g: R = C H , R2 = R3 = H
1
7
15
2
1
3
O
h: R = H, R = C H13, R = H
6
O
1
2
3
i: R = C H15, R = H, R = C H
7
3
7
ing trans epoxide. However, as with the series of the al-
kyl and cycloalkyl-substituted endoperoxides, their
activities varied greatly, for example 1q is in the 16–
31 lM range, while 1p was in the 250–500 lM range.
j: R = C H , R = C H , R3 = H
1
2
O
4
7
15
6 13
3
1
2
k: R = CH3, R = H, R = CH2OH
l: R = CH3, R = H, R = CH2OCOAd
m: R = CH3, R = H, R = CH2OCOCH2Ad
n: R = CH3, R = H, R = CH2OCOCH2Ad(3-Br)
o: R = CH3, R = H, R = (CH2)3OH
p: R = CH3, R = H, R = CH2OBn
q: R = CH2OBn, R = H, R3 = CH2OBn
1
2
3
b
c
1
2
3
1
2
3
1
2
3
In order to broaden the scope of functionality, phenyl-
substituted endoperoxide 1r and tricyclic epoxy-endo-
peroxide 4 were also evaluated as antifungal agents.
With their activity in the low-micromolar range, they
also showed potential as building blocks for derivatisa-
tion, in order to improve antifungal activity.
1
2
3
1
2
r: R = Ph, R2 = H, R3 = Ph
1
2 2
Scheme 1. Reagents: (a) MCPBA; (b) DCC, AdCH CO H, DMAP;
(
c) DCC, 3-BrAdCH CO H, DMAP.
2
2

38
T. D. Avery et al. / Bioorg. Med. Chem. 15 (2007) 36–42
Table 2. Hemolytic activities of selected novel endoperoxides
A mammalian cell-toxicity assay was performed on
compounds 1k, 1q, 3q, 1r, and 4, which demonstrated
relatively low IC50 values. This test was used to evaluate
mammalian cell growth inhibition characteristics of po-
tential lead compounds, which were compared to the
commercial drugs Fluconazole and Amphotericin B,
Figure 2.
a
Hemolysis (%)
Compound
IC50 (lM)
1
2
1
2
1
2
1
2
3
1
1
3
4
a
a
g
g
h
h
i
31–63
31–63
100
41
60
33
61
31
86
45
25
0
63–125
63–125
250–500
500–1000
250–500
500–1000
500–1000
16–31
Compounds 1q, 1r, and 4 showed significant growth
inhibitory activity levels at 0.1 mM and 1.0 mM while
compounds 1k and 3q were inhibitory at 1.0 mM but
only moderately toxic at 0.1 mM with 1k showing the
best profile. The solvent (ethanol) was not inhibitory
at levels of up to 5% and fluconazole was not inhibitory
at 1 mM. Amphotericin B was also used for comparison
and was not growth inhibitory at 0.1 mM. For the test-
ing of Amphotericin B at a level of 1.0 mM, the cells
were exposed to 5% DMSO, a level that caused about
i
i
k
q
q
16–31
63–125
16–31
2
1
0
a
Percent hemolysis of erythrocytes induced by 1 mM compound.
5
0% inhibition of cell growth. The Amphotericin B
Compounds 1a, 2a, 1c, 1e, 1g, 2g, 3g, 1k, 3p, 1q, 3q, 1r,
and 4 showed promising growth inhibition activity
against C. albicans when compared to those of commer-
cially available antifungal drugs, Ketoconazole and
Nystatin. The activity of these endoperoxides is still
however an order of magnitude higher than that of
Amphotericin B.
caused no additional growth inhibition compared to sol-
vent alone.
In conclusion, a range of novel endoperoxides and their
epoxy derivatives were assessed for antifungal activity
on C. albicans. A range of activities were observed with
some very promising results enabling determination of
broad structure activity relationships. Some of the more
promising candidates were evaluated for hemolytic
activity and mammalian cell toxicity. Compounds con-
taining alkyl substituents with corresponding good
IC50 values tended to be hemolytic. Diaryl endoperoxide
A selection of the more promising compounds were
evaluated for hemolytic activity at the upper concentra-
tion limit of the IC50 assay (1 mM) to ascertain their
potential as serum administered drugs, Table 2. As pre-
viously observed the epoxides showed much less hemo-
lysis activity than their parent endoperoxides,
1
r and tricyclic epoxy endoperoxide 4 were also evaluat-
8
ed to expand our knowledge of antifungal activities of
peroxides. Although good IC50 levels were observed,
both compounds were found to be toxic in the mamma-
lian cell toxicity assay.
however, the epoxides showed poorer antifungal activ-
ity than their parent endoperoxide (vide supra). Evalu-
ating the hemolysis data against the IC50 values, it
appeared that compounds 1a, 2a, 1g, 2g, 1k, 1q, 3q,
and 4 would be suitable for further investigation as
their IC50 values are much lower than the 1 mM con-
centration used during the hemolysis assay. Com-
pounds 1k, 1q, and 4 were particularly promising,
showing no hemolysis at this concentration in conjunc-
tion with a low IC50 value. It is suspected that high
hemolytic activity is associated with the extended
hydrocarbon chains, which are able to insert into the
Compound 1k was found to have good antifungal activ-
ity coupled with 0% hemolysis and only moderate toxic-
ity to mammalian cells. We feel that this compound and
possibly derivatives thereof may be good candidates for
further evaluation. Moreover other compounds tested
may represent good candidates for topical applications
as hemolytic activity is not necessarily detrimental to
being a good candidate in this case.
8
lipid bilayer.
100
0
1
.1mM
.0mM
8
6
4
2
0
0
0
0
0
4
1r
1k
3q
1q
Fluconazole Amphotericin
B
Figure 2. Mammalian cell toxicity of selected endoperoxides (means ± SE).

T. D. Avery et al. / Bioorg. Med. Chem. 15 (2007) 36–42
39
3. Experimental
3.5. 3-Heptyl-4-hexyl-3,6-dihydro-1,2-dioxine (1j)
3
.1. General methods
Yield: 584 mg, 34%; colourless oil; R 0.25 (1:19 ethylac-
f
1
etate/hexane); H NMR (300 MHz, CDCl ): d 0.86–0.90
3
Solvents were dried by appropriate methods wherever
needed. Thin-layer chromatography (TLC) used alu-
minium sheets coated with silica gel 60 F254 (40 ·
(m, 6H), 1.28–1.72, (m, 20H), 2.00 (t, J = 7.2 Hz, 2H),
4.27–4.31 (m, 1H), 4.40–4.47 (m, 1H), 4.56–4.63 (m,
1
3
1H), 5.59 (br s, 1H); C NMR (75 MHz, CDCl ): d
3
8
0 mm) and visualized under 254 nm light, or developed
14.1, 22.59, 22.63, 25.6, 27.3, 29.0, 29.2, 29.5, 31.1,
31.7, 31.8, 32.2, 70.0, 81.1, 117.3, 139.0; IR (neat):
in vanillin or permanganate dip. Flash chromatography
was conducted using silica gel 60 of particle size
À1
+
1671, 1348, 1059 cm ; MS m/z (+EI): 268 (M , 5),
1
.040–0.063 mm. Melting points were uncorrected. H
NMR and C NMR spectra were recorded in CDCl3
solution at either 300 or 600 MHz instrument, TMS
0
251 (44), 236 (16), 153 (100), 127 (27), 57 (56); HRMS
1
3
+
+
(+EI) (M+Na) found 291.2296; (M+Na) calcd for
C H O Na 291.2300.
1
7
32
2
(
0 ppm) and CDCl (77.0 ppm) were used as internal
3
standards. All yields reported refer to isolated material
judged to be homogeneous by TLC and NMR
spectroscopy.
3.6. ± (3R,6R)-3-[(Benzyloxy)methyl]-6-methyl-3,6-dihy-
dro- 1,2-dioxine (1p)
Yield: 2.27 g, 65%; colourless oil; R 0.4 (1:9 ethylace-
f
1
3
1
.2. General procedure for the synthesis of endoperoxides
1
a-k, 1n-r
tate/hexane); H NMR (300 MHz, CDCl ): d 1.22 (d,
3
1
J = 6.6 Hz, 3H), 3.57 (dd, J = 10.8, 3.9 Hz, 1H), 3.78
(dd, J = 10.8, 7.5 Hz, 1H), 4.54–4.70 (m, 3H), 4.76 (qq,
J = 6.6, 1.5 Hz, 1H), 5.87 (ddd, J = 10.5, 3.0, 1.5 Hz,
A solution of the appropriate parent 1,3-butadiene in
1
3
1H), 5.94 (ddd, J = 10.5, 1.5, 1.5 Hz, 1H); C NMR
CH Cl (30 mL/g) was photolysed with 3 · 500 W hal-
ogen lamps in the presence of Rose Bengal bis(triethyl-
ammonium) salt (30 mg/g of 1,2-butadiene) through
which was bubbled a constant stream of oxygen for
2
2
(75 MHz, CDCl ): d 17.8, 70.3, 73.5, 74.3, 78.3, 123.9,
3
127.6, 127.7, 128.3, 131.0, 138.2; IR (neat): 1496, 1453,
1366, 1091, 737, 720, 698 cm ; MS m/z (+EI): 221
À1
+
([M+H] , 17), 122 (23), 105 (57), 91 (100); HRMS
6
h. The reaction was performed in a Pyrex flask fitted
+
+
with an external cooling jacket. The solution was con-
centrated in vacuo and the resulting residue purified by
flash chromatography. For known endoperoxides see
(+EI) (M) found 220.1096; (M) calcd for C H O
13 16 3
220.1099.
8
15
16
15
8
the following: compounds 1a, 1c, 1g, 1h, 1i,
8
3.7. ± (3R,6S)-3,6-Di[(benzyloxy)methyl]-3,6-dihydro-
1,2- dioxine (1q)
1
6
8
11
15
8
1
k, 1l, 1r , 1e, 1f, and 1o.
3
(
.3. ± 3-(1-Adamantylmethyl)-3,6-dihydro-1,2-dioxine
1b)
Yield: 908 mg, 28%; colourless oil; R 0.48 (1:4 ethylac-
f
1
etate/hexane); H NMR (300 MHz, CDCl ): d 3.45 (dd,
3
J = 10.8, 6.6 Hz, 2H), 3.68 (dd, J = 10.8, 7.2 Hz, 2H),
4.53 (d, J = 12.0 Hz, 2H), 4.62 (d, J = 12.0 Hz, 2H),
4.76–4.79 (m, 2H), 4.97 (m, 2H), 7.25–7.34 (m, 10H);
Yield: 234 mg, 37%; colourless oil; R 0.49 (1:19
f
1
ether/hexane); H NMR (200 MHz, CDCl ): d 1.23
3
1
3
(
dd, J = 15.0, 3.2 Hz, 1H), 1.43 (dd, J = 15.0,
C NMR (75 MHz, CDCl ): d 69.9, 73.4, 78.4, 126.1,
3
8
4
.4 Hz, 1H), 1.74–1.56 (m, 12H), 1.96 (br s, 3H),
.42 (dddd, J = 16.4, 2.2, 2.2, 2.2 Hz, 1H), 4.62
127.6, 128.3, 137.9; IR (neat): 1496, 1453, 1363,
1090 cm ; MS m/z (+EI): 327 [(M+H) , 20], 181
À1
+
+
(
dddd, J = 16.4, 2.2, 2.2, 2.2 Hz, 1H), 4.85–4.76 (m,
(100); HRMS (+EI) (M+H)
(M+H) calcd for C H O 327.1596.
found 327.1603.;
+
1
5
H), 5.81 (dddd, J = 10.4, 2.2, 2.2, 2.2 Hz, 1H),
.92 (dddd, J = 10.4, 2.2, 2.2, 2.2 Hz, 1H);
2
0
23
4
1
3
C
NMR (50 MHz, CDCl ): d 28.6, 32.0, 36.9, 42.7,
3.8. General procedure for DCC coupling of carboxylic
1
acids to endoperoxide 1k
3
3
4
1
2
7.0, 69.5, 75.3, 123.4, 129.8; IR (neat): 1451, 1106,
À1
+
039, 996, 731 cm ; MS m/z (+EI): 234 (M , 8),
04 (16), 203 (53),177 (45), 149 (33), 135 (100);
To a solution of endoperoxide 1k (1 equiv) and DMAP
(cat.) in dichloromethane (30 mL/g of endoperoxide) at
0 ꢁC under nitrogen was added carboxylic acid (1 equiv)
followed by DCC (1 equiv). The mixture was stirred for
5 h after which time the precipitate was filtered off and
washed with cold dichloromethane. The volatiles were
removed in vacuo and the crude residue purified by col-
umn chromatography.
+
+
HRMS (+EI) (M) found 234.16097; (M) calcd for
C H O 234.16198.
1
5
22
2
3
(
.4. ± (3R,6S)-3,6-Diadamantyl-3,6-dihydro-1,2-dioxine
1d)
Yield: 43 mg, 74%; colourless solid; mp 109–111 ꢁC; R
f
1
.57 (1:19 ethylacetate/hexane); H NMR (300 MHz,
0
CDCl ): d 1.60–1.73 (m, 24), 1.98 (br s, 6H), 3.92 (s,
3.9. ± [(3R,6R)-6-Methyl-3,6-dihydro-1,2-dioxin-3-
yl]methyl 2-adamantylacetate (1m)
3
1
3
2
3
1
H), 6.07 (s, 2H); C NMR (75 MHz, CDCl ): d 28.4,
3
7.1 (masked carbon), 38.7, 85.8, 125.3; IR (nujol):
344, 1072, 1027, 995, 756 cm ; MS m/z (+EI): 354
À1
Yield: 379 mg, 81%; colourless oil; R 0.53 (1:4 ethylac-
f
+
1
(
M , 5), 336 (56), 279 (16), 219 (24), 135 (100); HRMS
etate/hexane); H NMR (300 MHz, CDCl ): d 1. 22 (d,
3
+
+EI) (M+Na) found 377.2458; (M+Na) calcd for
+
(
C H O Na 377.2457.
J = 6.9 Hz, 3H), 1.61–1.72 (m, 12H), 1.97 (br s, 3H),
2.12 (s, 2H), 4.20–4.31 (m, 2H), 4.54–4.60 (m, 1H),
2
4
34
2

40
T. D. Avery et al. / Bioorg. Med. Chem. 15 (2007) 36–42
4
1
.79 (qq, J = 6.9, 1.8 Hz, 1H), 5.87 (ddd, J = 9.9, 3.6,
.8 Hz, 1H), 6.00 (ddd, J = 9.9, 1.8, 1.8 Hz, 1H);
CDCl ): d 1.66–1.77 (m, 24H), 2.02 (br s, 6H), 3.44 (s,
3
13
1
3
C
2H), 3.66 (s, 2H); C NMR (75 MHz, CDCl ): d 28.1,
3
NMR (75 MHz, CDCl ): d 17.5, 28.6, 32.8, 36.7, 42.4,
36.4, 36.9, 38.7, 50.2, 84.6; IR (nujol): 1344, 1268,
1101, 1022, 991, 852 cm ; MS m/z (+EI): 370 (M , 3),
338 (20), 235 (14), 207 (50), 178 (40), 135 (100); HRMS
3
À1
+
4
1
3
8.8, 63.3, 74.3, 76.7, 122.9, 131.9, 171.6; IR (neat):
734, 1450, 1257, 1137, 1020, 725 cm ; MS m/z (+EI):
À1
+
06 (M , 3), 289 (22), 177 (19), 135 (100), 95 (29);
+
+
(+EI) (M+Na) found 393.2418; (M+Na) calcd for
C H O Na 383.1623.
+
+
HRMS (+EI) (M+Na) found 329.1740; (M+Na)
calcd for C H O Na 329.1729.
2
4
24
3
1
8
26
4
3.14. ± (1aS,2R,5aS)-2-Heptylperhydrooxireno[2,3-d][1,
2]dioxine (2g) and ± (1aR,2R,5aR)-2-heptylperhydroox-
ireno[2,3-d][1,2]dioxine (3g)
3.10. ± [(3R,6R)-6-Methyl-3,6-dihydro-1,2-dioxin-3-
yl]methyl 2-(3-bromoadamantyl)acetate (1n)
Yield: 214 mg, 72%; colourless oil; R 0.6 (1:4 ethylace-
f
Compound 2g: yield: 68 mg, 39%; colourless oil; R 0.5
f
1
1
(1:19 ethylacetate/hexane); H NMR (300 MHz, CDCl ):
tate/hexane); H NMR (300 MHz, CDCl ): d 1.22 (d,
3
3
J = 6.6 Hz, 3H), 1.60–1.69 (m, 6H), 2.16–2.33 (m,
d 0.88 (t, J = 6.9 Hz, 3H), 1.28–.73 (m, 12H), 3.26 (dd,
1
0H), 4.21 (dd, J = 12.0, 3.3 Hz, 1H), 4.31 (dd,
J = 12.0, 8.1 Hz, 1H), 4.56 (ddddd, J = 8.1, 3.3, 3.6,
.8, 1.8 Hz, 1H), 4.80 (qq, J = 6.6, 1.8 Hz, 1H), 5.87
ddd, J = 9.9, 3.6, 1.8 Hz, 1H), 6.00 (ddd, J = 9.9, 1.8,
J = 4.5, 1.2 Hz, 1H), 133.37 (d
t J = 4.5, 0.6 Hz,
1H), 4.28–4.42 (m, 3H); C NMR (75 MHz, CDCl3):
1
(
1
3
1
1
d 14.0, 22.6, 24.9, 29.0, 29.4, 30.6, 31.7, 48.7, 53.1, 69.2,
79.2; IR (neat): 11465, 1248, 1042, 914, 733 cm ; MS
À1
1
3
+
m/z (+EI): 200 (M , 3), 183 (45), 109 (33), 81 (42), 69
.8 Hz, 1H); C NMR (75 MHz, CDCl ): d 17.5, 32.3,
3
+
(71), 57 (88), 43 (100); HRMS (+EI) (M) found
4.6, 37.2, 40.1, 47.4, 48.3, 53.5, 63.4, 65.0, 74.2, 76.5,
22.6, 132.1, 170.7; IR (neat): 1732, 1681, 1654, 1636,
449, 1240, 1135, 1022, 815 cm ; MS m/z (+EI): 385
+
200.1412; (M) calcd for C H O 200.1412.
1
1
20
3
À1
+
(M , 9), 366 (55), 305 (100), 257 (26), 215 (28), 133
(36), 95 (77); HRMS (+EI) (M) found 385.1014; (M)
Compound 3g: yield: 30 mg, 17%; colourless oil; R 0.45
f
+
+
1
(1:19 ethylacetate/hexane);
H
NMR (300 MHz,
calcd for C H O Br 385.1014.
26
CDCl ): d 0.88 (t, J = 6.9 Hz, 3H), 1.27–1.51 (m, 10H),
1
8
4
3
1
3.52 (ddd, J = 4.5, 3.6, 1.5 Hz, 1H), 4.27–4.44 (m, 3H);
.69 (q, J = 7.2 Hz, 2H), 3.31 (dd, J = 4.5, 1.8 Hz, 1H),
3
operoxides 2a–r and 3b, f, g, i, j, l–n, p, q
.11. General procedure for the synthesis of epoxyend-
1
2
13
C NMR (75 MHz, CDCl ): d 14.0, 22.6, 25.2, 29.1,
3
29.5, 29.6, 31.7, 50.6, 50.8, 69.9, 77.9; IR (neat): 1466,
1378, 1247, 1044, 913, 734 cm ; MS m/z (+EI): 200
À1
To a solution of endoperoxide 1a–r (1 equiv) in CH Cl
2
2
+
(M , 1), 183 (100), 127 (98), 109 (47), 83 (52), 57 (83);
(
20 mL/g of endoperoxide) was added 70% m-chloroper-
+
+
benzoic acid (2 equiv), the reaction mixture was stirred
at ambient temperature until complete by TLC. Dichlo-
romethane was then added and the solution extracted
with satd Na S O followed by satd NaHCO . The
HRMS (+EI) (M) found 200.1413; (M) calcd for
C H O 200.1412.
1
1
20
3
3.15. ± (1aS,2R,5aS)-2-Heptyl-1a-hexylperhydrooxiren-
o[2,3- d][1,2]dioxine (2j) and ± (1aR,2R,5aR)-2-heptyl-
1a-hexylperhydrooxireno[2,3-d][1,2]dioxine (3j)
2
2
3
3
organic layer was dried over MgSO , filtered and vola-
4
tiles removed in vacuo. The crude epoxides were purified
by column chromatography. For known epoxyendoper-
8
15
15
oxides see the following: compounds 2a, 2c, 2h, 2i/
8
Compound 2j: yield: 221 mg, 42%; colourless oil; R 0.45
f
8
8
12
15
8
14
1
3
i, 2l/3l, 2r, 2e, 2f/3f, 2o, and 4.
(1:9 ether/hexane); H NMR (300 MHz, CDCl ): d 0.86–
3
0
J = 9.0, 3.6 Hz, 1H), 4.27–4.38 (m, 2H); C NMR
.90 (m, 6H), 1.29–1.90 (m, 22H), 3.26 (s, 1H), 4.18 (dd,
1
3
3.12. ± (1a(R/S),2R,5a(R/S)-2-(1- Adamantylmeth-
yl)perhydrooxireno[2,3-d][1, 2]dioxine 2b and 3b
(75 MHz, CDCl ): d 14.0, 14.0, 22.5, 22.6, 24.4, 25.7,
3
28.2, 29.1, 29.2, 29.5, 31.6, 31.8, 32.7, 55.1, 59.5, 70.0,
80.2; IR (neat): 1467, 1378, 1045, 911, 726 cm ; MS
À1
Characterised as a mixture. Yield: 207 mg, 97%; colour-
H
1
+
m/z (+EI): 284 (M , 4), 267 (25), 157 (63), 113 (100),
less solid; R 0.24 (1:1 dichloromethane/hexane);
f
+
109 (80), 85 (90); HRMS (+EI) (M) found 284.2351;
NMR (200 MHz, CDCl ): d 1.00–0.88 (m, 2H), 1.48–
3
+
(M) calcd for C H O 284.2351.
1
3
4
2
3
5
1
2
.35 (m, 2H), 1.73–1.54 (m, 24H), 1.94 (br s, 6H), 3.21–
.17 (m, 2H), 3.32–3.30 (m, 1H), 3.49–3.46 (m, 1H),
.28–4.20 (m, 2H), 4.40–433 (m, 2H), 4.52–4.48 (m,
1
7
32
3
Compound 3j: yield: 128 mg, 24%; colourless oil; R 0.4
f
1
3
1
H); C NMR (50 MHz, CDCl ): d 28.4, 28.4, 31.4,
(1:9 ether/hexane); H NMR (300 MHz, CDCl ): d 0.86–
3
3
1.9, 36.7, 36.7, 42.4, 42.5, 44.1, 45.0, 48.5, 50.8, 52.0,
4.4, 68.9, 69.5, 74.2, 76.0; IR (nujol): 1363, 1106,
0.91 (m, 6H), 1.21–1.45 (m, 18H), 1.50–1.63 (m, 2H),
1.84–1.96 (m, 2H), 3.27 (d, J = 3.6 Hz, 1H), 4.08 (br d,
J = 10.2 Hz, 1H), 4.21 (ddd, J = 13.5, 3.9, 1.5 Hz, 1H),
À1
+
048, 1032, 906, 842 cm ; MS m/z (+EI): 250 (M , 5),
1
3
49 (16), 218 (1), 177 (4), 149 (9), 135 (100); Anal. Calcd
4.51 (dd, J = 13.5, 0.9 Hz, 1H); C NMR (75 MHz,
CDCl ): d 13.97, 14.03, 22.5, 22.6, 23.4, 25.8, 29.1,
for C H O : C, 71.97; H, 8.86; found: C, 72.02; H, 8.64.
1
5
22
3
3
29.2, 29.3, 29.4, 31.6, 31.76, 31.83, 55.7, 59.0, 69.9,
80.2; IR (neat): 1467, 1378, 1044, 968, 903 cm ; MS
À1
3.13. ± (1aR,2S,5R,5aS)-2,5-Diadamantylperhydrooxi-
reno[2,3- d][1,2]dioxine (2d)
+
m/z (+EI): 284 (M , 4), 267 (21), 253 (66), 237 (51),
1
99 (36), 185 (46), 157 (100), 139 (33); HRMS (+EI)
+
+
Yield: 26 mg, 93%; colourless solid; mp 193–194 ꢁC; R
(M) found 284.2352; (M) calcd for C H O
3
f
17 32
1
.5 (1:19 ethylacetate/hexane); H NMR (300 MHz,
0
284.2351.

T. D. Avery et al. / Bioorg. Med. Chem. 15 (2007) 36–42
41
3.16. ± [(1aR,2S,5R,5aS)-5-Methylperhydrooxireno[2,3-
d][1,2]dioxin-2-yl]methanol (2k)
Compound 3n: yield: 18 mg, 12%; colourless oil; R 0.39
f
1
(dichloromethane); H NMR (300 MHz, CDCl ): d 1.35
3
(d, J = 6.9 Hz, 3H), 1.59–1.71 (m, 6H), 2.17–2.34 (m,
10H), 3.26 (dd, J = 4.2, 0.9 Hz, 1H), 3.31 (d,
Yield: 173 mg 66%; colourless oil; R 0.2 (2:3 ethylace-
f
1
13
tate/hexane); H NMR (300 MHz, CDCl ): d 1.29 (d,
J = 4.2 Hz, 1H), 4.31–4.54 (m, 4H);
C NMR
3
J = 6.6 Hz, 3H), 2.22 (br s, 1H), 3.28 (dd, J = 4.2,
0.9 Hz, 1H), 3.59 (t, J = 4.2 Hz, 1H), 3.89 (dd,
J = 12.0, 4.2 Hz, 1H), 4.11 (dd, J = 12.0, 8.1 Hz, 1H),
(75 MHz, CDCl ): d 15.9, 32.3, 34.6, 37.3, 40.2, 40.3,
3
47.4, 48.3, 49.2, 53.49, 53.54, 61.9, 74.8, 76.0, 170.6;
IR (neat): 1732, 1454, 1241, 1135, 1017, 923, 815
À1
+
cm ; MS m/z (+EI): 401 (M , 2), 384 (23), 321 (79),
4
0
5
1
.36 (ddd, J = 8.1, 4.2, 4.2 Hz, 1H), 4.53 (d q, J = 6.6,
1
3
.9 Hz, 1H); C NMR (75 MHz, CDCl ): d 14.9, 51.1,
303 (80), 193 (40), 147 (79), 133 (75), 94 (92), 43 (100);
3
+
+
1.3, 60.0, 73.6, 77.9; IR (neat): 3419, 1454, 1374,
252, 1004, 926, 886, 727 cm ; MS m/z (+EI): 147
HRMS (+EI) (M) found 401.0967; (M) calcd for
C H O Br 401.0964.
À1
1
8
26
5
+
+
(
[M+H] , 2), 146 (M , 2), 115 (60), 45 (100); HRMS
+
+
(
+EI) (M) found 146.0581; (M) calcd for C H O
4
3.19. ± [(1aS,2R,5S,5aR)-5-Methylperhydrooxireno[2,3-
d][1,2]dioxin-2-yl]methyl benzyl ether (2p) and ± [(1aS,
6
10
1
46.0579.
2S,5R,5aR)-5-methylperhydrooxireno[2,3-d][1,2]dioxin-
2-yl]methyl benzyl ether (3p)
3
.17. ± [(1aR,2S,5R,5aS)-5-Methylperhydrooxireno[2,3-
d][1,2]dioxin-2yl]methyl 2-adamantylacetate (2m) and ±
(1aS,2S,5R,5aR)-5-methylperhydrooxireno[2,3-d][1,2]di-
oxin-2yl]methyl 2-adamantylacetate (3m)
[
Compound 2p: yield: 585 mg, 55%; colourless oil; Rf
0.15 (1:4 ethylacetate/hexane); H NMR (300 MHz,
1
CDCl ): d 1.30 (d, J = 6.9 Hz, 3H), 3.25 (dd, J = 6.6,
3
Compound 2m: yield: 133 mg, 47%; colourless oil; Rf
0
d 1.29 (d, J = 6.6 Hz, 3H), 1.61–1.68 (m, 12H), 1.97
1.8 Hz, 1H), 3.55 (t, J = 6.6 Hz, 1H), 3.81 (dd,
J = 15.9, 8.1 Hz, 1H), 3.89 (dd, J = 15.9, 10.2 Hz, 1H),
4.41–4.53 (m, 2H), 4.58 (d, J = 18.0 Hz, 1H), 4.65 (d,
1
.35 (dichloromethane); H NMR (300 MHz, CDCl ):
3
1
3
(
3
br s, 3H), 2.12 (s, 2H), 3.25 (dd, J = 4.2, 1.2 Hz, 1H),
J = 18.0 Hz, 1H), 7.26–7.38 (m, 5H); C NMR (75
MHz, CDCl ): d 14.9, 51.6, 51.7, 67.4, 73.6, 73.7, 76.6,
1
.57 (t, J = 4.2 Hz, 1H), 4.36–4.53 (m, 4H); C NMR
3
3
(
75 MHz, CDCl ): d 14.9, 28.6, 32.8, 36.7, 42.3, 48.7,
127.7, 127.8, 128.4, 138.0; IR (neat): 1496, 1453, 1373,
1108, 741 cm ; MS m/z (+EI): 236 (M , 6), 105 (84),
3
À1
+
5
1
9
1.30, 51.32, 60.8, 73.6, 75.2, 171.6; IR (neat): 1733,
À1
+
450, 1255, 1137, 1026 cm ; MS m/z (+EI): 322 (M ,
+
91 (100), 77 (53); HRMS (+EI) (M) found 236.1045;
+
(M) calcd for C H O 236.1049.
), 305 (39), 279 (41), 206 (26), 135 (100); Anal. Calcd
1
3
16
4
for C H O : C, 67.06; H, 8.13; found: C, 66.97; H,
5
1
8
26
8
.16.
Compound 3p: yield: 301 mg, 28%; colourless oil; R 0.3
f
1
1:4 ethylacetate/hexane); H NMR (300 MHz, CDCl ):
(
3
Compound 3m: yield: 57 mg, 20%; colourless oil; R 0.29
f
d 1.35 (d, J = 6.9 Hz, 3H), 3.20 (d, J = 4.2 Hz, 1H), 3.41
(d, J = 4.2 Hz, 1H), 3.72 (dd, J = 10.5, 5.4 Hz, 1H), 3.84
(dd, J = 10.5, 5.7 Hz, 1H), 4.43–4.50 (m, 2H), 4.57 (d,
1
(
(
dichloromethane); H NMR (300 MHz, CDCl ): d 1.34
d, J = 6.9 Hz, 3H), 1.59–1.73 (m, 12H), 1.98 (br s, 3H),
3
2
J = 4.2 Hz, 1H), 4.31–4.54 (m, 4H);
.13 (s, 2H), 3.24 (dd, J = 4.2, 0.9 Hz, 1H), 3.33 (d,
C
J = 12.0 Hz, 1H), 4.61 (d, J = 12.0 Hz, 1H), 7.30–7.37
1
3
13
NMR
(m, 5H); C NMR (75 MHz, CDCl ): d 15.9, 50.0,
3
(
75 MHz, CDCl ): d 15.9, 28.6, 32.9, 36.7, 42.4, 48.7,
53.1, 68.3, 73.6, 74.6, 77.2, 127.6, 127.7, 128.4, 137.6;
IR (neat): 1496, 1453, 1370, 1113, 739 cm ; MS m/z
3
À1
4
1
3
9.4, 53.5, 61.7, 74.8, 76.1, 171.4; IR (neat): 1732,
452, 1257, 1198, 1137, 1019, 915 cm ; MS m/z (+EI):
22 (M , 19), 405 (30), 279 (34), 220 (82), 135 (100);
À1
+
(+EI): 236 (M , 1), 105 (48), 91 (100), 77 (24); HRMS
+
+
+
(+EI) (M) found 236.1047; (M) calcd for C H O
13 16 4
+
+
HRMS (+EI) (M+Na) found 345.1669; (M+ Na)
calcd for C H O Na 345.1678.
236.1049.
1
8
26
5
3.20. ± (1aR,2S,5R,5aS)-2,5-Di[(benzyloxy)methyl]per-
3
.18. ± [(1aR,2S,5R,5aS)-5-Methylperhydrooxireno[2,3-
d][1,2]dioxin-2yl]methyl 2-(3-bromoadamantyl)acetate
2n) and ± [(1aS,2S,5R,5aR)-5-methylperhydrooxiren-
hydrooxireno[2,3-d][1, 2]dioxine (2q) and ± (1aR,2R,5-
S,5aS)-2,5-di[(benzyloxy)methyl]perhydrooxireno[2,3-
d][1,2]dioxine (3q)
(
o[2,3-d][1,2]dioxin-2yl]methyl 2-(3-bromoadamantyl)ace-
tate (3n)
Compound 2q: yield: 437 mg, 60%; colourless oil; R 0.2
f
1
1:4 ethylacetate/hexane); H NMR (300 MHz, CDCl ):
(
3
Compound 2n: yield: 40 mg, 26%; colourless oil; R 0.44
f
d 3.46 (m, 2H), 3.74–3.77 (m, 4H), 4.52–4.67 (m, 6H),
1
13
(
(
dichloromethane); H NMR (300 MHz, CDCl ): d 1.29
d, J = 6.3 Hz, 3H), 1.59–1.72 (m, 6H), 2.16–2.34 (m,
7.26–7.38 (m, 10H); C NMR (75 MHz, CDCl ): d
3
3
49.9, 67.7, 73.7, 77.1, 127.8 (masked carbon), 128.4,
137.8; IR (neat): 1496, 1453, 1365, 1255, 1207, 1114,
1
0H), 3.25 (dd, J = 4.2, 1.2 Hz, 1H)1,3 3.57 (t,
J = 4.2 Hz, 1H), 4.42–4.54 (m, 4H); NMR
75 MHz, CDCl ): d 14.9, 32.3, 34.6, 37.3, 40.2 (masked
À1
+
1027, 907, 739 cm ; MS m/z (+LSI): 342 (M , 4), 251
C
+
(13), 181 (100), 154 (57); HRMS (+EI) (M+H) found
(
3
+
343.1545; (M+H) calcd for C H O 343.1545.
carbon), 47.4, 48.3, 51.3, 53.5, 61.0, 65.0, 73.7, 75.0,
70.8; IR (neat): 1735, 1448, 1241, 1164, 1132, 1026
cm ; MS m/z (+EI): 401 (M , <0.1), 321 (100), 303
2
0
23
5
1
À1
+
Compound 3q: yield: 169 mg, 23%; colourless oil; R 0.3
f
1
(1:4 ethylacetate/hexane); H NMR (300 MHz, CDCl ):
(
(
12), 175 (32), 147 (66), 133 (47), 91 (74); HRMS
+EI) (M) found 401.0963; (M) calcd for C H O Br
3
+
+
d 3.44 (s, 2H), 3.69 (dd, J = 10.8, 5.4 Hz, 2H), 3.82 (dd,
J = 10.8, 5.4 Hz, 2H), 4.51 (t, J = 5.4 Hz, 2H), 4.56 (s,
1
8
26
5
4
01.0964.

42
T. D. Avery et al. / Bioorg. Med. Chem. 15 (2007) 36–42
1
3
4
5
H), 7.29–7.39 (m, 10H); C NMR (75 MHz, CDCl ): d
growth inhibition. DMSO caused no inhibition at 1%
but significant inhibition at 5%.
3
0.3, 68.2, 73.6, 77.7, 127.7, 127.9, 128.5, 137.6; IR
neat): 1496, 1453, 1364, 1273, 1097, 740 cm ; MS
À1
(
m/z (+EI): 342 (M , 1), 251 (48), 91 (100); HRMS
+
+
+EI) (M+H) found 343.1541; (M+H) calcd for
+
(
C H O 343.1545.
Acknowledgments
2
0
23
5
We thank the University of Adelaide and CSIRO
through the granting of a Brailsford Robertson Award
for financial support. We thank Pfizer for the gift of
Fluconazole.
3
.21. Compounds
For testing in vivo stocks of the novel compounds made
up in ethanol or dimethyl sulfoxide (DMSO) at concen-
trations of up to 100 mM. Amphotericin B was pur-
chased from Sigma and fluconazole was a gift from
Pfizer. Fluconazole was dissolved in ethanol and
Amphotericin B was dissolved in DMSO.
Supplementary data
Experimental details for diene precursors to endoperox-
ides 1b, 1c, 1d and 1j. Supplementary data associated
with this article can be found, in the online version, at
doi:10.1016/j.bmc.2006.10.021.
3
.22. Yeast assays
Candida albicans strain JRW#5 is a clinical isolate ob-
tained from Dr. J. R. Warmington (Curtin University).
It was employed in drug sensitive assays to measure the
levels at which compounds caused 50% growth inhibi-
References and notes
9
tion (IC ) as previously described. The level of ethanol
0
5
1
. (a) Saito, I.; Nattala, S. The Chemistry of Peroxides;
Wiley: New York, 1983; (b) Casteel, D. A. Nat. Prod. Rep.
used to dissolve compounds caused no growth inhibition
to the yeast, but DMSO at a level of 5%, but not 1%,
caused growth inhibition.
1
992, 9, 289; (c) Dussault, P. Synlett 1995, 997; (d) Casteel,
D. A. Nat. Prod. Rep. 1999, 16, 55; (e) Holzwarth, M.;
Trendel, J-M.; Albrecht, P.; Maier, A.; Michaelis, W. J.
Nat. Prod. 2005, 68, 759.
3
.23. Erythrocyte lysis assay
2
3
. Wright, C. W. J. Ethnopharmacol. 2005, 100, 67.
. Tang, Y.; Dong, Y.; Vennerstrom, J. L. Med. Res. Rev.
8
Washed human erythrocyctes (400 lL, 0.4 · 10 cells)
in phosphate-buffered saline were incubated with the
compounds to a final concentration of 1 mM and
incubated 30 min at 37 ꢁC. Cells were pelleted and
the absorbance of the supernatant was compared with
that for a sample of freeze-thawed cells to determine
the percentage of hemolysis. By themselves, the sol-
vents (DMSO and ethanol) used to dissolve com-
pounds caused no hemolysis.
2
004, 24, 425.
4
. Gunasekera, S. P.; Gunasekera, M.; Gunawardana, G. P.;
McCarthy, P.; Burres, N. J. Nat. Prod. 1990, 53, 669.
. (a) Jung, M.; Ham, J.; Song, J. Org. Lett. 2002, 4, 2763; (b)
Yao, G.; Steliou, K. Org. Lett. 2002, 4, 485.
5
6
7
. Barrett, D. Biochim. Biophys. Acta 2002, 1587, 224.
. Kotoyiannis, D. P.; Lewis, R. E. Lancet 2002, 359, 1135.
8. Taylor, D. K.; Avery, T. D.; Greatrex, B. W.; Tiekink, E.
R. T.; Macreadie, I. G.; Macreadie, P. I.; Humphries, A.
D.; Kalkanidis, M.; Fox, E. M.; Klonis, N.; Tilley, L. J.
Med. Chem. 2004, 47, 1833.
3
.24. Mammalian cell toxicity assay
9
. Macreadie, P.; Avery, T.; Greatrex, B.; Taylor, D.;
Macreadie, I. Bioorg. Med. Chem. Lett. 2006, 16, 920.
The toxicity of compounds to mammalian cells was
determined using a Chinese Hamster Ovary (CHO) cell
line. Compounds were added to final concentrations of
1
1
1
0. Samaranayake, Y. H.; Samaranayake, L. P. Med. Micro-
biol. 1994, 41, 295.
1. Matsumoto, M.; Dobasshi, S.; Kuroda, K.; Kondo, K.
Tetrahedron 1985, 41, 2147.
2. Greatrex, B.; Jenkins, N.; Taylor, D.; Tiekink, E. J. Org.
Chem. 2003, 68, 5205.
13. Nahmany, M.; Melman, A. Org. Lett. 2001, 3, 3733.
14. Akbulut, N.; Balci, M. J. Org. Chem. 1988, 53, 3338.
0
.1 and 1.0 mM and the cells were cultured for in mini-
mal essential Eagle’s medium (MEM) Alpha medium.
After 3 days of incubation cells were visually examined
and then harvested to estimate the amount of growth
which was estimated from protein levels. Protein levels
were estimated using the Pierce BCA Protein Assay
Reagent kit. Ethanol at levels up to 5% caused no
1
1
5. Greatrex, B.; Taylor, D. J. Org. Chem. 2004, 69, 2577.
6. Greatrex, B.; Taylor, D. J. Org. Chem. 2005, 70, 470.