The activity of dispiro peroxides against Fasciola hepatica

Article abstract of DOI:10.1016/j.bmcl.2011.07.024

Dispiro 1,2,4-trioxanes and 1,2,4,5-tetraoxanes had superior efficacy against Fasciola hepatica than the corresponding ozonides (1,2,4-trioxolanes). For highest efficacy, spiroadamantane and carboxymethyl substructures were required. Three compounds compl

Full text of DOI:10.1016/j.bmcl.2011.07.024

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5320–5323
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Bioorganic & Medicinal Chemistry Letters
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The activity of dispiro peroxides against Fasciola hepatica
Xiaofang Wang a, Qingjie Zhao a, Mireille Vargas b,c, Yuxiang Dong a, Kamaraj Sriraghavan a,
Jennifer Keiser b,c, Jonathan L. Vennerstrom a,
⇑
a College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE, USA
b Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
c University of Basel, CH-4003 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Dispiro 1,2,4-trioxanes and 1,2,4,5-tetraoxanes had superior efficacy against Fasciola hepatica than the
corresponding ozonides (1,2,4-trioxolanes). For highest efficacy, spiroadamantane and carboxymethyl
substructures were required. Three compounds completely cured F. hepatica-infected mice at single oral
doses of 50 mg/kg and two were partially curative at single doses of 25 mg/kg.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 25 May 2011
Revised 1 July 2011
Accepted 6 July 2011
Available online 19 July 2011
Keywords:
Artemisinin
Peroxide
Fasciola hepatica
The liver flukes Fasciola hepatica and F. gigantica are pathogenic
trematodes infecting an estimated 2.4–17 million people in the
Andean countries, Cuba, Western Europe, Egypt and Iran.1 More-
over, the morbidity and mortality of fascioliasis in cattle and sheep
results in considerable economic loss.2 The benzimidazole tricla-
bendazole (Fig. 1) is the drug of choice used to treat veterinary
fascioliasis, but it is registered in only four countries for the treat-
ment of human fascioliasis.3,4 Evidence of drug resistance to tricla-
bendazole in veterinary medicine5,6 provides an impetus for the
discovery and development of new drugs against fascioliasis.
We have shown that semisynthetic artemisinins (Fig. 1) and
synthetic ozonides (Fig. 2) have good efficacy against
F. hepatica.7–10 It is postulated10,11 that such peroxidic compounds
possess antiplasmodial12,13 and flukicidal activities because both
plasmodia and Fasciola spp. degrade hemoglobin to generate free
heme, a likely target14 of bioactive peroxides. In an effort to iden-
tify more effective synthetic peroxides, a structurally diverse
ozonide library of OZ78 (cis-1a) analogs was recently studied.10 It
pharmacokinetic profiles of 80-aryl ozonides compared to those
of 80-alkyl ozonides.16
Griesbaum coozonolysis17 of oxime ether 618 and ketoester 7
afforded ozonide ester 8 as a 2.5:1 mixture of cis and trans isomers
(Scheme 1). Chromatographic separation into the individual iso-
mers followed by hydrolysis of the cis isomer afforded 4a in high
yield. Ozonides 1a–3a and 5a were obtained as previously
described.19–21
Acid-catalyzed condensation of b-hydroperoxy alcohols 9–11
with 2-adamantanone or cyclohexanone afforded trioxane esters
12–16 (20–56% yields) (Scheme 2) which were isolated as single
trans isomers after crystallization (vide infra). Hydrolysis of
12–16 afforded trioxane acids 1b–5b. b-Hydroperoxy alcohols
9–11 were formed in 52–98% yields by regioselective perhydroly-
sis22,23 of the corresponding epoxides, which in turn, were formed
H
Cl
was found that
a spiroadamantane substructure, an acidic
O
H
N
Cl
O
functional group (or ester prodrug), and the peroxide bond and
nonperoxide oxygen atom of the ozonide heterocycle, were all re-
quired for high efficacy against F. hepatica.10 We now report an
investigation of the 1,2,4-trioxane and 1,2,4,5-tetraoxane analogs
of ozonides (1,2,4-trioxolanes) 1a–5a (Fig. 2).15 Target peroxide
heterocycles 5a–c were designed on the basis of the superior
O
O
SMe
H
N
Cl
Triclabendazole
O
R
Artemisinin
Artemether
Artesunate
R =
R =
R =
O
OCH
3
OCO(CH ) COOH
2 2
⇑
Corresponding author. Tel.: +1 402 559 5362; fax: +1 402 559 9543.
E-mail address: jvenners@unmc.edu (J.L. Vennerstrom).
Figure 1. Triclabendazole and the artemisinins.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.024

X. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5320–5323
5321
OH
OH
OH
O
O
O O
X
66.5
62.9
8'
Y
Y
TPP
X
OOH
MeOOC
MeOOC
1a–1d, Y = CH2COOH
2a–2c, Y = COOH
3a–3c, Y = CH2COOH
4a–4c, Y = COOH
H
H
19
9
(major trans isomer)
O
O
8'
COOH
X
OH
61.6
5a–5c
OOH
X = O (a), OCH2 (b), OO (c), CH2O (d)
Figure 2. Target dispiro peroxides.
H
20
(trans isomer)
OMe
N
Scheme 3. b-Hydroperoxy alcohol stereochemistry. Assigned hydroxymethyl
group 13C NMR signals are indicated in ppm.
O O
a
6
+
COOEt
O
HOO
OOH
O O
O O
a
O
COOEt
8, 46%
R
b
7
b
R
23, 45%
24, 77%
2c, 98%
5c, 99%
4a, 99%
21, 22
21, 23 R = COOEt
Scheme 1. Reagents and conditions: (a) O3, CH2Cl2/cyclohexane, 0 °C then sg
chromatography; (b) aq NaOH/EtOH, 60 °C, 4 h, then 1 M HCl, 0 °C.
22, 24 R = C6H4COOMe
Scheme 4. Reagents and conditions: (a) 2-adamantanone, Re2O7, CH2Cl2, rt, 12 h;
(b) 15% aq KOH, EtOH/THF, 50 °C, 4–20 h, then AcOH.
predominantly as their cis isomers24 by treatment of their keto ester
precursors20,21 with the sulfur ylide formed from trimethylsulfoxo-
nium iodide and potassium tert-butoxide.25 Even though b-hydro-
peroxy alcohols 9–11 were formed as mixtures of cis and trans
isomers, the trans isomers predominated as demonstrated by the
triphenylphosphine reduction of 9 to its corresponding 1,2-diol 19
and NMR analysis of the latter (Scheme 3). Observation of a signal
at 66.5 ppm in the 13C NMR spectrum of 1926 is consistent with a
shielded axial hydroxymethyl group indicating that the hydroper-
oxide in 9 is equatorial. Similarly, we suggest that the signal at
62.9 ppm in the 13C NMR spectrum of 9 is that of a shielded axial
hydroxymethyl group. By way of comparison, Li et al.23 report
hydroxymethyl group 13C NMR signals at 61.6 and 67.2 ppm for
the isomers of b-hydroperoxy alcohol 20. Finally, trioxane acid 1d
was obtained by hydrolysis of trioxane ester 18; the latter was
formed in low yield by acid-catalyzed condensation of b-hydroper-
oxy alcohol 1722 with methyl 2-(4-oxocyclohexyl)acetate.
Tetraoxane acids 2c and 5c were obtained by hydrolysis of their
respective tetraoxane esters 23 and 24; the latter were formed in
45% and 75% yields by Re2O7 catalyzed condensation27 of
1,1-dihydroperoxide esters 21 and 22 with 2-adamantanone
(Scheme 4). 1,1-Dihydroperoxide esters 21 and 22 were obtained
in quantitative yields from the corresponding keto esters by treat-
ment with 50% aq. H2O2 and I2 catalyst.28 Tetraoxane acids 1c, 3c,
and 4c were synthesized as previously described.29,30
Target compound efficacy data against F. hepatica are shown in
Tables 1 and 2. At 8–13 weeks post-infection, rats were treated
OH
OOH
O O
O
O O
O
a
b
R
R
R
9-11
c
1b, 86%
2b, 91%
5b, 93%
c
14, 31%
15, 38%
16, 56%
12, 37%
13, 20%
3b, 95%
4b, 97%
9, 12, 14 R = CH2COOMe
10, 13, 15 R = COOEt
11, 16
R = C6H4COOMe
OH OH
O
O O
O
d
X
O
18, X = OMe, 35%
1d, X = OH, 86%
c
17
Scheme 2. Reagents and conditions: (a) 2-adamantanone, CSA, CH2Cl2, rt, 12 h; (b) cyclohexanone, CSA, CH2Cl2, rt, 12 h; (c) 15% aq KOH, EtOH/THF, 50 °C, 4 h, then AcOH; (d)
methyl 2-(4-oxocyclohexyl)acetate, CSA, CH2Cl2, rt, 12 h.

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X. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5320–5323
Table 1
Worm burden reductions in adult F. hepatica harbored in rats following the administration of dispiro peroxides at single oral doses of 100 mg/kg
O O
X
O
O
O
O
Y
Y
COOH
X
X
3a–3c, 4a–4c
1a–1d, 2a–2c
5a–5c
Compd
X
Y
Worm burden reduction (%)
Curesa
Control
ASb
1ac
1b
1c
1d
—
—
O
—
—
—
30
0/12
2/5
10/10
3/3
4/4
0/3
0/4
1/3
4/4
0/3
2/3
0/3
0/3
3/3
3/3
3/3
3/3
2/4
CH2COOH
CH2COOH
CH2COOH
CH2COOH
COOH
COOH
COOH
CH2COOH
CH2COOH
CH2COOH
COOH
COOH
COOH
—
100e
100e
100e
0
OCH2
OO
CH2O
O
OCH2
OO
O
OCH2
OO
O
OCH2
OO
O
2a
2b
52
61e
100e
0
2c
3ad
3b
95e
29
3c
4a
4b
4c
5a
5b
5c
26
100e
100e
100e
100e
92e
OCH2
OO
—
—
a
b
c
Cures = number of rats cured/number of rats treated.
AS = artesunate.
Data from Keiser et al.8
d
e
Data from Zhao et al.10
p <0.05 from the Kruskal–Wallis test comparing the median values of the responses between the treatment and control groups.
Table 2
with single 25–100 mg/kg oral doses of target compounds pre-
pared as suspensions in 7% (v/v) Tween 80 and 3% (v/v) EtOH. At
day six post-treatment, rats were sacrificed and adult flukes were
recovered from the bile ducts and livers. Target compound effica-
cies were evaluated by comparing the mean total worm burdens
of treated and untreated control rats. Statistical significance was
calculated using the Kruskal–Wallis test.
Worm burden reductions in adult F. hepatica harbored in rats following the
administration of selected dispiro peroxides at single oral doses of 50 and 25 mg/kg
Compd
Dose (mg/kg)
Worm Burden Reduction (%)
Cures
Control
1aa
1bb
1bb
1cb
1cb
2c
—
50
50
25
50
25
50
50
50
50
50
25
—
53
0/12
2/4
4/4
3/4
4/4
0/4
1/4
0/4
1/4
0/4
3/3
1/4
100c
88c
100c
71c
48
Like 1a, seven compounds were completely curative at
100 mg/kg doses (Table 1). These compounds were then tested
at lower doses of 50 and 25 mg/kg (Table 2). Several trends can
be seen from the combined efficacy data. First, we suggest that
the complete loss of efficacy for 1,2,4-trioxane 1d compared to
its active regioisomer 1b results from a different iron (II) reaction
profile. Previous investigations22 with the unsubstituted (Y = H)
analogs of 1d and 1b reveal that although both 1,2,4-trioxanes
undergo a preferred attack of iron (II) on the less hindered perox-
ide oxygen atom, 1d forms a higher proportion of inactive car-
bonyl-containing reaction products, and, unlike 1b, does not
form a secondary carbon-centered radical by b-scission of the spi-
roadamantane substructure. Second, for 1,2,4-trioxolane/1,2,4-tri-
oxane/1,2,4,5-tetraoxane compound sets 1a–c, 2a–c, 3a–c, 4a–c,
and 5a–c, the trioxanes and tetraoxanes were superior to the tri-
oxolanes. Third, the efficacies of 1a–c compared to 2a–c show the
superiority of an 80-carboxymethyl versus 80-carboxy substituent.
Fourth, the efficacies of 1a–c compared to 3a–c and 2a–c com-
pared to 4a–c show the superiority of a spiroadamantane versus
spirocyclohexane. Fifth, unlike the superior antimalarial efficacies
of 80-aryl versus 80-alkyl ozonides,16 the 80-aryl 5a–c were inferior
to the corresponding 80alkyl peroxide heterocycles 1a–c. Finally,
compounds 1b and 1c had the highest overall efficacies, but of
the two, as previously determined by Kirchhofer et al.31 1c had
the best efficacy against juvenile F. hepatica and is easier to syn-
thesize than 1b. Ongoing investigations will assess if the superior
efficacies of the 1,2,4-trioxanes and 1,2,4,5-tetraoxanes versus the
corresponding 1,2,4-trioxolanes are due to pharmacokinetic
differences.32
4b
71c
61c
19
4cb
5a
5bb
5bb
100c
0
a
b
c
Data from Zhao et al.10
Data from Kirchhofer et al.31
p <0.05 from the Kruskal–Wallis test comparing the median values of the
responses between the treatment and control groups.
Acknowledgment
This investigation received financial support from NIH
(R21AI076783) and the Swiss National Science Foundation (Project
No. PPOOA-114941).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.07.024.
References and notes
1. Keiser, 1.; Utzinger, J. Clin. Microbiol. Rev. 2009, 22, 466.
2. Schweizer, G.; Braun, U.; Deplazes, P.; Torgerson, P. R. Vet. Rec. 2005, 157, 188.
3. Fairweather, I. J. Helminthol. 2009, 83, 139.
4. Keiser, J.; Utzinger, J. Emerg. Infect. Dis. 2005, 11, 1507.

X. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5320–5323
5323
5. Moll, L.; Gaasenbeek, C. P.; Vellema, P.; Borgsteede, F. H. Vet. Parasitol. 2000, 91,
153.
6. Keiser, J.; Utzinger, J. Exp. Opin. Drug Discov. 2007, 2, S9.
7. Keiser, J.; Utzinger, J. Trends Parasitol. 2007, 23, 555.
8. Keiser, J.; Utzinger, J.; Tanner, M.; Dong, Y.; Vennerstrom, J. L. J. Antimicrob.
Chemother. 2006, 58, 1193.
9. Keiser, J.; Utzinger, J.; Vennerstrom, J. L.; Dong, Y.; Brennan, G.; Fairweather, I.
Trans. R. Soc. Trop. Med. Hyg. 2007, 101, 1219.
10. Zhao, Q.; Vargas, M.; Dong, Y.; Zhou, L.; Wang, X.; Sriraghavan, K.; Keiser, J.;
Vennerstrom, J. L. J. Med. Chem. 2010, 53, 4223.
11. Kaiser, M.; Wittlin, S.; Nehrbass-Stuedli, A.; Dong, Y.; Wang, X.; Hemphill, A.;
Matile, H.; Brun, R.; Vennerstrom, J. L. Antimicrob. Agents Chemother. 2007, 51,
2991.
trans-3-Carboxy-7,8,15-trioxadispiro[5.2.5.2]hexadecane (4b). mp 157–158 °C.
1H NMR d 1.20–2.40 (m, 22H), 2.53–2.64 (m, 1H), 3.61 (brs, 1H), 3.75 (brs, 1H);
13C NMR d 22.3, 22.7 (br), 25.5, 28.2 (br), 29.9 (br), 34.6 (br), 40.4, 64.6, 102.3,
181.1. Anal. Calcd for C14H22O5: C, 62.20; H, 8.20. Found: C, 62.26; H, 8.30.
trans-Adamantane-2-spiro-30-90-(40-carboxyphenyl)-10,20,40-
trioxaspiro[5.5]undecane (5b). mp 212–213 °C. 1H NMR d 1.38–2.20 (m, 20H),
2.64–2.74 (m, 1H), 2.86 (brs, 1H), 2.98 (brs, 1H), 3.76–4.02 (m, 2H), 7.30 (d,
J = 8.0 Hz, 2H), 8.04 (d, J = 8.0 Hz, 2H), 11.51 (brs, 1H); 13C NMR d 27.1, 28.8
(br), 33.0 (br), 33.4, 36.4 (br), 37.1, 43.7, 62.3, 77.6, 104.5, 126.9, 127.2, 130.5,
152.2, 171.0. Anal. Calcd for C24H30O5: C, 72.34; H, 7.59. Found: C, 72.22; H,
7.63.
Adamantane-2-spiro-30-90-(40-carboxyphenyl)-10,20,40,50-
tetraoxaspiro[5.5]undecane (5c). mp 194–195 °C; 1H NMR d 1.56–2.14 (m,
20H), 2.66–2.76 (m, 1H), 3.20 (brs, 1H), 3.32 (brs, 1H), 7.34 (d, J = 8.5 Hz, 2H),
8.04 (d, J = 8.5 Hz, 2H); 13C NMR d 27.0, 29.5 (br), 30.1 (br), 31.8 (br), 33.1, 34.2
(br), 36.9, 43.8, 107.3, 110.6, 127.1, 127.3, 130.5, 152.2, 171.4. Anal. Calcd for
12. White, N. J. Science 2008, 320, 330.
13. Vennerstrom, J. L.; Arbe-Barnes, S.; Brun, R.; Charman, S. A.; Chiu, F. C. K.;
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.
14. Robert, A.; Benoit-Vical, F.; Claparols, C.; Meunier, B. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 13676.
C23H28O6: C, 68.98; H, 7.05. Found: C, 68.90; H, 6.96.
16. Charman, S. A.; Arbe-Barnes, S.; Bathurst, I. C.; Brun, R.; Campbell, M.;
Charman, W. N.; Chiu, F. C. K.; Chollet, J.; Craft, C. J.; Creek, D. J.; Dong, Y.;
Matile, H.; Maurer, M.; Morizzi, J.; Nguyen, T.; Papastogiannidis, P.; Scheurer,
C.; Shackleford, D. M.; Sriraghavan, K.; Stingelin, L.; Tang, Y.; Urwyler, H.;
Wang, X.; White, K. L.; Wittlin, S.; Zhou, L.; Vennerstrom, J. L. Proc. Natl. Acad.
Sci. 2011, 108, 4400.
17. Griesbaum, K.; Liu, X.; Kassiaris, A.; Scherer, M. Libigs Ann./Recueil 1997, 1381.
18. Marques, C. A.; Selva, M.; Tundo, P.; Montanari, F. J. Org. Chem. 1993, 58, 5765.
19. Tang, Y.; Dong, Y.; Karle, J. M.; DiTusa, C. A.; Vennerstrom, J. L. J. Org. Chem.
2004, 69, 6470.
20. Dong, Y.; Tang, T.; Chollet, J.; Matile, H.; Wittlin, S.; Charman, S. A.; Charman,
W. N.; Santo Tomas, J.; Scheurer, C.; Snyder, C.; Scorneaux, B.; Bajpai, S.;
Alexander, S. A.; Wang, X.; Padmanilayam, M.; Rao, C. S.; Brun, R.;
Vennerstrom, J. L. Bioorg. Med. Chem. 2006, 14, 6368.
21. Dong, Y.; Wittlin, S.; Sriraghavan, K.; Chollet, J.; Charman, S. A.; Charman, W.
N.; Scheurer, C.; Urwyler, H.; Santo Tomas, J.; Snyder, C.; Creek, D. J.; Morizzi, J.;
Koltun, M.; Matile, H.; Wang, X.; Padmanilayam, M.; Tang, Y.; Dorn, A.; Brun,
R.; Vennerstrom, J. L. J. Med. Chem. 2010, 53, 481.
15. Melting points are uncorrected. 1H and 13C NMR spectra were recorded in
CDCl3 on a 500 MHz spectrometer. All chemical shifts are reported in parts per
million (ppm) and are relative to internal (CH3)4Si (0 ppm) for 1H and 77.0 ppm
for 13C NMR.
trans-Adamantane-2-spiro-30-90-carboxymethyl-10,20,40-
trioxaspiro[5.5]undecane (1b). mp 178–179 °C. 1H NMR d 0.96–2.20 (m, 21H),
2.29 (d, J = 5.0 Hz, 2H), 2.54 (brs, 1H), 2.95 (brs, 1H), 3.74 (s, 2H); 13C NMR d
27.1, 27.6 (br), 28.6 (br), 29.3 (br), 31.5 (br), 33.4, 36.1 (br), 37.1, 39.7, 62.7,
77.6, 104.4, 177.5. Anal. Calcd for C19H28O5: C, 67.83; H, 8.39. Found: C, 68.02;
H, 8.36.
Adamantane-2-spiro-30-90-carboxymethyl-10,20,50-trioxaspiro[5.5]undecane
(1d). mp 210–211 °C. 1H NMR d 1.20ꢀ2.00 (m, 19H), 2.04ꢀ2.26 (m, 2H), 2.27
(d, J = 6.0 Hz, 2H), 2.73 (brs, 1H), 2.92 (brs, 1H), 3.75 (brs, 1H), 4.07 (brs, 1H),
10.98 (brs, 1H); 13C NMR d 27.4, 27.5, 28.5 (br), 29.3 (br), 31.9, 33.6, 34.3 (br),
37.7, 40.5, 63.9, 81.3, 101.4, 178.3. Anal. Calcd for C19H28O5: C, 67.83; H, 8.39.
Found: C, 68.01; H, 8.18.
22. Tang, Y.; Dong, Y.; Wang, X.; Sriraghavan, K.; Wood, J. K.; Vennerstrom, J. L. J.
Org. Chem. 2005, 70, 5103.
23. Li, Y.; Hao, H.-D.; Wu, Y. Org. Lett. 2009, 11, 2691.
trans-Adamantane-2-spiro-30-90-carboxy-10,20,40-trioxaspiro[5.5]undecane
(2b). mp 180–181 °C. 1H NMR d 1.20–2.20 (m, 21H), 2.52–2.63 (m, 1H), 2.94
(brs, 1H), 3.62 (brs, 1H), 3.72 (brs, 1H); 13C NMR d 22.7 (br), 23.8 (br), 27.1, 27.9
(br), 28.5 (br), 30.0 (br), 33.0 (br), 33.3, 36.0 (br), 37.1, 40.5, 64.0, 76.9, 104.4,
181.2. Anal. Calcd for C18H26O5: C, 67.06; H, 8.13. Found: C, 67.30; H, 8.28.
Adamantane-2-spiro-30-90-carboxy-10,20,40,50-tetraoxaspiro[5.5]undecane (2c).
mp 175–176 °C. 1H NMR d 1.44–2.12 (m, 20H), 2.42–2.52 (m, 1H), 2.93 (brs,
1H), 3.16 (brs, 1H); 13C NMR d 23.6 (br), 24.5 (br), 27.0, 28.1 (br), 30.2 (br), 33.1,
34.3 (br), 36.9, 41.2, 107.0, 110.6, 179.8. Anal. Calcd for C17H24O6: C, 62.95; H,
7.46. Found: C, 62.51; H, 7.50.
trans-3-Carboxymethyl-7,8,15-trioxadispiro[5.2.5.2]hexadecane (3b). mp 196–
197 °C. 1H NMR d 0.96–1.22 (m, 2H), 1.32–2.00 (m, 15H), 2.29 (d, J = 6.5 Hz,
2H), 2.18 (brs, 1H), 2.50 (brs, 1H), 3.75 (s, 2H); 13C NMR d 22.3, 25.5, 28.4 (br),
33.3, 39.8, 63.3, 77.8, 102.3, 177.7. Anal. Calcd for C15H24O5: C, 63.36; H, 8.51.
Found: C, 63.59; H, 8.48.
24. Leslie, C. P.; Bentley, J.; Biagetti, M.; Contini, S.; Di Fabio, R.; Donati, D.; Genski,
T.; Guery, S.; Mazzali, A.; Merlo, G.; Pizzi, D. A.; Sacco, F.; Seri, C.; Tessari, M.;
Zonzini, L.; Caberlotto, L. Bioorg. Med. Chem. Lett. 2010, 20, 6103.
25. Cleij, M.; Archelas, A.; Furstoss, R. J. Org. Chem. 1999, 64, 5029.
26. Bellucci, G.; Chiappe, C.; Lo Moro, G. J. Org. Chem. 1995, 60, 6214.
27. Ghorai, P.; Dussault, P. H. Org. Lett. 2009, 11, 213.
28. Zmitek, K.; Zupan, M.; Stavber, S.; Iskra, J. Org. Lett. 2006, 8, 2491.
29. Opsenica, I.; Opsenica, D.; Smith, K. S.; Milhous, W. K.; Šolaja, B. A. J. Med. Chem.
2008, 51, 2261.
30. Amewu, R.; Stachulski, A. V.; Ward, S. A.; Berry, N. G.; Bray, P. G.; Davies, J.;
Labat, G.; Vivas, L.; O’Neill, P. M. Org. Biomol. Chem. 2006, 4, 4431.
31. Kirchhofer, C.; Vargas, M.; Braissant, O.; Dong, Y.; Wang, X.; Vennerstrom, J. L.;
Keiser, J. Acta Trop. 2011, 118, 56–62.
cis-3-Carboxy-7,14,15-trioxadispiro[5.1.5.2]pentadecane (4a). mp 161–163 °C;
1H NMR d 1.34–1.99 (m, 18H), 2.35–2.39 (m, 1H); 13C NMR d 27.7, 24.8, 25.9,
33.1, 34.5, 41.0, 107.7, 109.1, 181.3. Anal. Calcd for C13H20O5: C, 60.92; H, 7.87.
Found: C, 60.74; H, 7.79.
32. O’Neill, P. M.; Amewu, R. K.; Nixon, G. L.; Bousejra ElGarah, F.; Mungthin, M.;
Chadwick, J.; Shone, A. E.; Vivas, L.; Lander, H.; Barton, V.; Muangnoicharoen,
S.; Bray, P. G.; Davies, J.; Park, B. K.; Wittlin, S.; Brun, R.; Preschel, M.; Zhang, K.;
Ward, S. A. Angew. Chem., Int. Ed. 2010, 49, 5693.