Design and synthesis of novel artemisinin-like ozonides with antischistosomal activity

Article abstract of DOI:10.1002/hlca.200590229

To develop new drugs for prevention and treatment of schistosomiasis, a series of novel artemisinin-like ozonides 10 were synthesized via a facile three-step procedure starting with the degraded product of artemisinin (Scheme). The Criegee ozonolysis reac

Full text of DOI:10.1002/hlca.200590229

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Design and Synthesis of Novel Artemisinin-Like Ozonides with
Antischistosomal Activity
by Zhong-Shun Yanga), Wen-Min Wub), Ying Li*a), and Yu-Lin Wub)
a) Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, P. R. China
(phone: +86-21-50805832; fax: +86-21-50807088; e-mail: yli@mail.shcnc.ac.cn)
b) State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic
Chemistry, Chinese Academy of Sciences, Shanghai 200032, P. R. China
To develop new drugs for prevention and treatment of schistosomiasis, a series of novel artemisinin-like
ozonides 10 were synthesized via a facile three-step procedure starting with the degraded product of artemisinin
(Scheme). The Criegee ozonolysis reaction of the unsaturated lactone intermediates 14 is the key step which pro-
vided the target molecules 10. The in vivo pharmacological results suggested that this type of artemisinin ana-
logues exhibited moderate antischistosomal activity (Table).
Introduction. – Schistosomiasis is a chronic and debilitating parasitic disease caused
by flatworms belonging to the genus Schistosoma, which is widespread throughout
tropical and subtropical areas and ranks second behind malaria in terms of socio-eco-
nomic and public-health importance [1]. It is currently estimated that more than 600
million people in 74 countries live at risk of infection, and 200 million people are suffer-
ing from schistosomiasis [2].
There is yet no vaccine available [3], and the current mainstay of schistosomiasis
control is chemotherapy with praziquantel (1) as the drug of choice [2a][4] (Fig. 1).
But left with a single efficacious antischistosomal drug is a dangerous situation since
oxamniquine (2) is difficult to obtain, and metrifonate (3) was withdrawn from the mar-
ket in 1998 (Fig. 1). In view of rapid re-infection following treatment and concern about
the development of tolerance and/or resistance to praziquantel, there is an urgent need
Fig. 1. Three known antischistosomal drugs
© 2005 Verlag Helvetica Chimica Acta AG, Zürich

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for research and development of novel drugs for the prevention and treatment of schis-
tosomiasis.
Fortunately, artemisinin (4) [5] and its derivatives such as dihydroartemisinin (5)
[6], artemether (6) [7], arteether (7) [8], and artesunate (8) [9], besides the widely
acknowledged antimalarial effect, also exhibited good antischistosomal activity (Fig.
2). After massive clinic trials, the Chinese Ministry of Public Health approved arte-
mether and artesunate for chemoprophylaxis of oriental schistosomiasis in 1996 as
an additional tool for transmission control. Laboratory studies revealed that arte-
mether exhibited the highest activity against juvenile stages of the parasites, while
adult worms were significantly less susceptible [7b].
Fig. 2. Artemisinin and several of its important semi-synthetic derivatives
Recently, Vennerstrom and co-workers reported a large series of novel spiro 1,2,4-
trioxolanes (=ozonides) and dispiro 1,2,4-trioxolanes 9 which displayed excellent anti-
malarial and antischistosomal activities (Fig. 3) [10]. Among them, a drug candidate
OZ277 has entered phase-I clinical trials in the UK and would be promising to become
a new type of antimalarial and antischistosomal drug [11].
Fig. 3. Spiro[adamantane-2,3’-[1,2,4]trioxolanes] as antimalarials and schistosomicides
The pharmacological results suggested that the antischistosomal mechanism of arte-
misinins might be analogous to what has been proposed for the mechanism of action of
artemisinin derivatives on malaria parasites, which involves activation of artemisinins
within the parasites by intraparasitic heme-iron, leading to the cleavage of the endoper-
oxide bridge and generation of C-centered free radicals which attacked the targets,
probably some important proteins, to kill the parasites [12]. Therefore, the peroxidic
bond in the artemisinins and ozonides would be the key factor contributing to high anti-
parasitic activity.

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Based on the aforementioned knowledge/rationale, we designed and synthesized a
series of novel artemisinin-like ozonides and evaluated their in vivo antischistosomal
activity. The remarkable structure feature of these ozonides resides in the artful
replacement of the 1,2,4-trioxane moiety of the parent artemisinins by a 1,2,4-trioxo-
lane moiety.
Results and Discussion. – Chemistry. A series of artemisinin-analogous ozonides 10
were synthesized from diketo methyl ester 11 [13], the acid-catalyzed (TFA=CF3-
COOH) degradation product of artemisinin (4), via a facile three-step procedure
(Scheme). According to our previous work [14], compound 12 was obtained by intra-
molecular condensation of 11 in the presence of barium hydroxide, after acidification
with dilute hydrochloric acid to pH 4–5. But if the reaction mixture was acidified to
pH<3, then a lactone by-product 13 was formed, which was present as a co-crystal mix-
ture with the target compound, thus hampering the subsequent purification. At À208,
treatment of compound 12 with various Grignard reagents followed by acidification
with dilute sulfuric acid afforded the unsaturated lactone intermediate 14, commonly
accompanied by a corresponding conjugated-diene-acid by-product 15. Generally,
compounds 14 and 15 could be purified by acid–base methods without column chroma-
tography. Finally, the desired artemisinin-like ozonides 10 were prepared by Criegee
ozonolysis of intermediate 14 at À788. Of course, inevitably 10 was accompanied by
small amount of over-ozonolyzed product 16. The new ozonides 10 could be purified
by column chromatography followed by recrystallization from AcOEt/petroleum
ether. It was interesting that the structure of ozonide 10a not only is highly similar to
that of the parent artemisinin (4), but that 10a is also its homologue. In general,
these new ozonides were stable without any decomposition for several months at
room temperature.
When iBuMgBr was employed to react with compound 12, another rearrangement
product 17 was obtained besides the expected compounds 14e and 15c. This result may
be correlated to the relatively large steric bulk of the isobutyl substituent, which could
lead to an easier elimination of this group as compared to other substituents, but the
detailed reaction mechanism of the formation of 17 is not clear yet.
Antischistosomal Activity. The novel artemisinin-like ozonides 10a–e were tested in
vivo for their antischistosomal activity on Schistosoma japonicum with O-[(tert-
butoxy)carbonyl]dihydroartemisinin [15] as the positive control (Table). These prelimi-
nary results showed that ozonides 10 exhibited moderate antischistosomal activity at a
one-off dose of 300 mg/kg (p.o.). Among them, ozonide 10a displayed the highest worm-
reduction rate. Furthermore, it seemed that the activity was decreasing with increasing
bulk of the substituent R, which suggests that antischistosomal activity decreases when
the peroxide bond is sterically inaccessible to heme-iron species in parasites.
In addition, there was no indication of toxicosis after absorption of the above dose
of ozonides, as checked at different intervals for one month post medication.
Conclusions. – In summary, a set of novel artemisinin-homologous 1,2,4-trioxolanes
were synthesized, and their in vivo antischistosomal activity on mice model infected
with Schistosoma japonicum was evaluated. The ozonides 10a–e exhibited moderate
parasiticidal potency.

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Scheme. Synthesis of artemisinin-like ozonides
Table. Antischistosomal Activity of Ozonides 10 in vivo against Schistosoma japonicum at a Dose of 300 mg/kg
R
Animals
Total worms (meanÆs.d.)
Worm-reduction rate [%]
10a
10b
10c
10d
Me
Et
Pr
iBu
Bn
–
5
5
5
5
5
5
6
12Æ2
14Æ4
18Æ3
20Æ3
20Æ4
10Æ2
30Æ2
60
52
40
33
33
67
–
10e
Positive control
Blank control
–

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These results are expected to be useful in guiding the design of new antischistoso-
mal drugs of high efficacy and low toxicity. We expect that these novel ozonides could
well complement existing control strategies and have the potential to become an impor-
tant component of integrated control approaches to significantly reduce the current
burden of schistosomiasis in many parts of the world.
We thank the Shanghai Institute of Animal Parasitology, Chinese Academy of Agricultural Sciences, for car-
rying out the in vivo antischistosomal test.
Experimental Part
1. General. All commercially available reagents were used without further purification unless otherwise
stated. The solvents used were all AR grade and were distilled under positive pressure of dry N2 where neces-
sary. Yields were of purified compounds and were not optimized. Anal. TLC (reaction monitoring): home-made
HSGF254 precoated silica gel plates; visualization by UV or development with I2. M.p.: open capillary tubes;
Büchi 510 melting-point apparatus; uncorrected. IR Spectra (4000–600 cmÀ1); Perkin-Elmer 599B spectropho-
tometer; KBr pellets or thin films; in cmÀ1. 1H-NMR Spectra: CDCl3 solns. at r.t.; Bruker AMX spectrophotom-
eter; chemical shifts d in ppm downfield from SiMe4 as the internal standard, coupling constants J in Hz. EI-MS:
Finnigan MAT-95 mass spectrometer; in m/z (rel. %). Elemental analyses: CE-1106 elemental analyzer; all
results within Æ0.4% of the theoretical values.
2. Compound 14a–f, 15a–d, and 17. To a soln. of 12 (2.36 g, 10.0 mmol) in dry THF (50 ml), the Grignard
reagent, prepared from alkyl halide (40 mmol) and Mg powder (1.2 g, 50.0 mmol) in Et2O (80 ml) was added
dropwise at À208 under continuous stirring. After the addition, the mixture was stirred for 3 h at r.t. Then
the soln. was acidified with dil. sulfuric acid (3
M
) at À208, quenched with H2O (15 ml) and extracted with Et2O.
The combined extract was washed with 10% aq. Na2SO3 soln., 5% aq. NaOH soln., H2O, and brine, dried
(MgSO4), and evaporated, and the crude product 14 purified by flash chromatography or by recrystallization.
The aq. alkaline phase was acidified with 10% aq. HCl soln. to pH 5 and extracted with Et2O. The org. layer
was washed with H2O and brine, dried (MgSO4), and evaporated and the residue purified by column chroma-
tography or recrystallization to give product 15.
(3R,3aS,6R,6aS)-3,3a,4,5,6,6a,7,8-Octahydro-3,6,9-trimethyl-2H-naphtho[8a,1-b]furan-2-one (14a): Yield
75%. Colorless oil or crystals. M.p. 69–718 (from AcOEt/petroleum ether). IR (film): 2931, 2860, 1763 (C=
1
O), 1666 (C=C), 1452, 1379, 1331, 1217, 1163, 1013, 926, 910. H-NMR (CDCl3, 400 MHz): 5.63 (s, 1 H); 3.14
(quint., J=6.87, 1 H); 2.15–1.97 (m, 3 H); 1.92–1.84 (m, 1 H); 1.68 (s, 3 H); 1.74–1.60 (m, 3 H); 1.46–1.36
(m, 1 H); 1.24–1.17 (m, 2 H); 1.14 (d, J=7.14, 3 H); 1.09–0.99 (m, 1 H); 0.93 (d, J=6.59, 3 H). Anal. calc.
for C15H22O2 (234.34): C 76.88, H 9.46; found: C 76.72, H 9.60.
(aR,4R,4aS)-2,3,4,4a,5,6-Hexahydro-a,4,7-trimethylnaphthalene-1-acetic Acid (15a) [16]. Yield 22%. Col-
orless crystals. M.p. 135–1378. IR (KBr): 3300 (chel OH), 2970, 2925, 2825, 1705 (C=O), 1610 (C=C), 1460,
1
1380, 1240, 1080, 950. H-NMR (CDCl3, 60 MHz): 12.10 (s, 1 H); 6.13 (s, 1 H); 3.78 (q, J=7.00, 1 H); 1.82 (s,
3 H); 1.20 (d, J=7.00, 3 H); 1.03 (d, J=4.00, 3 H). Anal. calc. for C15H22O2 (234.34): C 76.88, H 9.46; found:
C 76.57, H 9.54.
(3R,3aS,6R,6aS)-9-Ethyl-3,3a,4,5,6,6a,7,8-octahydro-3,6-dimethyl-2H-naphtho[8a,1-b]furan-2-one (14b):
Yield 75%. White needles. M.p. 73–758 (from AcOEt/petroleum ether). IR (KBr): 2974, 2941, 2897, 1751
1
(C=O), 1668 (C=C), 1454, 1377, 1213, 1180, 906, 849. H-NMR (CDCl3, 600 MHz): 5.59 (s, 1 H); 3.14 (quint.,
J=6.84, 1 H); 2.14–2.06 (m, 2 H); 2.05–1.98 (m, 1 H); 1.96 (q, J=7.32, 2 H); 1.90–1.84 (m, 1 H); 1.67–1.60
(m, 3 H); 1.45–1.37 (m, 1 H); 1.24–1.14 (m, 2 H); 1.12 (d, J=7.43, 3 H); 1.06–0.99 (m, 1 H); 0.97 (t, J=7.57,
3H); 0.91 (d, J=6.35, 3 H). Anal. calc. for C16H24O2 (248.37): C 77.38, H 9.74; found: C 77.66 H 9.93.
(aR,4R,4aS)-7-Ethyl-2,3,4,4a,5,6-hexahydro-a,4-dimethylnaphthalene-1-acetic Acid (15b): Yield 24%.
White crystalline particles. M.p. 138–1408 (from AcOEt/petroleum ether). Yield: 24%. IR (KBr): 3300 (chel.
OH), 2968, 2924, 2825, 1705 (C=O), 1614 (C=C), 1458, 1419, 1380, 1242, 1078, 949, 675. 1H-NMR (CDCl3,
600 MHz): 6.09 (s, 1 H); 3.81 (q, J=6.84, 1 H); 2.18–1.98 (m, 7 H); 2.06 (q, J=6.84, 2 H); 1.72–1.66 (m, 1
H); 1.60 (t, J=10.99, 1 H); 1.29–1.22 (m, 1 H); 1.20 (d, J=7.32, 3 H); 1.19–1.10 (m, 2 H); 1.02 (t, J=7.33, 3
H); 0.98 (d, J=5.86, 3 H). Anal. calc. for C16H24O2 (248.37): C 77.38 H 9.74; found: C 77.51, H 10.02.
(3R,3aS,6R,6aS)-3,3a,4,5,6,6a,7,8-Octahydro-3,6-dimethyl-9-propyl-2H-naphtho[8a,1-b]furan-2-one (14c).
Yield 52%. Solidified colorless oil. IR (KBr): 2956, 2929, 2872, 1759 (C=O), 1664 (C=C), 1460, 1375, 1219,

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1184, 1171, 930, 914, 841, 723. 1H-NMR (CDCl3, 400 MHz): 5.62 (s, 1 H); 3.16 (quint., J=6.87, 1 H); 2.16–2.08
(m, 2 H); 2.07–1.99 (m, 1 H); 1.94 (t, J=7.70, 2 H); 1.91–1.85 (m, 1 H); 1.76–1.60 (m, 3 H); 1.47–1.37 (m, 3 H);
1.26–1.18 (m, 2 H); 1.15 (d, J=7.15, 3 H); 1.10–0.99 (m, 1 H); 0.94 (d, J=6.60, 3 H); 0.87 (t, J=7.36, 3 H). Anal.
calc. for C17H26O2 (262.39): C 77.82, H 9.99; found: C 78.09, H 10.28.
(3R,3aS,6R,6aS)-3,3a,4,5,6,6a,7,8-Octahydro-9-isopropyl-3,6-dimethyl-2H-naphtho[8a,1-b]furan-2-one (14d):
Yield 70%. Colorless oil. IR (film): 2956, 2860, 1767 (C=O), 1659 (C=C), 1462, 1379, 1217, 1171, 1010, 912,
882, 843, 721. 1H-NMR (CDCl3, 400 MHz): 5.62 (s, 1 H); 3.16 (quint., J=6.78, 1 H); 2.23–1.97 (m, 4 H);
1.94–1.86 (m, 1 H); 1.76–1.57 (m, 3 H); 1.48–1.38 (m, 1 H); 1.26–1.17 (m, 2 H); 1.15 (d, J=7.14, 3 H);
i
1.10–1.03 (m, 1 H); 1.00 (dd, J=6.87, 6 H); 0.93 (d, J=6.60, 3 H). EI-MS: 262 (38, M), 219 (88, [MÀ Pr]+),
218 (99, [MÀCO2]+), 203 (31, [MÀCO2]+), 189 (84, [MÀCH(Me)CO2H]+), 175 (100, [MÀMeÀCH-
(Me)CO2]+).
(3R,3aS,6R,6aS)-3,3a,4,5,6,6a,7,8-Octahydro-9-isobutyl-3,6-dimethylnaphtho[8a,1-b]furan-2-one
(14e):
Yield 27%. Colorless oil. IR (film): 2953, 2870, 1768 (C=O), 1662 (C=C), 1464, 1381, 1215, 1163, 914, 897.
1H-NMR (CDCl3, 600 MHz): 5.58 (s, 1 H); 3.13 (quint., J=6.84, 1 H); 2.12–2.05 (m, 2 H); 2.02–1.94 (m, 1
H); 1.89–1.82 (m, 2 H); 1.80–1.60 (m, 5 H); 1.45–1.37 (m, 1 H); 1.22–1.15 (m, 2 H); 1.13 (d, J=6.84, 3 H);
1.07–0.99 (m, 1 H); 0.91 (d, J=6.83, 3 H); 0.83, 0.81 (dd, J=6.35, 6.83, 6 H). EI-MS: 276 (24, M+), 232 (100,
i
[MÀCO2]+), 219 (44, [MÀ Bu]+), 203 (31, [MÀCH(Me)COOH]+), 189 (83, [MÀMeÀCH(Me)COO]+).
(3R,3aS,6R,6aS)-3,3a,4,5,6,6a,7,10-Octahydro-3,6-dimethyl-2H-naphtho[8a,1-b]furan-2-one (17): Yield
35%. White needles. M.p. 78–808 (from AcOEt-petroleum ether). IR (KBr): 3030, 2980, 2941, 2856, 1755
1
(C=O), 1649 (C=C), 1452, 1380, 1211, 1178, 1047, 928, 914, 883, 713. H-NMR (CDCl3, 600 MHz): 5.94–5.89
(m, 1 H); 5.87–5.83 (m, 1 H); 3.10 (quint., J=6.84, 1 H); 2.24–1.97 (m, 1 H); 2.13–2.03 (m, 2 H); 1.89–1.83
(m, 1 H); 1.73–1.67 (m, 3 H); 1.46–1.38 (m, 1 H); 1.26–1.14 (m, 2 H); 1.12 (d, J=7.32, 3 H); 1.07–1.00 (m, 1
H); 0.92 (d, J=6.84, 3 H). Anal. calc. for C14H20O2 (220.31): C 76.33, H 9.15; found: C 76.38, H 9.35.
(aR,4R,4aS)-2,3,4,4a,5,6-Hexahydro-7-isobutyl-a,4-dimethylnaphthalene-1-acetic Acid (15c): Yield 5%.
Colorless oil. IR (film): 3400 (chel. OH), 2953, 2926, 2860, 1705 (C=O), 1460, 1379, 1223, 1167, 926. 1H-
NMR (CDCl3, 600 MHz): 6.06 (s, 1 H); 3.80 (q, J=7.03, 1 H); 2.13–1.96 (m, 4 H); 1.95–1.80 (m, 2 H); 1.80–
1.40 (m, 4 H); 1.22 (d, J=7.32, 3 H); 0.98 (d, J=5.86, 3 H); 0.83, 0.82 (2d, J=6.35, 6 H). EI-MS: 276 (55,
i
M+), 231 (20, [MÀCOOH]+), 219 (26, [MÀ Bu]+), 203 [100, MÀCH(Me)COOH]+], 189 (22, [MÀMeÀCH-
(Me)COO]+).
(3aS,3R,6R,6aS)-9-Benzyl-3,3a,4,5,6,6a,7,8-octahydro-3,6-dimethyl-2H-naphtho[8a,1-b]furan-2-one (14f):
Yield 80%. Solidified colorless oil. IR (KBr): 3010, 2924, 2849, 1755 (C=O), 1668, 1495 (C=C), 1452, 1377,
1331, 1184, 1161, 910, 897, 735, 702. 1H-NMR (CDCl3, 400 MHz): 7.32–7.25 (m, 2 H); 7.24–7.18 (m, 1 H);
7.15–7.11 (m, 2 H); 5.67 (s, 1 H); 3.28 (s, 2 H); 3.10 (quint., J=6.88, 1 H); 2.16–2.04 (m, 2 H); 2.03–1.92 (m,
1 H); 1.90–1.82 (m, 1 H); 1.66–1.62 (m, 3 H); 1.48–1.38 (m, 1 H); 1.26–1.18 (m, 2 H); 1.14 (d, J=7.22, 3 H);
1.10–0.99 (m, 1 H); 0.92 (d, J=6.55, 3 H). Anal. calc. for C21H26O2 (310.44): C 81.25, H 8.44; found: C 81.21,
H 8.52.
(aR,4R,4aS)-7-Benzyl-2,3,4,4a,5,6-hexahydro-a,4-dimethylnaphthalene-1-acetic Acid (15d): Yield 10%.
Colorless oil. IR (KBr): 3400 (chel. OH), 3026, 2922, 2831, 1705 (C=O), 1603, 1495 (C=C), 1454, 1377, 1240,
1074, 937, 700. 1H-NMR (CDCl3, 400 MHz): 7.31–7.25 (m, 2 H); 7.22–7.16 (m, 3 H); 6.21 (s, 1 H); 3.80 (q,
J=7.05, 1 H); 3.38 (s, 2 H); 2.18–1.98 (m, 5 H); 1.74–1.67 (m, 1 H); 1.62 (t, J=9.82, 1 H); 1.22 (d, J=7.05, 3
H); 1.19–1.12 (m, 3 H); 0.98 (d, J=6.04, 3 H). EI-MS: 310 (60, M+), 219 (100, [MÀBn]+), 265 (14,
[MÀCOOH]+), 237 (44, [MÀCH(Me)COOH]+), 145 (84, [MÀBnÀCH(Me)COOH]+), 91 (97, Bn+).
3. Ozonides 10a–e and 16. Through a soln. of 14 (2.0 mmol) in dry pentane (30 ml) was bubbled ozone at
À788, until the soln. turned into light blue. After completion, the soln. was flushed with N2 for 2 min before
being evaporated at r.t. The residue was purified by flash chromatography followed by recrystallization to pro-
vide the desired ozonides 10a–e in 10–30% yield and over-ozonolyzed products 16a–c.
(3R,3aS,6R,6aS)-Octahydro-3,6,9-trimethyl-9,12-epoxy-12H-furo[3,2-k][2,3]benzodioxocin-2(3H)-one) (10a):
Yield 28%. White needles. M.p. 142–1448 (from AcOEt/petroleum ether) ([16]: 135–1408). IR (KBr): 2941,
2860, 1763 (C=O), 1452, 1381, 1227, 1182, 1149, 1088, 1013, 939. 1H-NMR (CDCl3, 300 MHz): 5.35 (s, 1 H);
2.85 (quint., J=7.29, 1 H); 2.39–2.16 (m, 2 H); 2.09 (dt, J=14.62, 3.20, 1 H); 1.82–1.54 (m, 5 H); 1.50 (s, 3
H); 1.49–1.33 (m, 2 H); 1.17 (d, J=7.29, 3 H); 1.15–1.08 (m, 1 H); 0.98 (d, J=6.46, 3 H). Anal. calc. for
C15H22O5 (282.34): C 63.81, H 7.85; found: C 64.08, H 7.75.
(3R,3aS,6R,6aS)-9-Ethyloctahydro-3,6-dimethyl-9,12-epoxy-12H-furo[3,2-k][2,3]benzodioxocin-2(3H)-one
(10b): Yield 15%. White catkin/flossy crystals. M.p. 143–1458 (from AcOEt/petroleum ether). IR (KBr): 2972,
2939, 2860, 1763 (C=O), 1464, 1377, 1188, 1122, 1086, 1024, 957, 679. 1H-NMR (CDCl3, 300 MHz): 5.36 (s, 1 H);
2.84 (quint., J=7.22, 1 H); 2.32 (q, J=6.65, 1 H); 2.28–2.12 (m, 1 H); 2.03 (dt, J=14.53, 2.99, 1 H); 1.80 (q,

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J=7.61, 2 H); 1.74–1.56 (m, 5 H); 1.56–1.42 (m, 1 H); 1.40–1.28 (m, 1 H); 1.16 (d, J=7.29, 3 H); 1.14–1.06 (m,
1 H); 0.99 (t, J=7.50, 3 H); 0.98 (d, J=6.32, 3 H). Anal. calc. for C16H24O5 (296.36): C 64.84, H 8.16; found: C
65.08, H 7.95.
(3R,3aS,6R,6aS)-Octahydro-3,6-dimethyl-9-propyl-9,12-epoxy-12H-furo[3,2-k][2,3]benzodioxocin-2(3H)-
one 10c: Yield 10%. White needles. M.p. 144–1468 (from AcOEt/petroleum ether). IR (KBr): 2939, 2860, 1774,
1
1761 (C=O), 1454, 1380, 1188, 1148, 1122, 989, 957, 920, 677. H-NMR (CDCl3, 400 MHz): 5.35 (s, 1 H); 2.84
(quint., J=7.25, 1 H); 2.32 (q, J=6.69, 1 H); 2.26–2.14 (m, 1 H); 2.03 (dt, J=14.71, 3.20, 1 H); 1.76–1.70 (m,
3 H); 1.70–1.57 (m, 3 H); 1.57–1.40 (m, 4 H); 1.40–1.30 (m, 1 H); 1.17 (d, J=7.29, 3 H); 1.14–1.08 (m, 1 H);
0.98 (d, J=6.60, 3 H); 0.92 (t, J=7.36, 3 H). Anal. calc. for C17H26O5 (310.39): C 65.78, H 8.44; found: C
65.77, H 8.40.
(3R,3aS,6R,7S)-Hexahydro-3,6-dimethyl-2-oxo-7-(3-oxohexyl)benzofuran-7a(2H)-carboxaldehyde (16a):
Yield 12%. Colorless oil. IR (film): 2960, 2875, 2720 (C(O)ÀH), 1790 (C=O, lactone), 1732 (C=O, aldehyde),
1709 (C=O, ketone), 1456, 1379, 1183, 1032, 953, 926, 875. 1H-NMR (CDCl3, 400 MHz): 9.74 (s, 1 H); 2.61–2.48
(m, 2 H); 2.46–2.31 (m, 1 H); 2.34 (t, J=7.22, 2 H); 1.89–1.62 (m, 6 H); 1.62–1.52 (m, 2 H); 1.48–1.38 (m, 1 H);
1.16–1.06 (m, 2 H); 1.10 (d, J=7.15, 3 H); 0.99 (d, J=6.47, 3 H); 0.89 (d, J=7.36, 3 H). EI-MS: 294 (1, M+), 266
(12, [MÀCO]+), 265 (36, [MÀCHO]+), 251 (12, [MÀPr]+), 237 (100, [MÀCHOÀCO]+).
(3R,3aS,6R,7S)-Hexahydro-3,6-dimethyl-7-(4-methyl-3-oxopentyl)-2-oxobenzofuran-7a(2H)-carboxalde-
hyde (16b): Yield 21%. Colorless oil. IR (film): 2933, 2860, 2720 (C(O)ÀH), 1778 (C=O, lactone), 1732 (C=O,
1
aldehyde), 1705 (C=O, ketone), 1466, 1383, 1342, 1182, 1032, 955, 926, 878, 733. H-NMR (CDCl3, 400 MHz):
9.75 (s, 1 H); 2.63–2.51 (m, 2 H); 2.49–2.37 (m, 2 H); 1.92–1.64 (m, 4 H); 1.64–1.54 (m, 2 H); 1.50–1.40 (m, 1
H); 1.24–1.19 (m, 2 H); 1.11 (d, J=7.01, 3 H); 1.07 (d, J=6.88, 6 H); 1.00 (d, J=6.46, 3 H). EI-MS: 294 (1, M+),
i
266 (20, [MÀCO]+), 265 (40, [MÀCHO]+), 251 (24, [MÀ Pr]+), 237 (100, (MÀCHOÀCO]+).
(3R,3aS,6R,6aS)-Octahydro-3,6-dimethyl-9-(2-methylpropyl)-9,12-epoxy-12H-furo[3,2-k][2,3]benzodioxo-
cin-2(3H)-one (10d): Yield 30%. White needles. M.p. 150–1528 (from AcOEt/petroleum ether). IR (KBr): 2959,
2860, 1770 (C=O), 1468, 1359, 1219, 1188, 1146, 1119, 1036, 995, 957, 922. 1H-NMR (CDCl3, 400 MHz): 5.34 (s, 1
H); 2.84 (quint., J=7.25, 1 H); 2.34 (q, J=6.56, 1 H); 2.28–2.14 (m, 1 H); 2.04 (dt, J=15.99, 3.80, 1 H); 1.91–
1.80 (m, 1 H); 1.76–1.60 (m, 5 H); 1.60–1.56 (m, 2 H); 1.54–1.46 (m, 1 H); 1.44–1.31 (m, 1 H); 1.17 (d, J=7.29,
3 H); 1.15–1.07 (m, 1 H); 0.98 (d, J=6.46, 3 H); 0.94 (d, J=6.60, 6 H). Anal. calc. for C18H28O5 (324.42): C 66.64,
H 8.70; found: C 66.97, H 8.37.
(3R,3aS,6R,6aS)-9-Benzyl-octahydro-3,6-dimethyl-9,12-epoxy-12H-furo[3,2-k][2,3]benzodioxocin-2(3H)-
one (10e): Yield 15%. White needles. M.p. 148–1508 (from AcOEt/petroleum ether). Yield: 15%. IR (KBr):
2945, 2860, 1772 (C=O), 1498 (C=C), 1456, 1244, 1186, 1146, 1113, 1086, 1003, 957, 754. 1H-NMR (CDCl3,
300 MHz): 7.38–7.20 (m, 5 H); 5.35 (s, 1 H); 3.06 (s, 2 H); 2.81 (quint., J=7.08, 1 H); 2.28 (q, J=6.74, 1 H);
2.19–2.03 (m, 1 H); 1.95 (br. d, J=14.58, 1 H); 1.80–1.58 (m, 5 H); 1.47–1.37 (m, 1 H); 1.35–1.23 (m, 1 H);
1.15 (d, J=7.15, 3 H); 1.11–1.02 (m, 1 H); 0.95 (d, J=6.46, 3 H). Anal. calc. for C21H26O5 (358.43): C 70.37,
H 7.31; found: C 70.72, H 7.14.
(3R,3aS,6R,7S)-Hexahydro-3,6-dimethyl-2-oxo-7-(3-oxo-4-phenylbutyl)benzofuran-7a(2H)-carboxalde-
hyde (16c): Yield 12%. Colorless oil. IR (film): 3100, 3030, 2929, 2860, 2720 (C(O)ÀH), 1778 (C=O, lactone),
1
1732 (C=O, aldehyde), 1713 (C=O, ketone), 1600, 1497 (C=C, Ar), 1454, 1381, 1182, 1032, 953, 735, 702. H-
NMR (CDCl3, 400 MHz): 9.66 (s, 1 H); 7.35–7.17 (m, 5 H); 3.67 (s, 2 H); 2.66–2.50 (m, 2 H); 2.50–2.30 (m,
2 H); 1.90–1.60 (m, 6 H); 1.44–1.34 (m, 1 H); 1.10 (d, J=7.14, 3 H); 1.07–0.95 (m, 1 H); 0.91 (d, J=6.46, 3
H). EI-MS: 342 (10, M+), 314 (30, [MÀCO]+); 285 (44, [MÀCOÀCHO]+), 251 ([64, MÀBn]+), 223 (60,
[MÀCOÀBn]+), 195 (56, [MÀPhCH2COÀCO]+), 179 (88, [MÀBnÀCH(Me)COO]+), 91 (100, Bn+).
4. Mice and Schistosoma Parasites. Mice (Kunming strain) of both sexes, weighing 18–24 g, were purchased
from the Shanghai Animal Centre, Chinese Academy of Sciences, Shanghai, China. They were maintained on a
rodent food and H2O ad libitum. An Anhui isolate of S. japonicum, released from laboratory-bred Oncomelania
hupensis snails, was provided by Shanghai Institute of Animal Parasitology, Chinese Academy of Agricultural
Sciences. Each mouse was infected with 50–55 cercariae via the shaved abdominal skin.
5. Assessment of Drug Effects. Ozonides 10a–e were suspended in 1% (carboxymethyl)cellulose (CMC)
soln. All reagents used were of anal. grade. To five mice as a group, at 30 days post-infection, the ozonides
were administered intragastrically at a one-off dose of 300 mg/kg. At 30 days post-medication, mice were killed
by exsanguination, and the Schistosoma were collected by perfusion with ice-cold HBSS from the mesenteric
veins and livers. Worm motor activity, alterations in the tegument, damage to the gut, and parasite survival
were assessed by careful examination of the worms under an inverted microscope. Parasite death was defined
as no motor activity during 2 min of observation and extensive tegumental alterations, i.e., >50% of the tegu-
ment characterized by vesiculation, collapse, and peeling.

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ELVETICA
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ACTA – Vol. 88 (2005)
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Received June 27, 2005