Study on the mechanism of action of artemether against schistosomes: The identification of cysteine adducts of both carbon-centred free radicals derived from artemether

Article abstract of DOI:10.1016/S0960-894X(03)00293-2

Reaction of the antimalarial and anti-schistosome drug artemether (1) and catalytic amount of ferrous ion in the presence of excess cysteine gave two adducts of cysteine and previous postulated primary and secondary carbon-centred free radicals besides th

Full text of DOI:10.1016/S0960-894X(03)00293-2

Bioorganic & Medicinal Chemistry Letters 13(2003) 1645–1647
Study on the Mechanism of Action of Artemether against
Schistosomes: The Identification of Cysteine Adducts of Both
Carbon-Centred Free Radicals Derived from Artemether
Wen-Min Wu,a Yan-Li Chen,a Zili Zhai,b Shu-Hua Xiaob and Yu-Lin Wua,*
aState Key Laboratory of Bio-Organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, Shanghai 200032, China
bInstitute of Parasitic Diseases, Chinese Center for Diseases Control and Prevention, Shanghai 200025, China
Received 6 January 2003; accepted 14 March 2003
Abstract—Reaction of the antimalarial and anti-schistosome drug artemether (1) and catalytic amount of ferrous ion in the pres-
ence of excess cysteine gave two adducts of cysteine and previous postulated primary and secondary carbon-centred free radicals
besides their other rearrangement products. This piece of further evidence for the presence of carbon-centred radicals, especially the
secondary carbon-centred free radical for the first time by the isolation of its coupling adduct, will be help to understand the
mechanism of action of artemether and other qinghaosu derivatives against parasites.
# 2003Elsevier Science Ltd. All rights reserved.
Artemether (1), a derivative of qinghaosu (artemisinin,
2), was firstly reported as a more active antimalarial
compound than its parent one by Li et al. in 1979.1 Now
it has been used as a very effective drug to fight the most
serious parasitic disease worldwide, malaria, especially
for the multi-drug resistant malarial parasite. During
the last decade it was found that artemether also pos-
sessed potent effect against the juvenile stages of
another parasite schistosome and could be used as a
prophylactic drug for prevention of schistosomiasis.2
Recently we performed a comparative study on adult
Schistosoma japonicium survival after incubation in two
media, Hank’s balanced salt solution (HBSS) and
RPMI 1640, supplemented with calf serum, antibiotics,
artemether and haemin. It was interesting to observe
that worm mortalities were consistently higher and
occurred faster in HBSS in comparison with those in
RPMI 1640. A control experiment (without the incu-
bation of S. japonicium) showed that decomposition of
artemether to free radical reaction products in RPMI
1640 was much faster than in HBSS. In this respect, it
was indicated that these free radical reaction products
of artemether exhibited no damage to the schistosomes.
It was suggested that the worms needed to ingest arte-
mether and haemin to the gut. Then the free radical
reaction took place in the gut. The radical was toxic to
the worms and killed the worms eventually.3 For the
detection of these products of minor quantity in the
incubation media, we prepared their authentic samples
from the free radical reaction of artemether and ferrous
ion. Herewith, we would like to report the chemistry of
isolation and identification of these products.
The reaction of qinghaosu and its derivatives with fer-
rous ion produced mainly two products, that is com-
pounds 3 and 4, which were proposed to be formed
through a primary carbon-centred free radical and a
secondary carbon-centred free radical, respectively.4ꢀ7
Lately the reaction of qinghaosu and its 12-deoxy-12-
aryl derivatives with catalytic amount of Fe(II/III) (fer-
rous or ferric ion) in the presence of cysteine or reduced
glutathione also could take place and produced a
hydrogen attracted product and a cysteine adduct or a
glutathione adduct of the primary carbon-centred free
*Corresponding author. Tel.: +86-21-641-63300x1415; fax: +86-21-
631-66128; e-mail: ylwu@pub.sioc.ac.cn
0960-894X/03/$ - see front matter # 2003Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00293-2

1646
W.-M. Wu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1645–1647
radical.8ꢀ10 At almost the same time the heme–qin-
ghaosu adduct and heme–artemether derived from the
primary carbon-centred free radical was also reported
from French laboratory.11
the foregoing fraction 6 (in 1.4% yield) was quite dif-
ferent with those adducts derived from the primary car-
bon-centred free radical of qinghaosu and its
derivatives. However, it contained the cysteine segment
1
and the artemether skeleton too as shown by H NMR
Considering the presence of cysteine in the incubation
medium RPMI 1640, the reaction of artemether was
carried out with catalytic amount of ferrous sulfate and
excess cysteine. Thus the reaction was run in aqueous
acetonitrile (1:1) at room temperature for 4 h, when all
starting artemether was consumed. Working-up gave an
organic phase and an aqueous one. From the organic
phase two known tetrahydrofuranyl product (3b) and
3-hydroxyl product (4b) was isolated in 26 and 44%
yield, respectively. Besides, a hydrogen-attracted pro-
duct 512 of the proposed primary carbon-centred free
radical was also isolated in 10% yield. From the aqu-
eous phase both adducts 613 and 714 of cysteine with the
proposed secondary and primary carbon-centred free
radical could be detected by TLC developed by using
nBuOH/AcOH/H2O as eluent and visualized by spray-
ing with ninhydrin as a pink spot, and were isolated by
reversed phase column chromatography. The structure
of major adduct 7 (in 3.2% yield) was easily determined
by its high resolution ESI mass spectrum and NMR
spectra, which had similar pattern with corresponding
adduct of other qinghaosu derivative.10 The structure of
and 13C NMR spectra. The NMR pattern of cysteine
part was similar to that of adduct 7 and the pattern of
the artemether part was similar to that of compound 4b
except the OMe signal. These NMR data and the
molecular weight 388 measured by ESI-MS showed this
compound was the adduct from the proposed secondary
carbon-centred free radical. The configuration of C-12-
hydroxyl was determined to be a because of the axial–
axial coupling constant (8.6 Hz) between H-11 and H-12.
The configuration of cysteine residue at C-3was proposed
to be b according to the coupling pattern of H-3. The
proton at C-3had chemical sift 2.97 as a doublet-doublet
peak with J value of 5.6 and 11.3Hz, which meant that H-
3was an axial proton and in a orientation calculated by
the molecular modelling (AM1).
The hydrogen-attracted product and the cysteine adduct
of primary carbon-centred free radical from qinghaosu
or its derivatives have been identified in our labora-
tory.8,10 However, the cysteine adduct of secondary
carbon-centred free radical from qinghaosu series was
isolated for the first time. Therefore, up to now, the

W.-M. Wu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1645–1647
1647
postulated primary and secondary carbon radicals were
confirmed not only by ESR spectra,4,5 but also by the
identification of both free radical adducts. On the other
side, with these standard samples in our hand we have
detected the products after incubation of artemether
with or without S. japonicium in different media. It was
found that RPMI 1640 contained a number of amino
acids including cysteine, therefore, artemether already
converted to its free radical reaction products and
cysteine adducts before it reached to the gut of parasite.
The observation and deduction of biological respect
were reported in detail elsewhere.3
F.; Mareda, J.; Millasson-Schmidt, P.; Brunner, G.; Burger,
U. Helv. Chim. Acta 1996, 79, 1475.
8. Wu, Y.; Yue, Z.-Y.; Wu, Y.-L. Angew. Chem., Int. Ed.
1999, 38, 2580.
9. Wang, D.-Y.; Wu, Y.-L. Chem. Commun. 2000, 2193.
10. Wang, D.-Y.; Wu, Y.-L.; Wu, Y.-K.; Liang, J.; Li, Y. J.
Chem. Soc., Perkin Trans. 1 2001, 605.
11. Robert, A.; Dechy-Cabaret, O.; Cazelles, J.; Meunier, B.
Acc. Chem. Res. 2002, 35, 167.
12. Compound 5: Mp: 66–68 ꢁC. ½aŠD20=+86 (c 0.59, CHCl3),
IR (KBr) n: 3552, 2989, 1758, 1466, 1240, 1094 cmꢀ1 1H
.
NMR (300 MHz, CDCl3) d: 0.89 (3H, d, J=7.3Hz), 0.94 (3H,
d, J=6.5 Hz), 0.97 (3H, t, J=5.9 Hz), 2.12 (3H, s), 2.48 (1H,
m), 3.43 (3H, s), 4.59 (1H, d, J=4.0 Hz), 6.29 (1H, s). EI–MS
(m/z): 281, 267, 209, 163. Anal. calcd for C16H28O5: C, 63.97;
H, 9.40. Found: C, 63.94; H, 9.42.
Acknowledgements
13. Compound 6: Syrup, ½aŠ2D5=ꢀ24.2 (c 0.5, CH3CN/
H2O=1:1). IR (KBr) n: 3420, 2930, 1634, 1386. 1H NMR
(400 MHz, D2O) d: 0.96 (3H, d, J=6.2 Hz, 13-H3), 1.00 (3H, d,
J=7.2 Hz, 14-H3), 1.77 (3H, s, 15-H3), 1.95 (2H, m), 2.20 (1H,
m), 2.35 (1H, m), 2.97 (1H, dd, J=5.6 Hz, 11.3 Hz, 3-H), 3.19
(1H, dd, J=7.0 Hz, 14.5 Hz, 30-H), 3.25 (1H, dd, J=4.6 Hz,
14.6 Hz, 30-H), 3.99 (1H, dd, J=4.4 Hz, 7.0 Hz, 20-H), 4.93(1H,
d, J=8.6 Hz, 12-H), 5.59 (1H, s, 5-H). 13C NMR (75 MHz,
This work was supported by the National Natural
Science Foundation of China, the Chinese Academy of
Sciences, and the State Ministry of Science and
Technology.
D2O–CD3COCD3) d: 13.73 (CH3), 18.72 (CH3), 22.83(CH ),
3
References and Notes
22.90 (CH2), 30.93 (CH2), 32.68 (CH2), 33.35 (CH), 34.22
(CH2), 35.12 (CH), 41.89 (CH), 47.72 (CH), 49.68 (CH), 54.86
CH), 83.93 (C), 95.92 (CH), 97.19 (CH), 111.18 (C), 172.81
(CO). ESI-MS (m/z): 388 (M+1), 775 (2M+1). ESI-HRMS
calcd for C18H30NO6S: 388.1788. Found: 388.1771.
1. Li, Y.; Yu, P.-L.; Chen, Y.-X.; Li, L.-Q.; Gai, Y.-Z.; Wang,
D.-S.; Zheng, Y.-P. Kexue Tongbao 1979, 24, 667. Li, Y.; Yu,
P.-L.; Chen, Y.-X.; Li, L.-Q.; Gai, Y.-Z.; Wang, D.-S.; Zheng,
Y.-P. Yaoxue Xuebao 1981, 16, 429.
2. Xiao, S.-H.; Booth, M.; Tanner, M. Parasit. Today 2000,
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J.; Mei, J.-Y.; Scorneaux, B.; Chollet, J.; Zhai, Z., Parasit.
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14. Compound 7: Syrup, ½aŠ2D5=+27.6 (c 1.0, CH3CN/
H2O=1:1), ½ꢀŠ25=+33.9 (c 0.8, H2O). IR (KBr) n: 3408, 2878,
D
1762, 1633, 1224 cmꢀ1. 1H NMR (400 MHz, D2O) d: 0.96 (3H,
d, J=7.3Hz, 14-H 3), 0.99 (3H, d, J=6.4 Hz, 13-H3), 2.26 (3H,
s, CH3CO), 2.38 (1H, m), 2.59 (2H, m), 2.84 (1H, m), 3.13
(1H, dd, J=7.1 Hz, 14.8 Hz, 30-H), 3.20 (1H, dd, J=4.4 Hz,
14.8 Hz, 30-H), 3.46 (3H, s, OCH3), 3.96 (1H, dd, J=4.4 Hz,
7.2 Hz, 20-H), 4.78 (1H, d, J=4.0 Hz, 12-H), 6.33 (1H, s, 5-H).
13C NMR (100 MHz, D2O–CD3COCD3) d: 11.96 (CH3), 20.28
(CH3), 20.77 (CH3), 24.34 (CH2), 27.92 (CH2), 31.28, 32.01
(CH2), 33.24 (CH2), 35.14 (CH2), 35.14, 47.57, 53.51, 53.62,
55.46, 73.20 (C), 87.02, 103.76, 171.77 (CO), 172.35 (CO). ESI-
MS (m/z): 420 (M+1), 839 (2M+1). ESI-HRMS calcd for
C19H34NO7S: 420.2050. Found: 420.2039.