A possible antimalarial action mode of qinghaosu (artemisinin) series compounds. Alkylation of reduced glutathione by C-centered primary radicals produced from antimalarial compound qinghaosu and 12-(2,4-dimethoxyphenyl)-12-deoxoqinghaosu

Article abstract of DOI:10.1039/b006906j

Antimalarial compound qinghaosu (1) and its phenyl derivative 2 were reacted with reduced glutathione (GSH) and Fe(II/III) to give, besides other known degradation products, an interesting adduct from a primary C-centered free radical and GSH. Because GSH plays a very important role in the cell cycle, this finding may eventually lead to a new understanding of its mode of action.

Full text of DOI:10.1039/b006906j

A possible antimalarial action mode of qinghaosu (artemisinin) series
compounds. Alkylation of reduced glutathione by C-centered primary radicals
produced from antimalarial compound qinghaosu and
1
2-(2,4-dimethoxyphenyl)-12-deoxoqinghaosu
Dong-Ye Wang and Yu-Lin Wu
State Key Laboratory of Bio-organic & Natural Products Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China. E-mail: ylwu@pub.sioc.ac.cn
Received (in Cambridge, UK) 23rd August 2000, Accepted 3rd October 2000
First published as an Advance Article on the web 31st October 2000
Antimalarial compound qinghaosu (1) and its phenyl
derivative 2 were reacted with reduced glutathione (GSH)
With the adduct of cysteine and the primary free radical
derived from qinghaosu and its derivatives in hand, it would be
interesting to observe if an adduct could form from glutathione
and this free radical. Malaria parasite infected red cells have a
high concentration of the reduced glutathione (GSH, the main
reducing agent in physiological systems).10 It was also reported
that an excess GSH in the parasite may be responsible for
protecting the parasite from the toxicity of heme.11 In general,
GSH takes part in many biological functions, including the
detoxification of cytosolic hydrogen peroxide and organic
peroxides and then protects cells from oxidative stress.
Therefore, depletion of GSH or inhibiting glutathione reductase
in tumor or parasite cell will induce oxidative stress and then kill
these cells,12 which may explain the mechanism of some
antitumor or antiparasite agents. In addition, an alternative
promising approach to finding new antitumor or antiparasite
agents is provided.
and Fe(II/III) to give, besides other known degradation
products, an interesting adduct from a primary C-centered
free radical and GSH. Because GSH plays a very important
role in the cell cycle, this finding may eventually lead to a
new understanding of its mode of action.
Qinghaosu (artemisinin, 1) originally isolated from a traditional
Chinese herb,1 and its derivatives are the most promising
antimalarial medicines. Their unique 1,2,4-trioxane peroxy
Here we report that the C-centered radicals of compound 2
and qinghaosu (1) can abstract a hydrogen atom from GSH, and,
the novel adducts between 2, 1 and GSH are characterized.
The 12-(2,4-dimethoxyphenyl)-12-deoxoqinghaosu (2) pre-
pared from dihydroqinghaosu is a UV detectable, stable,
lipophilic and hence easily handled qinghaosu derivative. It is
also a more active antimalarial agent than qinghaosu and
artermether. Therefore compound 2 was firstly used as the
substrate to react with GSH in the presence of catalytic amounts
of ferrous sulfate. Thus, a mixture of GSH, AcONa, a catalytic
structure has been proved to be essential for antimalarial
activity2 and is entirely different from that of previous
generations of antimalarial drugs such as quinine and chlor-
oquine. Cell-biological studies showed that qinghaosu’s tox-
icity appeared to be the consequence of the membrane damage
4 2
amount of FeSO ·7H O and 0.5 equivalents of compound 2 in
3
that might result from protein alkylation. On the other hand,
aq. MeCN was stirred at rt for 3 days. From the organic extract,
alkylation of a simplified heme in vitro has also been reported,4
but this does not provide a full description of the antimalarial
mechanism of qinghaosu and its derivatives. We have reported
the reaction mechanism of the Fe(II)-induced cleavage of the
peroxide bond in qinghaosu and its derivatives and certified the
presence of C-centered radicals derived from this cleavage and
the following radical rearrangement.5 At the same time the
DNA damage associated with this process has also been
starting material 2 (20%) and known compounds 3, 4, 5, and 6
9
(43%, 26%, 2%, and 4%, respectively) were obtained. On the
other hand adduct 713 (1%) was isolated from the aqueous layer
by column chromatography (RP-C-18) (Scheme 1).
It is reasonable to propose that adduct 7 is formed by coupling
the primary C-centered free radical and GSH. Adduct 7 gives a
typical ESI mass spectrum peak at m/z = 735 (M + Na 2 1) and
all the corresponding proton resonances in the NMR spectra.
Adduct 7 tends to rearrange to give compound 8 in acidic
medium, partial rearrangement could be observed even in
deuteriated water in the NMR tube.
,6
5
7
observed in our laboratory. A parallel correlation between the
chemical reactivity of Fe(II)-induced cleavage of the peroxide in
qinghaosu’s derivatives and their antimalarial activity in vivo
8
and also the intermolecular interaction —abstraction of a
The successful isolation of adduct 7 encouraged us to explore
the reaction of GSH with qinghaosu–Fe(II/III) and search for the
hydrogen atom from cysteine and attachment to the cysteine
sulfur atom—of the primary C-centered radicals involved in the
degradation of qinghaosu have been found in our previous
work. Continuing these explorations we have synthesized a pair
of C-11 epimers of 12-(2,4-dimethoxyphenyl)-12-deoxoqing-
haosu (2) and found once again the parallel relationship of
reactivity of their Fe(II)-induced cleavage and antimalarial
activity. In the presence of cysteine and catalytical ferrous or
ferric ion a stable product from cysteine and the primary free
radical from phenyl qinghaosu 2 could be isolated and
products from both the organic and water phases. The reaction
of qinghaosu (1) with 2 equivalents of GHS and 0.04 equivalent
Fe(II) was performed at 37 °C under argon. After 12 hours all
8
qinghaosu was consumed to yield known compounds 9 (5%),
6
6
10 (41%), 11 (26.4%), and 12 (1%, a C-3 epimer of known
1
4
compound), all collected from the organic phase, and 13 (4%)
collected from the aqueous phase of the reaction mixture after
work-up (Scheme 2). The reaction also proceeds wider acid
conditions in the range pH 1–7, giving slight variations in the
yields of 9 and 13.
9
identified. These experimental results provided further evi-
dence for the participation of a primary carbon centered free
radical in the antimalarial action of qinghaosu compounds.
The most interesting adduct 13 is a highly polar, water
soluble compound, which was recovered from the reaction
DOI: 10.1039/b006906j
Chem. Commun., 2000, 2193–2194
This journal is © The Royal Society of Chemistry 2000
2193

elucidated by NMR and other spectroscopy. In Plasmodium
parasite the GSH concentration is maintained by means of the
glutathione reductase catalyzed reduction of glutathione dis-
ulfide (GSSG) at the expense of NADPH. GSH plays an
important role in protecting the parasite from oxidative stress.
The formation of a GSH–qinghaosu adduct reduces the amount
of GSH and the adduct itself might also cause inhibition of the
glutathione reductase in the parasite. Therefore the present
finding may lead to a new understanding of the mode of action
of these antimalarial drugs.
This work was supported by the Chinese Academy of
Sciences (no. KJ951-A1-504-04), the National Natural Science
Foundation (no. 29572075, 09561423, 29832020, 39870899),
and the Ministry of Science and Technology of China (no.
9
70211006-6). We also thank Dr Yikang Wu for the helpful
discussion.
Notes and references
1
(a) J.-M. Liu, M.-Y. Ni, Y.-F. Fan, Y.-Y.Tu, Z.-H. Wu, Y.-L. Wu and
W.-S. Chou, Huaxue Xuebao, 1979, 37, 129; (b) D. L. Klayman,
Science, 1985, 228, 1049; (c) A. R. Butler and Y.-L. Wu, Chem. Soc.
Rev., 1992, 85.
Scheme 1
2
(a) Y. Li, P.-L. Yu, Y.-X. Chen, L.-Q. Li, Y.-Z. Gai, D.-S. Wang and
Y.-P. Zheng, Yaoxue Xuebao, 1981, 16, 429; (b) Y.-L. Wu and Y. Li,
Med. Chem. Res., 1995, 5, 569.
3
4
5
6
S. R. Meshnick, T. E. Taylor and S. Kamchonwongpaisan, Microbiol.
Rev., 1996, 60, 301, and references cited therein.
(a) A. Robert and B. Meunier, Chem. Soc. Rev., 1998, 27, 273; (b) A.
Robert and B. Meunier, J. Am. Chem. Soc., 1997, 119, 5968.
W.-M. Wu, Z.-J. Yao, Y.-L. Wu, K. Jiang, Y.-F. Wang, H.-B. Chen, F.
Shan and Y. Li, J. Chem. Soc., Chem. Commun., 1996, 2213.
W.-M. Wu, Y.-K. Wu, Y.-L. Wu, Z.-J. Yao, C.-M. Zhou, Y. Li and F.
Shan, J. Am. Chem. Soc., 1998, 120, 3316, and references cited
therein.
7
8
9
D.-Y. Wang, Y.-K. Wu, Y.-L. Wu, Y. Li and F. Shan, J. Chem. Soc.,
Perkin Trans. 1, 1999, 1827.
Y. Wu, Z.-Y. Yue and Y.-L. Wu, Angew. Chem., Int. Ed., 1999, 38,
2
580.
D.-Y. Wang, Y.-L. Wu, Y. Wu, J. Liang and Y. Li, J. Chem. Soc., Perkin
Trans. 1, submitted for publication.
1
1
0 H. Atamna and H. Ginsburg, Eur. J. Biochem., 1997, 250, 670.
1 (a) H. Ginsburg, O. Famin, J. Zhang and M. Krugliak, Biochem.
Pharmacol., 1998, 56, 1305; (b) O. Famin, M. Krugliak and H.
Ginsburg, Biochem. Pharmacol., 1999, 58, 59.
1
2 R. H. Schirmer, J. G. Muller and R. L. Krauth-Siegel, Angew. Chem.,
Scheme 2
Int. Ed. Engl., 1995, 34, 141.
3 Compound 7: mp: > 210 °C. H NMR (600 MHz, D
1
1
2
O) d: 7.37 (d, 1H,
J = 7.8 Hz, Ar-H), 6.60 (d, 1H, J = 8.4 Hz, Ar-H), 6.56 (d, 1H, J = 2.4
Hz, Ar-H), 6.15 (s, 1H, H-5), 4.70 (m, 1H, H-12), 4.50 (m, 1H, H-6A),
mixture by chromatography on reverse phase silica gel (RP-
C18, H
BuOH–AcOH–H
in EtOH as a pink spot. The mass spectrum (ESI) exhibited
2
O). On thin-layer chromatography (TLC, with 3+1+1 n-
3
3
.76 (6H, 2OCH ), 3.74 (2H, 2H-9A), 3.08 (m, 1H, H-2A), 3.02 (m, 1H,
2
O) 13 could be visualized by 0.5% ninhydrin
H-7A), 2.80 (m, 1H, H-7A), 2.64 (m, 1H, H-3), 2.57 (m, 1H, H-11), 2.47
(m, 1H, H-3), 2.40 (m, 2H, 2H-4A), 2.22 (m, 1H, H-2), 2.04 (s, 3H, Ac),
1.98 (m, 2H, 2H-3A), 1.78–1.61 (m, 4H, H-9b, H-8a, H-7, H-8b), 1.39
(m, 1H, H-10), 1.26 (m, 1H, H-2), 1.12 (m, 1H, H-1), 1.05 (m, 1H,
3
+
+
typical peaks at m/z 548 (M + H) , 570 (M + Na) , and 586 (M
+
+
K) . Its high resolution mass spectrum (SI) exhibited the
+
H-9a), 0.85 (d, 3H, J = 6.6 Hz, CH at C-10), 0.50 (d, 3H, J = 6.6 Hz,
peaks at m/z 570.2090 (M + Na) , and 592.1916 (M 2 H +
+
1
CH
3
at C-11); MS (ESI) m/z: 779 (M 2 1 + 3Na), 757 (M + 2Na), 735
2
Na) . NMR analyses were performed in D
2
O, including H
(M + 1 + Na).
13
1
and C NMR, DEPT, DQ-COSY, NOESY, HMQC ( J) and
long-range ( J) proton-carbon correlation (HMBC). Compound
1
1
4 Compound 13 H NMR (600 MHz, D
H, H-6A), 3.85 (m, 3H, 2H-9A and H-2A), 3.14 (dd, 1H, J = 14.1, 5.1 Hz,
H-7A), 2.93 (dd, 1H, J = 14.1, 8.7 Hz, H-7A), 2.62 (m, 4H, 2H-4A, 2H-3),
.47 (m, 1H, H-11), 2.24 (m, 2H, 2H-3A), 1.98 (m, 1H, H-9ß), 1.82-1.91
2
O) d: 9.98 (s, 1H, CHO), 4.63 (m,
3
1
1
3 showed several cross-peaks (H-7A/H-3, H-6A/H-3) in NOESY
and (H-7A/C-3, H-3/C-7A) in HMBC spectrum. The data
undoubtedly showed that a s-bond was present between the
sulfur of the glutathione residue and C-3 in the qinghaosu part.
Compound 13 was therefore the result of an adduct between
2
(m, 3H, H-10, H-7, H-8a), 1.79 (m, 1H, H-8b), 1.63 (m, 1H, H-2), 1.56
(m, 1H, H-2), 1.39 (m, 1H, H-1), 1.31 (m, 1H, H-9a), 1.16 (d, 3H, J =
3 3
7.2 Hz, CH at C-11), 1.04 (d, 3H, J = 6.6 Hz, CH
at C-10); 13C NMR
·
3
(75 MHz, CDCl ) d: 209.99 (HCNO), 187.32 (C-12), 178.97 (C-10A),
primary carbon-radical derived from qinghaosu and GS .
1
77.59 (C-5A), 176.72 (C-1A), 174.67 (C-8A), 86.01(q, C-6), 56.95 (C-1 or
In conclusion, qinghaosu (1) and its phenyl derivative 2 could
be degraded in the presence of reduced glutathione (GSH) and
a catalytical amount of Fe(II/III). Besides known products
previously isolated from the reaction of qinghaosu and its
derivative with ferrous ion, an interesting adduct from the
primary C-centered free radical derived from qinghaosu and S-
centered free radical from GSH was isolated and structurally
C-2A), 56.86 (C-2A or C-1), 55.75 (C-6A), 53.45 (C-7), 46.50 (C-11),
4
3
6.13 (C-9A), 37.89 (C-10), 37.52 (C-9), 35.44 (C-7A), 34.67 (C-4A or C-
), 34.11 (C-4A or C-3), 30.43 (C-2), 28.94 (C-3’ or C-8), 28.88 (C-3A or
+
C-8), 23.00 (CH
3 3
at C-1), 20.26(CH at C-11); MS (ESI) m/z: 548 (M
+
37 10 3
1), 570 (M + Na); HRMS (SI) m/z: Calcd for C23H O N S + Na:
570.2092; Found: 570.2090; Calcd for C23H37O10N S 2 H + 2Na:
3
592.1911; Found: 592.1916.
2194
Chem. Commun., 2000, 2193–2194