Cleavage of qinghaosu (artemisinin) induced by non-iron transition-metal ions in the presence of excess cysteine

Article abstract of DOI:10.1002/hlca.200390249

In the presence of an excess of cysteine, a catalytic amount of a non-iron transition-metal ion (Co²⁺, Cu²⁺, Ni²⁺, Ti ⁴⁺, and Mn²⁺) may also induce cleavage of qinghaosu (artemisinin; 1) to give those end products previously reported for Fe ²⁺-mediated degradation.

Full text of DOI:10.1002/hlca.200390249

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Helvetica Chimica Acta Vol. 86 (2003)
Cleavage of Qinghaosu (Artemisinin) Induced by Non-Iron Transition-Metal
Ions in the Presence of Excess Cysteine
by Yikang Wu* and He-Hua Liu
State Key Laboratory of Bio-organic and Natural Products Chemistry,
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences,
354 Fenglin Road, Shanghai 200032, China
(e-mail: yikangwu@pub.sioc.ac.cn)
In the presence of an excess of cysteine, a catalytic amount of a non-iron transition-metal ion (Co2‡, Cu2‡
,
Ni2‡, Ti4‡, and Mn2‡) may also induce cleavage of qinghaosu (artemisinin; 1) to give those end products
previously reported for Fe2‡-mediated degradation.
The mode of antimalarial action of qinghaosu (QHS; 1) type compounds (1,2,4-
trioxanes) has been an enduring subject of research [1] since the late 1970s as a
consequence of wide-spreading [2] of multidrug-resistant malaria cases. It was noticed
in the 1970s that the peroxy bridge in QHS was essential for its antimalarial activity.
However, the potential reason for the importance of the peroxy bridge remained
entirely unclear until the early 1990s, when Posner and Oh [3] showed, through a
simplified analog, that ferrous ion could induce cleavage of such compounds and, thus,
generate radicals. The radical-generating ability was also demonstrated by many others
[4] to be common to all 1,2,4-trioxanes, which, along with the well-established
biological notion that radicals may cause damage to cell components, promoted the
concept that QHS kills the malaria parasite through the formation of transient radicals.
As a prerequisite for in vivo generation of radicals from QHS, there must exist some
one-electron reducing species inside the intra-erythrocytic malaria parasite. Until now,
much attention [1a] has been attracted to the intraparasitic free heme and hemozoin
(oxidized and polymerized heme) because they are present in substantial quantities.
Alternatively, those intraparasitic redox enzymes/functional proteins might [5] also
cleave QHS and, thus, could be potential targets of QHS. In that context, the central
transition-metal ion(s) of the redox centers may not necessarily be limited to Fe2‡,
because many known metalloenzymes [6] do have non-iron metals at their active
centers. It is, therefore, very interesting to know whether transition-metal ions other
than Fe2‡ may also induce a similar cleavage of QHS and what the outcome would be.
The cleavage reactions were carried out at 358 for 12 h under N2 MeCN/H2O 1:1 (v/v)
containing QHS (0.50 mmol), cysteine hydrochloride (1.00 mmol), Et3N, and added
metal ion (4.0 ¥ 10À4 mmol). The main results are summarized in Table 1. Remarkable
differences were indeed observed with different metal ions under otherwise identical
conditions. One of the most obvious differences lay in the rates. With Co2‡ or Cu2‡
(added as CoCl2 or CuSO4, resp.), the reaction proceeded almost as fast as with Fe2‡.
At the end of the 12 h reaction, there was no QHS (1) left. The reaction with Ti4‡

Helvetica Chimica Acta Vol. 86 (2003)
3075
Scheme
(added as TiCl4), however, was substantially slower (ca. 14% of 1 remained
unchanged). The cleavage-induced by Ni2‡ or Mn2‡ (added as NiSO4 or MnSO4,
resp.) was rather sluggish and incomplete, with a large amount of QHS recovered
unchanged at the end of the 12-h reaction.
To obtain the redox profiles of the metal chelates under the cleavage reaction
conditions, we also recorded cyclic voltammograms (CV) (Fig. 1) in the absence of
QHS. By comparison with the cyclic voltammogram of cysteine (not shown) under the
same conditions (except without adding any metal ion and QHS), it appeared that the
curves in the left-hand region of each CV (‡1.2 to ‡ 0.6 V) were associated with the
redox of cysteine itself. The curves in the À 0.4 to À 1.0 V region should be related to
the redox of the metal ions (chelates). The CVs for the Ni2‡ and Mn2‡ chelates were
quite similar within this region, with no distinct peaks at all. This might explain the
difficult degradation of QHS when these two metal ions were involved under the given
conditions. The cyclic voltammogram of the Co2‡ system was probably the closest one
to the system with the Fe2‡ ion. Therefore, the comparable rates of the Co2‡ and Fe2‡
systems seemed also to be reasonable. Cu2‡ Chelate(s) did not show obvious peaks in
the À 0.4 to À 1.0 V region. However, there were indeed clearly defined peaks in the
lower trace in the ‡ 0.4 V to 0 V region (corresponding to the electron release from the
chelates), which should be enough to reduce QHS (‡0.87 V [8]). It seemed that the
ease with which the cleavage reaction took place was not related to the redox
potentials. Possible involvement of inner-sphere electron transfer from the transition-
metal ion was previously proposed for a similar phenomenon [9].

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Helvetica Chimica Acta Vol. 86 (2003)
a
Table 1. Results of Cleavage (cf. Scheme) of QHS (1) Induced by Various Transition-Metal Ions
)
Products [mmol]
1
6
7
8b)
9
10
Fe2‡ c
Co2‡
Ti4‡
)
0
0.014
ca. 0
ca. 0
0.00628
ca. 0
ca. 0
0.053
0.0388
0.0367
0.0310
ca. 0
0.129
0.122
0.118
0.188
0.1050.020
0.110
0.219
0.260
0.226
0.207
0.085
ca. 0
0.070
ca. 0
0.333
0.384
0.0791
0.0498
0.068
Cu2‡
Ni2‡
0.0411
Mn2‡ d
)
ca. 0
0.00443
0.0164
a) The reactions were carried out at 358 for 12 h under N2 in MeCN/H2O 1 : 1 (v/v; 10 ml) starting with
0.50 mmol of 1, 1.00 mmol of cysteine hydrochloride, 150 ml of Et3N, and 4.0 ¥ 10À4 mmol of the indicated metal
ion (added as CoCl2, TiCl4, CuSO4, NiSO4, and MnSO4, resp.). The amounts of the products (except 8) were
estimated from the molar ratios measured by means of the integrals of the acetal (or aldehyde) signals in the
1H-NMR spectra of the crude product mixtures. b) Estimated from the missing amount in mmol (which agreed
d
well with reversed-phase HPLC isolation results). c) Taken from [5b], for comparison. ) Mn2‡ has previously
been used to replace Fe2‡ as the central ion of a simplified heme analog in cleavage of QHS, entirely for
experimental convenience, see [7]. However, neither the product distribution for the cleavage reaction nor the
potential biological significance of the replacement of Fe2‡ with Mn2‡ was given.
The differences between the metal ions were also observed in the product
distribution. The general trends were similar to that observed with Fe2‡, with most
products formed from 1 via the O(2) radical route (via 2 and 4). The O(1) products (via
3 and 5) were essentially 7 (accounting for only 7 8% of the total yields) when using
Co2‡ or Ti4‡ as the central ion. Essentially no 6 was detected (except in the run with
Cu2‡) (see Scheme).
The relative proportions of the O(2) products 8 10 also varied with the central ion
(see Table 2). Formation of 8 was always significant. When Ni2‡ and Mn2‡ ions were
involved, the highest proportion of 8 in the total O(2) products was recorded. Use of
Cu2‡ as the central ion also led to ca. 41% of 8 in the total O(2) products. These data
indicated that the S-alkylation also took place readily when non-iron transition-metal
ions were employed as the central ion in the chelates (see Scheme). By analogy, if the
cleavage is induced by an in vivo SÀM (M ˆ transition-metal ion) type redox center
rather than the simple cysteine chelates, a substantial portion of 4 would also undergo
similar attacks and irreversibly inactivate the redox center.
a
Table 2. The O(2) Product Ratios of Cleavage of QHS (1) Induced by Various Transition-Metal Ions
)
Added metal ions
Product ratios [%]
8/(8 ‡ 9 ‡ 10)
9/(8 ‡ 9 ‡ 10)
10/(8 ‡ 9 ‡ 10)
Fe2‡
Co2‡
Ti4‡
Cu2‡
Ni2‡
Mn2‡
29.8
26.6
30.0
40.6
63.2
84.1
50.6
56.4
57.4
44.7
12.0
3.4
19.6
17.2
12.6
14.7
24.7
12.5
a) For further information, see Footnote a in Table 1.

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Figure. Cyclic voltammograms of the solutions of cysteine hydrochloride (1.00 mmol) and Et3N (150 ml) in
MeCN/H2O 1 :1 (v/v) containing 4.0 ¥ 10À4 mmol of a) Fe2(SO4)3, b) CoCl2, c) TiCl4, d) CuSO4, e) NiSO4, and
f) MnSO4

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Figure (cont.)

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Figure (cont.)

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Compared with the formation of 8 and 9, hydrogen abstraction (10) appeared to be
much less favorable in most cases. This is consistent with the fact that in the absence of
an excellent hydrogen donor (e.g., SH of cysteine), the hydrogen abstraction has never
been observed.
Hypotheses play a very important role in the study of the antimalarial mechanism of
QHS. The objectivity of a hypothesis, therefore, inevitably has a strong influence on the
investigation. By putting the problem in a broader perspective, we may avoid omitting
critical factors in the hypothesis in the first place and, hence, help us focus future
endeavors on the most-promising directions.
This work was assisted by the National Natural Science Foundation of China (20025207, 20272071,
29832020), the Life Science Special Fund of CAS supported by the Ministry of Finance of China (Stz 98-3-03),
the Chinese Academy of Sciences (CAS −Knowledge Innovation× Project, KGCX2-SW-209), the Qinghaosu
Science and Technology Foundation, and the Major State Basic Research Development Program (G2000077502).
The authors also thank Prof. Ying Li for a generous gift of QHS.
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Received March 31, 2003