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Current Medicinal Chemistry 2001, 8, 1803-1826

1803

Why Artemisinin and Certain Synthetic Peroxides are Potent Antimalarials. Implications for the Mode of Action

Charles W. Jefford*

Implications for the Mode of Action Charles W. Jefford * Department of Organic Chemistry, University of

Department of Organic Chemistry, University of Geneva, CH-1211 Geneva, Switzerland

Abstract: The discovery that the sesquiterpene peroxide yingzhaosu A (13 ) and 1,2,4- trioxane artemisinin (14 ) are active against chloroquine-resistant strains of Plasmodium falciparum, has opened a new era in the chemotherapy of malaria. In vitro and in vivo tests with synthetic structurally simpler trioxanes clearly demonstrate that much of the skeleton of 14 is redundant and that chirality is not required for activity. In addition, structure-activity relations and the search for the pharmacophore reveal that high antimalarial activity can be displayed by molecules which do not resemble the geometry of 13 and 14 at all. The possible mode of action of 13 , 14 , and synthetic peroxides is examined. They are believed to kill intraerythrocytic Plasmodium by interacting with the heme discarded by proteolysis of ingested hemoglobin. Complexation of heme with the peroxide bond followed by electron transfer generates an oxy radical that evolves to the ultimate parasiticidal agent. Experiments with ferrous reagents indicate that active peroxides including 14 and its congeners kill the parasite by alkylation with a sterically non-encumbered C- centered radical. However, another possibility is the involvement of a Fe(IV)=O species as the toxic agent. The review covers our own and other contributions to this timely topic and evaluates the different mechanisms proposed for the mode of action of peroxidic antimalarials.

INTRODUCTION

Background to the Disease

Malaria is an age-old disease and one of the commonest causes of illness in the world. It is estimated that about 2.5 billion people living in malarious zones are at risk and that 300-500 million clinical cases of malaria occur each year [1]. The disease is transmitted in a two-stage process by the bite of an infected female anopheline mosquito which can transfer to the human host four species of parasite, Plasmodium malariae, P. ovale, P. vivax and P. falciparum [2]. The parasites first invade the liver and then as merozoites penetrate erythrocytes where they proliferate and eventually burst the parasitized cells releasing about 25 daughter merozoites which then invade more erythrocytes so initiating a new infectious cycle. Some merozoites evolve into male and female gametes which undergo fertilization after being drawn into the gut of a second mosquito when it bites the infected host. The resulting sporozoites are then injected via the salivary glands into a fresh host through another bite thereby maintaining transmission.

The regions most affected by malaria are the northern part of South America, Central America, Africa below the Sahara, the Indian subcontinent, South East Asia, Vietnam, Indochina, Indonesia and the southern rim of the Pacific basin. Most patients suffer from uncomplicated malaria, characterized by fever, anemia and debilitation. However,

*Address correspondence to this author at the Department of Organic Chemistry, University of Geneva, CH-1211 Geneva, Switzerland; Ph.:

+41-22-7763316; Fax: +41-22-7763601; e-mail: Charles.Jefford@chiorg.unige.ch

0929-8673/01 $28.00+.00

chronic infections can lead to kidney failure, hyperactive malarial splenomegaly and Burkitt’s carcinoma. Most malaria is caused by P. vivax and is essentially benign, but Plasmodiun falciparum is virulent. It is the most dangerous because the level of parasitemia is much higher than for the other three species. Also more than 80% of the hemoglobin within the cell gets degraded to heme by the parasite. Moreover, the parasitized red blood cells in the late maturation stage lose their platelet-like form and no longer freely circulate in the blood. They become knobbly and stick together blocking the microvasculature in vital organs such as the brain; with coma and death being the usual sequel. In tropical Africa alone falciparum malaria claims each year the lives of 1.5-3 million children most of whom are under five years of age.

Treatment and Prevention of Malaria

The prevention and treatment of malaria today constitutes an acute challenge for modern medicine and public health management because many of the traditional quinoline-based drugs are becoming increasingly ineffective in certain parts of the world owing to multi-drug resistance [3]. In order to put the problem in perspective a short overview of the development of traditional antimalarials is presented.

Jesuit missionaries in Peru around 1630 discovered that the bark of the cinchona tree allayed fever. A few years later exportation of the bark to Europe at Rome’s behest led to its inclusion in the pharmacopoeia as a cure for fever [4]. In 1820 the active principle was found in to be the alkaloid quinine (1). This finding biased subsequent drug development in the years prior to 1939 towards chemically related remedies such as chloroquine (2), amodiaquine (3),

© 2001 Bentham Science Publishers Ltd.

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Current Medicinal Chemistry, 2001, Vol. 8, No. 15

Charles W. Jefford

MeO

H H HO N N
H
H
HO
N
N

Et 2 N

Me NH Cl N
Me
NH
Cl
N
HO NH Et 2 N Cl N
HO
NH
Et 2 N
Cl N
1 Me Et 2 N NH Cl N
1
Me
Et 2 N
NH
Cl
N

4

Fig. (1). Some traditional quinoline antimalarials.

mepaquine (4) and pamaquine (5). 4-Aminoquinoline and 9- acridine derivatives (2-4) and 1 act as blood schizonticides, whereas the 8-amino derivative 5 acts in the liver [5] Fig. (1). It should be mentioned that the mode of action of these quinoline derivatives was not known at the time. Consequently, the design of antimalarial drugs in the early

2

MeO

OMe

N HN Me
N
HN
Me

5

3

NEt 2

Unfortunately, in 1960 or thereabouts, the incidence of P. falciparum started to alarmingly increase. At the same time new chloroquine-resistant strains arose [9].

As a stopgap measure, mefloquine or Lariam (10), either used alone or in conjunction with 7 and 9 (Fansimef), was

HN

Cl Et CHMe 2 HN NH N NH NH H 2 N N NH 2
Cl
Et
CHMe 2
HN
NH
N
NH
NH
H 2 N
N
NH 2
6
7
O Cl HN S O MeO O N H S NH 2 2 N MeO
O
Cl
HN
S
O
MeO
O
N
H
S
NH 2
2 N
MeO
O
N
8
9

NH 2

Fig. (2). Some traditional inhibitors of dihydrofolate reductase and synthetase.

post-World War II years, based purely on mechanism, was a notable advance [6,7]. Proguanil (6), pyrimethamine (7), dapsone (8) and sulfadoxine (9) were rationally developed to curtail the growth of the malarial parasite in the liver and blood stages by virtue of their selective inhibition of dihydrofolate reductase and synthetase Fig. (2).

developed Fig. (3). However, mefloquine, since it too is a quinoline derivative, soon elicited resistant strains. Originally, the designers of Fansimef believed that 7 and 9 (termed Fansidar) would prevent resistance developing against 10, but the validity of this idea has been questioned

[10].

Disadvantages

of

Traditional

Nitrogen

Containing

In any event, the risk of severe cutaneous reaction (Stevens-Johnson syndrome) by Fansidar rules out its

Drugs

prophylactic use against falciparum malaria [11].

With this stock of cheap drugs available for prevention and cure, malaria was regarded as a vanquished disease [8].

Furthermore, Fansidar is inefficacious against P. vivax and carries a significant risk of mortality. In an endeavor to fill the niche left by progressively obsolescent chloroquine,

HO N H N CF 3 CF 3
HO
N
H
N
CF 3
CF 3

10

Fig. (3). Some successor antimalarials.

O OH O
O
OH
O

11

Cl

Bu

Bu

OH N Cl CF 3 Cl
OH
N
Cl
CF 3
Cl

12

Implications for the Mode of Action

Current Medicinal Chemistry, 2001, Vol. 8, No. 15

1805

recourse was recently made to antique drugs such as atovaquone (11) and proguanil (6), combined together as Malarone, and a derivatized phenanthrene isostere halofantrine (12) [12].

Apart from the diminished effectiveness of the aforementioned nitrogen heterocycles due to resistance by P. falciparum, there are certain personal disadvantages and risks for the patient or user. Chloroquine is limited in its geographical use, only working in the Middle East, Mexico and Central America. Mefloquine is expensive, 100 times mores so than chloroquine, and has resulted in seizures and psychiatric disorders. Halofantrine is equally expensive, unsuitable for prophylaxis, and has led to cases of cardiotoxicity. Even quinine is never totally effective, and its toxic side effects deter its usage [13].

THE ADVENT OF PEROXIDIC ANTIMALARIALS

Yingzhaosu and Artemisinin

Against this disheartening backdrop of increasingly unsatisfactory N-heterocyclic drugs coupled with the rising incidence of the deadly falciparum malaria, the advent of two non-nitrogenous lead compounds was not only timely, but signaled a new era of antimalarial chemotherapy [14, 15]. It was discovered in 1979 that two sesquiterpene peroxides obtained from Chinese medicinal plants, yingzhaosu A (13) and qinghaosu or artemisinin (14), possessed powerful antimalarial activity [16] Fig. (4). Yingzhaosu A (13), a typical 1,2-dioxane, occurs as a decomposition product arising in the stored roots of a sparsely growing vine, Artabotrys uncinatus (Lam.) Merr. [17]. Artemisinin (14) is a complex tetracyclic 1,2,4-trioxane found as the active principle of Artemisia annua Linn., a wild shrub of wide- spread habitat [18-20].

Me Me O O OH
Me
Me
O
O
OH

Me

OH

Me

13

O O O 4 C 11 B O O 6 Me D 3 A Me
O
O
O
4
C
11 B
O O
6
Me
D
3
A
Me

Me

Fig. (4). Yingzhaosu A and artemisinin.

Although the evidence is largely anecdotal, 13 is active against P. berghei, while the activity of 14 is securely

documented, being highly active against P. vivax and chloroquine-resistant P. falciparum. Rather than taking 13 and 14 for what they are, namely prototypes, tremendous efforts were deployed to devise syntheses and to make semi- synthetic first generation derivatives as potential drug candidates [21, 22]. The different aspects of artemisinin and its congeners have been well covered in many reviews [23- 28]. A recent review is devoted to peroxidic antimalarials in general [29].

Semi-synthetic Derivatives

In the case of yingzhaosu A (13), a synthetic route to the natural product based on (-)-carvone(15) as starting material [30] was adapted for preparing in some 10 or so steps essentially the surrogate molecules arteflene or Ro 42-1611 (16) and Ro 41-3823 (17) as pure enantiomers in addition to other analogues [31] Fig. (5). In comparison, derivatives of artemisinin were much easier to come by, merely entailing

the initial reduction of the freely available and crystalline natural product to the epimeric a and b -lactols (18) which were then converted to esters and ethers by standard procedures Fig. (6). Scores of semi-synthetic derivatives of 18 were produced of which the most important are b -arteether

(19), b -artemether (20), sodium b -artelinate

artesunic acid (22). All have antimalarial activities commensurate with that of the parent 14 [32, 33].

(21) and a -

Despite many successful syntheses, those presently devised for 13 and 14 are impractical either in terms of length or cost. For example, the surrogate arteflene has not been further promoted as a commercial pharmaceutical product, mainly for reasons of cost. Even the extra couple of steps required to convert 14 into 19-22 probably add too much to the price of the products for their intended use in third world markets. The idea of using the lead or even a first generation product as the actual commercial entity is

shortsighted. The usual proceeding for creating a new drug is

first to identify the pharmacophore of the providentially

provided natural product by stripping away the extraneous

bits. Once the essential structural elements are laid bare, the

next step is to elucidate the mode of action. With such information in hand, it should be possible to design more

14 potent, cheaper, synthetically accessible, and more biologically suitable antimalarials, and to gain, into the bargain, mechanistic insights that might reveal other therapeutic applications.

CF 3

Me O O
Me
O
O

O

Me
Me

O

Me

10 or so steps

Me

CF 3

and

15

16

O O
O
O

Me

(CH 2 ) 10 Me

Me

O

17

Fig. (5). Synthesis of arteflene (Ro 42-1611) and Ro 41-3823 from carvone.

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Current Medicinal Chemistry, 2001, Vol. 8, No. 15

Charles W. Jefford

14

HO O O O MeO O Me O O Me O O Me Me 18
HO
O
O
O
MeO O
Me
O O
Me
O O
Me
Me
18
Me
19

Me

C 6 H 4 CO 2 Na O O EtO O O O Me Me
C 6 H 4 CO 2 Na
O
O
EtO
O O O
Me
Me
O O
Me O O
Me
Me
20 Me
21
COOH O O O O O O Me Me 22
COOH
O
O
O
O
O O
Me
Me
22

Me

Fig. (6). Some semisynthetic derivatives of artemisinin (14 ).

LOOKING FOR THE PHARMACOPHORE The 1,2,4-trioxane ring per se is or rather was a relatively unknown chemical entity. Before 1957 none was

reported in the literature. Subsequently and especially since the discovery of artemisinin, many methods for their synthesis were devised [36]. As a result, numerous trioxanes of simplified, but varied structure were prepared and screened for in vitro activity against P. falciparum using the Desjardins test [37, 38]. In this test concentrations of the drug (expressed in ng/ml) which inhibit growth of the parasite by 50 and 90% are estimated (IC 50 and IC 90 ).

Tricyclic Trioxanes

On inspecting the artemisinin skeleton several questions arise. How many and which of the four rings (A,B,C, and D) are necessary for high activity? Is the boat conformation of the trioxane ring really necessary? Would a peroxide do as well? Is chirality important? What sort of peripheral substituents are required to bolster activity? It should be mentioned straightaway that the peroxide linkage in 14 is a critical element, since its metabolite deoxyartemisinin (23) having one O-atom less is without activity [34] Fig. (7). Moreover, the lactone function is a double liability in that it offers a site for hydrolytic degradation and also constitutes an inherent therapeutic weakness as attested by the 8-fold greater activity of deoxoartemisinin (24) [35].

O O O Me O Me 23
O
O
O
Me
O
Me
23
O O Me Me O Me O Me 24
O
O
Me
Me
O
Me O
Me 24

The synthesis and testing of many simpler tricyclic trioxanes reveal that certain rings in 14 are redundant. The high activity of the dimethyl derivative 25 shows that ring D can be dispensed with altogether or rather that the spirocyclic pentane ring serves in its stead Fig. (8). Clearly, the lactone function in ring B is superfluous. Further evidence that the B ring is unnecessary is attested by the high activity of the

endo-methoxy trioxanes 26 and 27. The pair of exo-methoxy epimers 28 and 29 performs less well, some five-fold. In

both instances, the phenyl group at the bridgehead boosts the activity over the methyl derivative. The peroxide 30 is devoid of activity; so there are limits to what extent the

basic sesquiterpene skeleton can be modified [39-41].

Fig. (7). Deoxyartemisinin and deoxoartemisinin.

Several other active, tricyclic trioxanes e.g. 31-33, have been synthesized, tested, and confirm that the full tetracyclic

O O B C Me O A O
O
O
B C
Me
O
A O

Me

MeO O C O O D A
MeO
O
C
O O
D
A

R

MeO O C O O D A
MeO
O
C
O O
D
A

25

26

R = Me

28 = Me

R

27

R

= Ph

29 = Ph

R

R

Me H O O O D A
Me
H
O
O O
D
A

30

PhCH 2 O O O O 31
PhCH 2 O
O
O O
31

Me

Fig. (8). Some tricyclic trioxanes.

OTs O MeO O O 32
OTs
O
MeO
O O
32

Me

OH O MeO Me O O Ph 33
OH
O
MeO
Me
O O
Ph
33

OMe

Implications for the Mode of Action

Current Medicinal Chemistry, 2001, Vol. 8, No. 15

1807

array of 14 is not required for high activity [42-44]. In the foregoing examples where tricyclic portions of the artemisinin architecture are mimicked, it could be argued that artemisinin-like activity is retained, because only the useless bits of the molecule were jettisoned. But as it will be seen later, molecules far removed from artemisinin (14) in shape and geometry can surpass it in activity. The same is true for yingzhaosu A (13); its structure does not need to be imitated in order for its effectiveness to be equalled.

Bicyclic Trioxanes

As the foregoing tricyclic trioxanes are not really so easy to prepare, a program was undertaken to devise syntheses of simpler bicyclic versions. For reasons of procedural convenience, three types of bicyclic trioxane were prepared, exemplified by the cis-fused dihydronaphthalene, cyclohexene and cyclopentene compounds 34, 37, and 41 Fig. (9). In spite of apparent structural similarities, the first two, and their derivatives 35, 36, 38-40, created something of a surprise by displaying little activity. The first set, 34-

[41, 45-46]. However, switching the parent ring from dihydronaphthalene to cyclohexene brought about a change in the right direction; the second set, 37-40, gave IC 50 values going from 729 to 104 ng /ml. The antimalarial activity, poor to start with, improved progressively on attaching spirocyclic rings at the C3 position. A dramatic jump was achieved in the case of the cyclopentene homologues 41-44; their IC 50 values against the W2 clone ranged from 5.31 to 0.49 ng/ml, demonstrating unequivocally that the carbocyclic framework of artemisinin is simply not required for activity. It is worth mentioning by way of reference that IC 50 values for artemisinin are usually about 1-2 ng/ml, so the preceding values are indicative of worthy contenders.

Unlike artemisinin, the cis-fused bicyclic structures of 41- 44 are flexible, permitting the trioxane ring to undergo inversion between two chair conformations. Clearly, the cis- fused, almost planar cyclopentene and the spirocyclopentane rings are crucial features of the pharmacophore in these fully synthetic analogues (v. infra). Another structural requirement which proved of supreme importance for attaining high

O O O 3 2
O
O
O
3
2

R 1

R

34

35

36

Me

Me

R 1 = R 2 = Me

R

R

Ph Ph O O O R 1 3 2 R 37 R 1 = R
Ph
Ph
O
O
O
R 1
3
2
R
37
R 1 = R 2 = Me
38 R 1 , R 2
=
(CH 2 )O(CH 2 ) 2

39

40

R 1 , R 2 = (CH 2 ) 5

R 1 , R 2 = (CH 2 ) 4

1 = H, R 2 = CH 2 C 6 H 4 OMe-4

1 = H, R 2 = (CH 2 ) 6 C 6 H 3 Cl 2 -3,4

Fig. (9). Some bicyclic trioxanes.

Ph Ph O R 1 O O 3 2 R 41 R 1 = R
Ph
Ph
O
R 1
O
O
3
2
R
41
R 1 = R 2 = Me

42

43

44

R 1 , R 2 = (CH 2 )O(CH 2 ) 2

R 1 , R 2 = (CH 2 ) 5

R 1 , R 2 = (CH 2 ) 4

36, was essentially inactive in the in vitro screen against the chloroquine-resistant W2 clone of P. falciparum, indicating that the trioxane ring is necessary, but not sufficient in itself

activity in the 41 series is the presence of two alkyl substituents at the C3 position and preferably spirocyclic, spirocyclopentyl being the best. Monosubstituted methyl,

O O N H HN O H H 5 N N O O O 5
O
O
N
H HN
O
H
H
5 N
N
O
O
O
5 O
6 O
C 6 H 4 F-p
O
C 6 H 4 Me
O O
C 6 H 4 F-p
C 6 H 4 F-p
O
O O
C 6 H 4 F-p
C 6 H 4 Me
45
46
47
OH H O C 6 H 4 Me O O C 6 H 4 Me
OH
H
O
C 6 H 4 Me
O
O
C 6 H 4 Me
48
OH H O C 6 H 4 Me O O C 6 H 4 Me
OH
H
O
C 6 H 4 Me
O
O
C 6 H 4 Me
OR H O C 6 H 4 F-p O O C 6 H 4 F-p
OR
H
O
C 6 H 4 F-p
O
O
C 6 H 4 F-p

49

50

R = CH 2 C 6 H 4 CO 2 Na

51

R

=

CO(CH 2 ) 2 CO 2 Na

52

R

= P(O)(ONa) 2

Fig. (10). Some synthetic trioxanes substituted at C5.

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Current Medicinal Chemistry, 2001, Vol. 8, No. 15

Charles W. Jefford

Table 1.

In vitro and In vivo Activity of Peroxides 16 and 17 and Trioxanes 44 and 53

Ph

C 6 H 4 F-p

Me Me

Ph O O O rac. O O O 44 53
Ph
O O
O
rac.
O O
O
44
53
O O CF 3 O Me CF 3 P. falciparum K1 strain, ng/ml 16
O
O
CF 3
O
Me
CF 3
P. falciparum
K1 strain, ng/ml
16
O O O Me(CH 2 ) 10 Me
O
O
O
Me(CH 2 ) 10
Me

17

C 6 H 4 F-p

rac.

Controls

ART CLQ MFQ

ED50

27.6

31.1

9.51

5.57

4.6

97.9

7.6

P. berghei, mg/kg/day x 4 ED50 p.o. ED90 p.o.

10.4

5.9

25.0

2.5

5.0

1.9

1.3

18.0

7.4

75.0

6.0 -

14.0

2.3

1.8

ED50 s.c

2.7

3.4

4.0

2.5

0.9

1.0

1.2

ED90 s.c.

3.9

4.1

8.0

6.0

2.5

1.2

1.8

ethyl, propyl or t-butyl derivatives regardless of configuration at C3, are all poorly active in vitro.

The activity of the best candidate 44 is also sensitive to substitution in other parts of the ring. Replacing the phenyl by tolyl or p-fluorophenyl substituents alters activity slightly. Substitution on the double bond, e.g. 45, and substitution at the C5 position in either the exo or endo configuration with neutral groups, e.g. 46-49, leads to EC 50 values of about 9-14 ng/ml, again for the W2 clone, which are about 3-4 times less active than the parent cyclopent-5,6- enes Fig. (10). When ionic water soluble groups are appended at the C5 position, as illustrated by trioxanes 50- 52, activity disappears entirely [48, 49]. The conclusion is that the configuration at C5 does not impinge strongly on activity, whereas the ionic nature of the substituent does.

As yingzhaosu A (13) is a fugitive species semi-synthetic derivatives have not been prepared. So arteflene (Ro 42- 1611) (16) and Ro 41-3823 (17) have to serve as indices of activity for the natural product [50]. It is therefore worth noting that the wholly synthetic cyclopentene-trioxanes 44 and its p-fluorophenyl analogue 53 exhibit ED 50 values of

9.5 and 5.6 against the chloroquine-resistant K1 strain of P. falciparum, values which are superior to those of 27.6 and 31.1 ng/mls shown by 16 and 17 respectively (Table 1). The p-fluorophenyl trioxane 53 also performs 2-4 times better than 16 against P. berghei both at the ED 50 and ED 90 levels by oral administration. A brief description of the in vivo test protocol is given later (see Table 2). The in vitro and in vivo results demonstrate that the yingzhaosu skeleton does not have to be copied to confer high activity. It demonstrates as well that 53 is a potential competitor to 16.

Chirality and Configuration

As all the synthetic trioxanes discussed so far are racemic mixtures, only one enantiomer being depicted for the sake of clarity, it might be asked if their IC 50 values should be halved to double their potency to what it appears to be on the assumption that only one of the enantiomers is the actual therapeutic agent. However, it can be concluded with confidence that in every case both enantiomers are equally parasiticidal on the basis of the proof offered by the exo 5- hydroxy enantiomers 54 and 55 Fig. (11). Each turned out

Ph Ph OH R S Ph Ph S O O O OH O O O
Ph
Ph
OH
R
S Ph
Ph
S
O O O
OH
O O
O
R
(+)
O
(-)
O
54
55
Me Me Me O Me Me Ph O S Me CO O Ph O O
Me
Me
Me
O
Me
Me
Ph
O
S
Me
CO
O
Ph
O
O
S
R
S Ph
S
Ph
CO
O O O
O
O O
O
R
O (-)
(+)
O
56
57

Fig. (11). Pairs of enantiomeric and diastereomeric synthetic trioxanes.

Implications for the Mode of Action

Current Medicinal Chemistry, 2001, Vol. 8, No. 15

1809

to have the same activity. Even the diastereomeric camphanoate derivatives 56 and 57 have similar activities to each other and also to 54 and 55, all compounds having IC 50 values lying between 1.5 and 2.4 ng/ml. In other words, they are all indistinguishable from artemisinin in their potency [51].

Further confirmation is tellingly provided by the in vivo activities of the pure enantiomers of the p-fluorophenyl derivative of cyclopentene-trioxane 58 and 59 when compared to that of the racemic mixture 53 (Table 2) [52]. In the Peters 4-day test against murine malaria, batches of mice are initially infected with chloroquine-sensitive P. berghei N or chloroquine-resistant P. yoelii NS lines respectively and dosed each day for 4 days subcutaneously (sc) or orally (po) with the test antimalarial [53]. On the 5th day the parasitemia is read and the effective dose which suppresses 50% and 90% estimated. It is immediately seen that the racemate 53, the (+) and (-)-enantiomers, 58 and 59, have essentially identical ED 50 values against the sensitive strain in both the sc and po modes of administration (Table 2). The same is true for the ED 90 values. A similar alignment of ED 50 and ED 90 values is also evident for the resistant line. It is also to be noted that the synthetic trioxanes (53, 58 and 59) are superior to artemisinin in the resistant line. Moreover, 53 offers a bonus in being gametocytocidal and sporontocidal [54].

The foregoing results from the two sets of enantiomerically pure synthetic trioxanes convey an unambiguous message. Configuration and chirality play no role in the mode of action. The trioxane ring in the synthetic bicyclic molecules have interconverting chair conformations, so a fixed boat conformation is not a requirement.

Nonetheless, other molecular features are obviously important and must intervene during the parasiticidal event. However, before they can be identified and their significance ascertained, the mode of action needs to be understood.

MODE OF ACTION

The above mentioned natural and synthetic trioxanes and peroxides are chemically very different from the traditional aminoalkyl substituted quinoline remedies such as quinine and chloroquine. The former are neutral species whereas the latter are bases. Both classes of drug are schizonticides acting on the parasite within the infected erythrocyte, but they do so by entirely different mechanisms. Curiously enough, the receptor in both cases has been identified as heme. Since heme is achiral it is understandable that the configuration of an enantiomerically pure antimalarial has no bearing on potency.

In order to be effective, an antimalarial must first of all diffuse into the red blood cell and interact with heme. It is now known from a large body of evidence that artemisinin (14), its lactol derivatives 18-22 and peroxidic antimalarials kill the malarial parasite in the blood of the host by a stepwise process of chemical induction Fig. (6). During the trophozoïte stage of the intraerythrocytic cycle the parasites invade the red blood cells and ingest hemoglobin to provide amino acids for growth. After proteolysis the spent prosthetic group, heme, because of its solubility and toxicity to the parasite, is eliminated immediately by oxidation and polymerization to the insoluble malarial pigment, hemozoin [55]. When the host is treated with quinine or chloroquine, it seems that polymerization of heme is pre-empted by

Table 2.

In vivo Antimalarial Activity of Some Cyclopentenotrioxanes

RR +

SS

(racemic)

53

C 6 H 4 F-p R C 6 H 4 F-p O O O (+)
C 6 H 4 F-p
R
C 6 H 4 F-p
O
O
O
(+)
R
58
C 6 H 4 F-p S S C 6 H 4 F-p O O O
C 6 H 4 F-p
S
S
C 6 H 4 F-p
O O
O
( _ )
59
 

ED 50

ED 90

ED 50

ED 90

ED 50

ED 90

P.

berghei N

sc

2.5

6.0

2.1

3.6

1.8

3.2

mg/kg/day x 4 po

 

2.5

6.0

2.6

4.8

2.1

3.6

sc

P. yoeliiNS mg/kg/day x 4 po

 

4.5

7.6

1.8

3.4

1.5

2.8

5.6

10.0

1.4

5.0

1.1

3.1

 

Chloroquine

Quinine

Artemisinin

ED 50

ED 90

ED 50

ED 90

ED 50

ED 90

P.

berghei N

sc

1.8

3.1

65

170

0.9

2.3

P.

yoeliiNS

sc

2.4

56

128

290

5.8

10.0

1810

Current Medicinal Chemistry, 2001, Vol. 8, No. 15

Charles W. Jefford

O O Ar Ar O O O Ar Ar 53 62 Me Me Me Me
O
O
Ar
Ar
O
O
O
Ar
Ar
53
62
Me
Me
Me
Me
HO 2 C
HO 2 C
N N
HO
N O
N
2 C
N Fe
HO 2 C
N Fe
N
N
Me
Me
Me
Me
60
61
O
64
63

Fig. (12). Oxygen atom transfer from a trioxane to heme, and then to cyclohexene.

chelation and its toxic effect potentiated so that the parasite is killed [56-58]. During the mortal event chloroquine remains chemically unchanged neither reacting with nor killing the parasite. Instead, it lets heme do the job. Treatment of the host with an antimalarial peroxide also interrupts the aforementioned detoxification process. But it does it in a different way from the quinoline-type drugs. It closely coordinates with heme and then reacts with it to produce a lethal agent.

The nature of the latter, and how it forms took some time to be delineated. Experiments performed with hemin and artemisinin confirmed their interaction to form an unknown adduct after generating an unidentified oxy radical [59-62]. Artemisinin was thought to alkylate heme and parasite proteins, but no structures were proposed [63]. It was also discovered that the administration of radiolabeled b -arteether,

dihydroartemisinin, and arteflene to P. falciparum-infected erythrocytes resulted in the transfer of label to six malarial proteins [64]. It was therefore concluded that in some undefined manner, not necessarily involved with parasite death, reaction with specific malarial proteins had occurred. Nonetheless, signs were seen that the parasiticidal process somehow involved transient radicals. Gradually, by devising model experiments with simpler peroxides and 1,2,4 - trioxanes, the nature of the lethal agent and the mechanistic details of the mode of action became clearer and more complete.

In recent years numerous model experiments have been performed and even today some of the results and interpretations presented as the final word are open to question as they are at variance with the canons of contemporary organic chemistry.

O FeCl 2 .4H 2 O (0.35 equiv) R(CH 2 ) 4 O Ar O
O
FeCl 2 .4H 2 O
(0.35 equiv)
R(CH 2 ) 4
O
Ar
O
Ar
O
MeCN, 22 o , 2h
HO
O
Ar
Ar
53 Ar = C 6 H 4 F-p
65
R = Cl
66%
66
R
= OH
8%
2+
67
R = H
2%
Fe
3+
_
Fe
Cl, H 2 O
+
H
O
O
O
. O
Ar
Ar
O +
O
_
Fe 2+
_
_
O
O
O Ar
3+
Ar
Ar
Fe
73 O
75
77
O
Ar
.
Ar
O
O
_
O
O
Ar
Ar
74
76

Ar

Fig. (13). Ferrous ion-induced rearrangement of trioxane 53 to propionates.

Implications for the Mode of Action

Current Medicinal Chemistry, 2001, Vol. 8, No. 15

1811

Are Antiparasitic Trioxanes Oxygen Atom Donors?

Originally it was thought, erroneously as it transpired, that racemic synthetic trioxanes such as 53 and artemisinin (14), as they appear to be loaded with excess oxygen, might behave as oxygen atom transfer agents. The idea was fostered by the fact that deoxyartemisinin (23) was the main metabolite and that heme might behave like a P450-type monooxygenase [65, 66]. It was postulated that active trioxanes would first transfer an oxygen atom to heme (60) to form the corresponding iron-oxene intermediate (61) which in turn would destroy a neighboring parasite by monooxygenating its protein Fig. (12). Heme might be expected to convert 14 into 23 during the parasiticidal event. Similarly, 53 would give the dioxolane 62. The idea was tested by using ferrous chloride tetrahydrate in acetonitrile as a model for heme. As a simple chemical model for the parasite cyclohexene (63) was chosen as it should be easily epoxidized to 64 by the ferryl equivalent of 61. Actual treatment of 53 and 14 with ferrous chloride caused rapid unraveling of the trioxane rings [67, 68] Fig. (13). Three products were formed from 53; the cyclopentenyl esters of d - chloropentanoic (65), d -hydroxypentanoic (66) and pentanoic acids (67) in yields of 66, 8 and 2% respectively [62]. The racemic diphenyl cyclopentene-trioxane 44 under the same conditions gave analogous results. Repetition of both experiments in the presence of cyclohexene (63) led to basically the same result. Neither dioxolane 62 nor epoxide 64 was formed.

In the case of artemisinin (14), only isomerization occurred [68]. Exposure to FeCl 2 in MeCN for no more than

15 min gave furan-acetate 68 and the hydroxy- deoxyartemisinin 69 in 78 and 17% yield Fig. (14). Repeating the experiment in the presence of cyclohexene (63) merely altered the yield of the two products to 84 and 1-8% respectively. No traces of 64 or deoxyartemisinin (23) were detected. b -Artemether (19) gave a similar result [68] Fig. (15). It reacted in 5 min. isomerizing to the corresponding furan-acetate 70, the hydroxy-deoxyartemsinin derivative 71, and an epimeric mixture of formyl diketones 72 in yields of 32, 23, and 16%. Running the reaction in cyclohexene improved the yields of the same three products to 36, 30 and

19%.

The Intermediacy of Carbon Radicals

The foregoing product compositions are strongly indicative of the intermediacy of radicals in their formation. In fact, there are many precedents for the redox behavior of ferrous ion towards cyclic peroxides which induces decomposition by forming radicals [69-72]. In the present instance, ferrous ion functions in the customary manner complexing with the O-O bond of 53 with concomitant single electron transfer Fig. (13). Scission of the resulting oxy radical 73 is driven by the formation of the ester function creating simultaneously the terminal pentanoate radical 74. Recuperation of the electron by ferric ion furnishes 75, which probably for reasons of entropy is unable to cyclize to the lactone 76. Instead, ambient chloride ion and to a lesser degree water, attack 75 giving the chloro and hydroxy products 65 and 66. Overall, the conversion of 53 to 65 and 66 can be considered as an isomerization with adjunction of

O O O O O O O Me O O Me FeCl 2 .4H 2
O
O
O
O
O
O
O
Me
O
O
Me
FeCl 2 .4H 2 O, MeCN
Me
O
O-O
Me
Me
O
+
Me
O
25 o , 5-15
min
OH
68
Me
14
Me
Me
69
2+
without cyclohexene
cyclohexene (1.18 equiv.)
78%
2+
17%
Fe
Fe
84%
1-8%
O
O
O
O O O
Me
2+
Fe
.
4 Me
O
2+
C-C scission
Fe
.
Me
O O
Me
O
3
H 78
80
Me
Me
2+
O
O
O
O
O
O=Fe
O
O
O
O O
O
O
2+
2+
Fe
Me
Fe
Me
.
Me
O
Me
Me
6 O O
Me
OH
.
Me
OH
Me
OH
1,5 shift
3
H
O
Me
Me
Me
O=Fe 2+
Me
79
81
82
83
2+
Fe

Fig. (14). Ferrous ion-induced isomerization of artemisinin via radical intermediates.

1812

Current Medicinal Chemistry, 2001, Vol. 8, No. 15

Charles W. Jefford

O O O MeO O Me FeCl 2 .4H 2 O MeCN O Me MeO
O
O
O
MeO
O
Me
FeCl 2 .4H 2 O
MeCN
O
Me
MeO
MeO
O
CHO
Me
O
O
O-O
+
+
O
Me
Me
O
Me
O
Me
25 o , 5 min
Me
OH
Me
19
Me 70
Me
71
Me
72
2+
without cyclohexene
cyclohexene (1.18 equiv.)
32%
23%
16%
Fe
36%
30%
19%
2+
2+
Fe
Fe
_ +
O
O
O MeO, CO, H
O
MeO
O MeO
O 2+
MeO O
O
O
. Me
.
Me Fe
O
Me
O
Me
Me
O O
+ Me
OO
Me
Me
2+
2+
Fe
Fe
Me
Me
Me
Me
97
98
99
100

Fig. (15). Ferrous ion-induced rearrangement of b -artemether.

a molecule of hydrogen chloride or water. The origin of the pentanoate 67 can be attributed to the abstraction of a hydrogen atom by 74 from the solvent.

Treatment of 53 with ferrous bromide in tetrahydrofuran (THF) as solvent gave mainly the bromo analogue of 65 [73, 74] Fig. (13). No dioxolane 62 was observed. Indirect confirmation of the radical 74 was obtained by carrying out the same experiment with ferrous sulfate and cupric acetate in methanol. The terminal olefin 77 was the exclusive product. As soon as 74 forms it is oxidized by cupric ion to the corresponding masked primary cation 75 which promptly eliminates a proton.

In just the same way as before ferrous ion complexes with the peroxide bond of artemisinin (14) Fig. (14). Although not specified in the preceding example, the ferrous ion after single electron transfer forms a covalent bond with the oxygen anion of the sundered peroxide bond giving a pair of equilibrating ferric monodentate oxygen radicals 78 and 79 which are formed in different proportions. They evolve differentlytoo. The main course is followed by 78 which cleaves to the pendent primary ethyl radical 80; the driving force being the acquisition of thermodynamic stability by formation of the acetate group. Expulsion of the contiguous ferric ion as ferrous ion and union of the two radical centers creates the tetrahydrofuran 68.

The evolution of the minor oxy radical 79 was originally puzzling Fig. (14). A 1,5 hydrogen atom shift to produce the

secondary C-centered radical 81 was proposed [75]. Thereafter, the cyclic enol ether 82 was supposed to arise by the excision of O=Fe 2+ [76]. The latter, if not dispersed or destroyed, could then react again with 82 to afford the epoxide 83. Alternatively, 83 could be formed directly by loss of ferrous ion from 81. Finally, opening of the epoxide 83 by internal nucleophilic attack by the tertiary hydroxyl group accounts for the formation of the minor product 69 [77]. The intermediacy of O=Fe 2+ may have been invented to account for deoxyartemisinin (23), the human metabolite of 14, which is formed in addition to 68 and 69 when artemisinin was exposed to ferrous bromide in THF (v. infra). Although C-centered radicals like 78 and 79 had been previously invoked [75] as possible agents responsible for the high antimalarial activity of the tricyclic tosylate (32), the later predilection for O=Fe 2+ as the toxic agent may have been influenced by the large body of research carried out with

P450-monooxygenases.

The 1,5 H Shift

Despite their plausibility, some of the foregoing steps seemed unsatisfactory and deserve comment. The 1,5 hydrogen transfer, although favored over 1,3 and 1,4 transfers, must meet certain geometric criteria for it to occur [78]. It appeared from an examination of the calculated and X-ray structures of 14 taken as a model for the Fe 3+ -bound complex that the interatomic distance between H-C(3) and O-C(6) in 79 is 2.478-2.803 Å [68]; a distance greater than

78

O O Fe 2+ O O O O 2+ 2+ Fe Me Fe . Me
O
O
Fe 2+
O O
O O
2+
2+
Fe
Me Fe
. Me
Me
O O
.
Me
O
H
OH
Me
Me
84
85

69

Fig. (16). Incorrect rearrangement of oxy radical 78 via 1,3 and 1,2 shifts.

Implications for the Mode of Action

Current Medicinal Chemistry, 2001, Vol. 8, No. 15

1813

the critical distance of 2.1 Å above which migration is thought not to be possible [79]. The distance between H- C(3) and O-C(4) in 78 was less (2.467-2.560 Å), but still exceeded 2.1 Å. As there was no clear-cut preference for 1,5 over 1,3-H shift, the latter was favored for plotting a path from 78 to 69 Fig. (16). Consequently, a 1,3-H shift followed by a 1,2 shift of a hydroxyl group (78Æ 84Æ 85) were proposed to give the tertiary radical which then was supposed to close to 69 by loss of ferrous ion.

Unfortunately, the preceding path is the wrong one as attested by the subsequent isolation of epoxide 83 and the trapping of the secondary radical 81 (v. infra). The findings of a recent study using density functional theory also provided a mechanistic corrective [80]. Taking 6,7,8- trioxybicyclo[3.2.2]nonane (86) as the model for 14, formal addition of a hydrogen atom affords the pair of oxy radicals 87 and 89 Fig. (17). Calculation confirmed that the interatomic distance between the oxygen radical and the contiguously oriented 1,5 disposed hydrogen atom in the ground state of 87 is 2.340 Å, a distance definitely greater than the critical value of 2.1 Å. However, the activation energy for 1,5 H-transfer to give the secondary carbon radical

stable than its acetal precursor 89 by 12.2 kcal/mol. These calculations of course strictly apply to the artificial oxy radicals 87 and 89, but nonetheless they are relevant to the 1,5 H shift 79Æ 81 and the C-C scission 78Æ 80. The conclusion is ineluctable. Converting an oxy radical to a C- centered radical by shifting a H-atom or cleaving a C-C bond is going to be easy. More importantly, the C-centered radicals 80 and 81 derived from artemisinin, once formed, will be unable to regress to their oxy radical predecessors because the energy of activation in the back reaction is now far higher than it was in the forward reaction. In other words, these C-centered radicals are kinetic products and their formation is irreversible.

A further indication that ester formation is the driving

force for producing an active primary C-centered was provided by calculations on the 1-methoxycyclopentyl-1- oxyl radical (91) and its scission product the d -radical (92) [52]. Their geometries were optimized by semi-empirical unrestricted Hartree-Fock calculations according to the PM3 method Fig. (18). The respective heats of formation, -59.5 and -76.6 kcal/mol, confirm that the conversion 91Æ 92 is strongly exothermic by 17.1 kcal/mol. A precedent for the

O O OH OH O 1,5 shift . O O . O H HO 86
O
O OH
OH
O
1,5 shift
.
O O
. O
H HO
86
87
88
.
O
O
O
O
C-C scission
.
HO
HO
89
90

Fig. (17). Rearrangement and cleavage of oxy radicals derived from 86 .

88 turned out to be low, only 6.4 kcal/mol. Moreover, the geometry of the reacting atoms in the transition state leading to 88 revealed that a collinear arrangement is not attainable, which means that it is evidently not a requirement. The carbon radical 88 was found to be more stable than the oxy radical 87 by 4.5 kcal/mol.

Carbon Radicals as Kinetic Products

The evolution of the acetal-type radical 89 was similarly favored. The activation energy for its cleavage to the formyl primary carbon radical 90 was computed to be 7.8 kcal/mol. Again, the C-centered radical 90 was predicted to be more

foregoing hypothetical reaction is the ready cleavage of 1- methylcyclopentyl hydroperoxide by ferrous ion to hexa-5- one-1-yl radical [81].

In similar fashion, the hydroxy oxy radical 93 derived by

adding a hydrogen atom to artemisinin serves as a model for both ferric derivatives 78 and 79 Fig. (19). The heats of formation for 93 and its rearranged products the primary and secondary radicals 94 and 95 were found to be -199.4, -216.3, and 219.3 kcal/mol respectively [68]. In other words, the act of creating the primary and secondary radicals releases 16.9 and 19.9 kcal/mol. Thus, both the 1,5 shift and C-C scission are strongly exothermic processes. By extrapolation, the rearrangements 78Æ 80 and 79Æ 81

H H H H H H H H H O . H O H .
H
H
H
H
H H
H
H H
O
.
H
O
H
.
H
H
H
H
O
H
H
H
O H H

H

91

92

H

Fig. (18). Cleavage of the methoxycyclopentyloxyl radical.

1814

Current Medicinal Chemistry, 2001, Vol. 8, No. 15

Charles W. Jefford

O O O O O O . Me O . Me OH O Me OH
O
O
O
O O O
.
Me
O
.
Me
OH
O Me
OH
Me
93
Me
94

Me

+

O O O HO Me OH . Me 95
O
O
O
HO
Me
OH
.
Me
95

Me

Fig. (19). Cleavage and rearrangement of the hypothetical oxy radical from artemisinin.

should be exothermic to the same degree and therefore irreversible. These conclusions, as it will be seen later, have special relevance to the mode of action of peroxidic antimalarials.

Is O=Fe 2+ an Intermediate?

No direct evidence has been reported for the existence of 82, while that in support of O=Fe 2+ is circumstantial. The experiments, the rationalization of which led to the proposal of such exotic species, were conducted by subjecting artemisinin (14) to ferrous bromide in THF alone or in the presence of various easily oxidizable or aromatizable addends as possible mechanistic witnesses [27, 76]. Under these conditions, 68 and 69 were formed as before in a similar ratio, but in diminished yields of 29 and 10%, the balance of material being deoxyartemisinin (23) (59%). The proportion of 23 went up on adding progressively more 1,4- cyclohexadiene. Hexamethyl Dewar benzene (HMDB) also boosted production of 23, becoming partially aromatized to hexamethylbenzene in the process. Consequently, it was inferred that 23 arose by protonation and intramolecular closure of the putative cyclic enol ether 82, increasingly furnished by loss of O=Fe 2+ from 81 Fig. (14). A supplementary avenue to 23 was assumed to issue from 81 by abstraction of hydrogen from 1,4-cyclohexadiene.

However, 23 could have arisen by a less exotic and mechanistically more likely route. The reduction of artemisinin (14) or perhaps, more appropriately, the ferric oxy radicals 78 and 79 by abstraction of hydrogen from THF or added 1,4-cyclohexadiene without a hydrogenation catalyst (none is needed) to give the dihydroxy derivative 96 followed by dehydration to 23 offers a reasonable explanation Fig. (20). Ferrous ion or a transient radical, rather than O=Fe 2+ , may well have catalyzed the aromatization of HMDB. Other addends such as methyl phenyl sulfide and tetralin, the oxygenation of which was presented as proof for O=Fe 2+ , may have abstracted oxygen directly from 14 or could have been oxidized adventitiously. The recent

O O O H 2 O HO Me Me OH 14 or 78 23 Me
O
O
O
H 2 O
HO
Me
Me
OH
14 or 78
23
Me
96
Fig. (20). Reduction of artemisinin or its oxy radical 78 to the
diol 96 and deoxyartemisinin 23 .

observation that large amounts of diol are formed when simple bicyclic endoperoxides are treated with FeBr 2 /THF attests to its reductive nature [82]. Finally, it is difficult to explain why O=Fe 2+ , a non-selective oxidant, if really present, which has no trouble in epoxidizing the enol ether 82, fails to react with admixed 1,4-cyclohexadiene to give its epoxide.

Lastly, it should be mentioned that the simple extrusion and transfer of an oxygen atom from a peroxide to a ferrous cation might be mechanistically difficult. In order to be an effective oxidant, the high valent iron-oxene species may need to be perferryl, the species formed in the P450 enzyme catalytic cycle, rather than ferryl [83]. If this be the case, the second step after coordination with ferrous ion, the rupture of the FeO-R bond to produce the perferryl species O=Fe 3+ requires the departure of R as an anion, not a radical, which is more costly in energy and less likely.

In view of the inertness of cyclohexene in the FeCl 2 - induced reactions with 53 and 14 together with the non- formation of deoxyartemisinin (23) or the dioxolane (62), the original proposal that only C-centered radicals are involved is undoubtedly correct and does not need to be modified to fit a P450 mono-oxygenase mechanism in spite of its attractiveness [84]. The FeCl 2 -induced reaction of b - artemether (19) with and without cyclohexene follows the same course and can be similarly interpreted Fig. (15). Deoxy-b -artemether is not formed. The furan-acetate 70 and hydroxy-deoxyartemisinin 71 arise in the standard way via cleavage and rearrangement of the pair of ferric oxy radicals 97 and 98. The third product the mixture of diketones 72 must have arisen by the formal loss of a molecule of methyl formate. How this happens is explicable in terms of the

elimination of ferrous ion in a counterclockwise or clockwise radical fragmentation of 97 or 98. The resulting methoxymethyl formate 99 then disintegrates by loss of a proton, carbon monoxide, and methoxide ion, giving the penultimate diketo-aldehyde 100, which finally isomerizes to

72.

The final piece in the mechanistic jigsaw puzzle was the isolation of the unstable epoxide 83, albeit in a tiny amount

(1%), from the reaction mixture obtained by treating 14 with one equivalent of ferrous sulfate in aqueous acetonitrile. This valuable piece of information confirms the sequence 79Æ 81Æ 83Æ 69 [85, 86]. However, it must be noted that the key experiment of actually isomerizing 83 to 69 appears not to have been done or at least reported. Proof for a secondary carbon radical, presumably 81, was secured by its capture with 2-methyl-2-nitrosopropane and the identification of the resulting nitroso radical by its ESR spectrum. Why the

Implications for the Mode of Action

Current Medicinal Chemistry, 2001, Vol. 8, No. 15

1815

O O O O O O O O Me O . Me O + O
O
O
O
O
O
O
O
O
Me
O
.
Me
O
+
O
O
O O
.
Me
OH
Me
Me
H
Ph
Ph
Ph
Me
Me
Me
.
+
N
N
N
N
N
N
Mn
Ph
Mn
Ph
Mn
N
Ph
N
Ph
N
N
N
N
Ph
Ph
Ph

Me

Ph

Ph

101 102 O O O O O Me O Me O O Me OH Me
101
102
O
O
O
O
O
Me
O
Me
O
O
Me
OH
Me
OH
Ph
Ph
Me
Me
+
N
N
N Mn
Ph
Ph
N Ph
N
H H
N
N
Ph
N
Ph
Ph
104
105

103

Fig. (21). Alkylation of Mn(II)TPP by artemisinin.

more active primary radical 80 was not trapped as well or instead of 81 was not commented on.

By recourse to a hydrophobic model which parallels the biological event, primary carbon radicals formed from artemisinin (14) could be trapped [87]. Instead of heme, which is notoriously unstable, managanous tetraphenylporhyrin (Mn(II)TPP), generated in situ by the interaction of Mn(III)TPPPOAc with tetrabutylammonium borohydride, was allowed to react with 14 Fig. (21). Single electron transfer occurred in the usual way by breaking the O- O bond. The resulting manganese(III) derivative 101 cleaved to the primary radical 102. Thereafter, intramolecular addition to one of the nearby pyrrole rings affords the pyrrole radical 103, then the cation 104 by internal transfer of an electron. Reduction by borohydride to the dihydropyrrole and demetallation afforded the covalent adduct 105. By applying the same procedure, the primary radicals from b - artemether (19) and the cyclopentene-trioxane 53 were similarly produced and captured by the pyrrole ring [88, 89].

It is reasonable to assume that in the biological context too the decomposition of artemisinin (14) and b -artemether

(19) proceeds via primary and secondary carbon radicals. Metabolic studies with 14 and b -arteether confirm the formation of products like 70 and 71 as well as deoxyartemisin (23) and its ethoxy derivative [90]. The origin of the deoxy compounds could be due to enzymatic deoxygenation which has nothing to do with the parasiticidal event. For example, an in vitro study of the metabolism of b -arteether in rat liver cytosol revealed that deoxy-b -artether was formed directly under the influence of an NADH-dependent cytosolic enzyme [91]. As a chemical equivalent of reduction by a Zn-containing NADH dehydrogenase zinc dissolving in acetic acid was taken as an appropriate reagent [92]. Adding an equivalent of Zn powder to a solution of 14 in AcOH with stirring at room temperature gave after a few hours a quantitative yield of deoxyartemisinin (23) [68]. Under the same conditions 19 gave deoxy-b -artemether (108) as the sole product in 68% yield Fig. (22). These results indicate that deoxygenation involves a two-electron reduction, which is a characteristicof zinc, but not of ferrous ion. First, Zn donates two electrons to the O-O bond of 19 to form the bidentate intermediate 106. The latter thanks to the oxophilic nature of zinc rearranges to 107. Finally, deoxyartemether (108) is formed

O Zn O O O O Zn=O O MeO O MeO O MeO MeO Me
O
Zn
O
O
O
O
Zn=O
O
MeO
O
MeO
O
MeO
MeO
Me
Zn
Me
Me
+
O-O
O O
Me
Me
Me
O
Me
O
_
Zn
O
Me
Me
Me
Me
19
106
107
108

Fig. (22). Deoxygenation of b -artemether with zinc.

Me

1816

Current Medicinal Chemistry, 2001, Vol. 8, No. 15

Charles W. Jefford

Table 3.

Product Yields (%) Obtained by Exposure of Artemisinin (14) to Fe(II) Reagents

Entry

Fe(II) reagent

Reaction speed

Furan 68

Pyran 69

Deoxo 23

Other 83, 109

Ref.

1

FeBr 2 , THF

15

min

29

10

59

0

[76]

2

FeBr 2 , THF, CHD 40%

15

min

17

4

71

0

[76]

3

FeCl 2 .4H 2 O, MeCN

5

min

77

11

0

0

[76]

4

FeCl 2 .4H 2 O, MeCN

5

min

78

17

0

0

[68]

5

FeCl 2 .4H 2 O, MeCN, CH

5

min

84

8

0

0

[68]

6

FeCl 2 , imidazole, MeCN

5

min

78

16

6

0

[109]

7

Hemin, PhCH 2 SH, THF

15-40 min

63

5.5

1.0

0

[76]

8

FeSO 4 , H 2 O, MeCN

hours

25

78

0

1-2

[86]

by extrusion of ZnO Zn(OAc) 2 .

which

dissolves

in

AcOH

giving

likely that 23, arising by reduction, stems from a reaction

which is separate from that of the catalyzed isomerization to the furan-acetate 68 and the hydroxypyran 69. Consequently,

 

a

doubt exists about the validity of any mechanism that is

Comments

on

the

Proposed

Unified

Mechanistic

partly based on 23. A scheme to be valid, and indeed to be

Framework

unified, can only take into account those products that share

Having in hand sufficient proof for the intermediacy of the epoxide 83, the primary and secondary radicals 80 and 81 together with their formation as kinetic products, and an absence of proof for the cyclic enol ether 82 and the ferryl species O=Fe 2+ , all the pieces are in place for defining the ultimate mechanism for the ferrous ion-cleavage of artemisinin and its congeners. A recent attempt, while ambitious and somewhat audacious, falls short of this goal [86]. A so-called unified mechanistic framework was formulated on the basis of an imperfect understanding of the data and some of the principles governing organic reactions. The data taken for consideration, although disparate, were quite limited (Table 3). With such a small sample a reliable interpretation is already compromised. It is immediately seen that deoxyartemisinin (23) is only produced in THF and also to a very minor degree when imidazole was present (entries 1, 2, 6, and 7). As mentioned earlier, it seems very

a common mechanism.

The reaction speeds (they cannot be called rates), namely, the time required to complete the reaction, are very rough estimates (Table 3). All that can be said is that FeSO 4 (entry 8) is much slower than the rest, which are more or less equally rapid in their action. Its slowness differentiates it from the other reagents by permitting other products to be identified, notably the epoxide 83 and one of its rearrangement products (109). As the formation of 109 was only alluded to, a word on how it arises is appropriate. The isomerization of 83 to hydroxypyran 69 was correctly detailed Fig. (23). Protonation of 83 to 110 followed by opening of the epoxide transfers positive charge to the acetal function as indicated by the species 111. Annihilation of charge on the oxonium ion by internal attack of the hydroxyl group then gives 69. An alternative course is to open the protonated epoxide 110 to form a five-membered ring. Attack

O + O O O O O O O O Me Me Me Me OH
O +
O
O O
O O
O
O
O
Me
Me
Me
Me
OH
Me
OH
Me
OH
O
O
H
OH
Me
Me
+ Me
83
110
111
O
O
O O
O
O O
O
O
H
OR
O
H
Me
Me
O
Me
O
Me
Me
Me
O
O
H
Me
+
Me
Me
110
112
109
R
= H
113
R
= Ac

69

Fig. (23). Opening of the epoxide 83 to give either a six or five-membered ring product.

Implications for the Mode of Action

Current Medicinal Chemistry, 2001, Vol. 8, No. 15

1817

by the hydroxyl group on the least substituted terminus of the epoxide entity leads to the furan 112. Deprotonation and cleavage gives 109, one of the observed decomposition products which was subsequently identified as its acetate. In fact, this dichotomy of epoxide opening is a characteristic feature in the rearrangement of artemisinins and depends on the nature of the adjacent ring atoms (v. infra).

The main mechanism proposed incorporates as its core the pair of equilibrating oxy radicals and 78 and 79 (produced from 14) which are assumed to be in equilibrium with their derived carbon radicals 80 and 81 Fig. (24). It should be remarked at the outset that the subsidiary equilibrating oxy radicals 114 and 115 shown as arising from 78 and the conversion of 114 to 116 are purely hypothetical in the case of artemisinin as no products formed from them were observed. They are meant to serve as reference structures for reactions of derivatives. The core equilibrium is supposed to be displaced to favor different amounts of the products 68, 69, and 23 depending on the kind of ferrous reagent and solvent employed. Two factors were invoked to account for the product composition. The better described is the solvent factor. The basic idea is that the preferential breakage of one over the other of the two bonds in Fe-O-R as in 80 and 81 is susceptible to the nature of the solvent. It is suggested that the Fe-O bond is stronger in THF than in MeCN, therefore favoring homolysis of the O-R bond, whereas the reverse is true in aqueous MeCN. This particular solvent effect was used to explain why 23 is formed as the major product in THF, but not observed in MeCN (Table 3, entry 1). Such an explanation is difficult to accept since radical reactions are generally insensitive to solvent polarity [93].

The second factor is the nature of the counter ion in the ferrous reagent which is assumed to affect the ability of ferrous ion to deliver an electron to the s * orbital of the O-O

bond. It is argued, without substantiation, that FeSO 4 , being less active than FeBr 2 (Table 3, entries 8 and 1) slows down "the steps of" (the rate of conversion of?) 81 to 69 and of 80 to 68, but aq. MeCN "works the opposite way" (speeds them up?). Put another way, it can be supposed that this means that the ratio of the equilibrating species 81 to 80 is about the same (3:1) when produced by either FeSO 4 or FeBr 2 in their respective solvents. Decomposition of 80 just gives 68 while 81 bifurcates to 23 and 69 in THF, but goes completely to 69 in MeCN. When FeCl 2 .4H 2 O in MeCN is used as reagent (entry 4), no 23 is formed as the solvent does not facilitate Fe-OR bond cleavage. The ratio of 68 to 69 now changes to 4.6:1. Apparently in this case the effects of solvent and reagent do not cancel out which means that the equilibrium shifts away from 81 towards 80 because as "the 1,5 shift is not so fast" 79 gives 81 less quickly. The major and minor radicals 80 and 81 then react giving 68 and 69 in the aforementioned ratio.

The trouble with the preceding interpretations is that they are based on incorrect premises and fuzzy factors. Intermediates 80 and 81 are stable carbon radicals evincing little tendency to revert to the oxy radicals 78 and 79. Deoxyartemisinin (23) is the product of a separate reaction. It should be pointed out too that trying to rationalize product ratios like 2.7:1 and 1:3 (cf. Table 3, entries 4 and 8), which reflect transition states not differing greatly in energy, is not a reliable undertaking.

The above mechanism was also deemed to be applicable to certain artemisinin derivatives. To simplify discussion, just two are considered here. b -Artemether (19) and its b - benzyloxy analogue on treatment with FeSO 4 in aqueous MeCN gave derivatives analogous to 68 and 69 in ratios of 1 to 1.2 and 1.8 to 1 respectively. The switch in ratios was attributed to slower 1,5 shifts due to conformational changes caused by the "larger" benzyloxy substituent. In reality the

O O O O O O O O Me O O O O 2+ 2+
O
O
O
O
O
O
O
O
Me
O
O
O
O
2+
2+
2+
Fe
.
Me
.
Fe
Fe
Me
O
2+
Me
Fe
O
.
C-C
6
1,5 shift
Me
O
Me
O
O
Me
O O
Me
OH
.
scission
80
78
79
Me
81
Me
Me
Me
2+
2+
2+
Fe
O=Fe
Fe
.
O
O
O
O
O
O
O
Me
O
O
O
O
O
Me
2+
Fe
O
O
Me
Me
OH
Me
O
Me
O
Me
Me
OH
O
Me
68
Me
114
Me
82
Me
83
2+
2+
Fe
.
Fe
O
O
O
O
O
O
O
O
O
O
O
2+
O
O
Fe
O
Me
Me
O
Me
Me
Me
O
Me
Me
O
Me
O
OH
Me
Me
Me
Me
116
115
23
69

Fig. (24). The main mechanism for the Fe(II)-induced reaction of artemisinin and its congeners (ref. [86]).

1818

Current Medicinal Chemistry, 2001, Vol. 8, No. 15

Charles W. Jefford

difference in steric compression on the rigid tetracyclic skeleton between such remote substituents must be negligible.

Arguments have been mustered to provide purportedly correct mechanisms for the reactions of certain carba artemisinins [94]. The action of FeBr 2 /THF on deoxoartemisinin (24) produced the completely unraveled formyl diketone (117) and the deoxodeoxyartemisinin (118) in yields of 79 and 8% Fig. (25). The first step is the setting up of the pair of equilibrating oxy radicals 119 and 120. Thereafter, their evolution was ascribed to the differential effect of THF. Cyclization 119Æ 122 was retarded, allowing cleavage 120Æ 123, which is favored, to take over. Closure of the hydroxyl group onto the double bond in 123 would afford118 in the postulated way. Fragmentation of 119 by circular movement of four electrons brings about cleavage to 117. It was not made clear why so little of the deoxygenated product was formed compared with the case of artemisinin. A simpler explanation is that 120 fragments just as well as 119 which necessarily bypasses the potential excision of O=Fe 2+ , while deoxygenation to 118 is probably the consequence of an independent reduction process arising from 119 and 120 through the diol 121.

The dideoxoartemisinin (124) seemed to present difficulties of interpretation even though the result was simple enough Fig. (26). With the same reagents as before just a single product was obtained, the hydroxy furan 125. The mechanism proposed [86] starts by adjunction of ferrous ion giving the pair of ferric oxy radicals 126 and 127. This time there is no avenue of cleavage open to 126 because thermodynamic stabilization cannot accrue by creation of a carbon-carbon double bond. The reaction proceeds entirely

from 127 which undergoes 1,5 H shift to 128. Even though it is in THF, a solvent alleged to favor excision of O=Fe 2+ , 128 fails inexplicably, if the unified mechanism is to be believed, to react in this sense and loses Fe 2+ instead forming the epoxide 129. The next step proposed is far- fetched as it entails opening of the epoxide with FeBr 2 to furnish 130. In order to get the groups in the right positions, the two ligands are now obliged to exchange with each other giving 131. The goal, the hydroxy furan 125, is finally reached by nucleophilic displacement of bromide ion by the oxide substituent. Of course, this tortuous route is not followed at all, because there is a rational shortcut. Protonation of the epoxide 129 will engender backside attack by the hydroxyl group to form the furan ring 125 directly. This is the expected closure. Closure to the six-membered ring only occurs when positive charge is formally located at the methylated terminus by an acetal function, e.g. 83Æ 110Æ 111Æ 69 Fig. (23).

It is to be noted that the hypothetical equilibrium between 78, 114 and 115 is unobserved. The path from 78 to 114 and then to 116 only becomes a reality when the carbonyl is absent, for example in the case of b -artemether (19) and deoxoartemisinin 24. Rather than halt at the 114 stage, a likelier course, as suggested above, is fully concerted cyclic fragmentation to the analogues of 116.

In conclusion, it appears on reviewing the selection of results (Table 3), that, apart from the presence of the odd product 23, there is not much to choose between them. Thus, the expansion of the mechanistic scheme to embrace the controversial formation of 23 is superfluous. To get a better notion of the scope of the mechanism more results are needed. New experiments need to be done and old

O O O O O O O Me O Me Me O-O Me FeBr 2
O
O
O
O
O
O
O
Me
O
Me
Me
O-O
Me
FeBr 2 , THF
Me
Me
O
+
25 o , 5 min
Me
24
Me
117
Me
118
2+
2+
Fe
2+
H 2 O
Fe
Fe
O
O
O
O
O
O
.
Me
Me
HO
Me
. O
Me
O O
Me
6 O
Me
OH
Fe 2+
Fe 2+
Me
Me
Me

119

120

121

O O O Me O
O
O
O
Me
O

Me

122

Me

O O Me OH
O
O
Me
OH

Me

123

Me

Implications for the Mode of Action

Current Medicinal Chemistry, 2001, Vol. 8, No. 15

1819

O O Me O-O Me Me FeBr 2 , THF O Me OH 25 o
O
O
Me
O-O
Me
Me
FeBr 2 , THF
O
Me
OH
25
o , 5 min
Me
124
Me
125
2+
O
Fe
O
O
O
.
Me
.
H
Me
OH
Me
Me
O
O
Me
O
O
Me
O
O
Me
.
2+
2+
2+
O
Fe
Fe
Fe
Me
Me
Me
Me
126
127
128
129
O
O
Me
FeBr 2
Me
FeBr 2
FeBr
Me
OH
OFeBr
Me
O
OH
129
125
Br
Br
Me
Me
130
131

Me

+

H

Fig. (26). Rearrangement of dideoxoartemisinin by FeBr 2 . Mechanistic avenues.

experiments carefully repeated with a variety of pure solvents and pure redox couples, not only with Fe(II), but also with Mn(II), Ru(II), and Ti(II) reagents, to secure incontrovertible results before proposing a unified mechanistic framework.

Peroxides Generating Hindered and Substituted Carbon Radicals have Poor Antimalarial Activity

It is now clear that artemisinin gives with ferrous reagents and presumably with heme, two types of radical, primary and secondary. An obvious question to ask is what is the relative parasiticidal power of the two? Is one more active than the other? Are other types of carbon radical equally as active? Another question concerns their susceptibility to steric hindrance.

A pertinent first answer to such questions was provided by the methylated tricyclic trioxanes 132-134 [95] Fig. (27). The b -methyl derivative 132 was about as twice as active as

artemisinin in vitro, having an IC 50 value of 4.5 ng/ml against the W2 clone. The a -methyl and dimethyl derivatives 133 and 134 were inactive. Two conclusions can be drawn. The first is that 133 and 134 are unable to access heme failing to elicit electron transfer, whereas 132 sits nicely on top of heme, generating one or both of the tertiary and secondary radicals 135 and 136 with perhaps the latter being the more effective at killing the parasite. The second is that coordination with heme takes place with all three trioxanes, but as 133 cannot undergo 1,5 shift only the secondary radical 136 forms. Similarly, the only option open to 134 is cleavage to the unreactive tertiary radical 137. Therefore, it appears that hindrance to coordination with heme is the determining factor for antimalarial activity in this instance. Nevertheless, the electronic nature of the alkyl radical is important too.

Which of the alkyl radicals, secondary or tertiary, is the most effective is answered by in vitro tests performed on derivatives of the tricyclic trioxane 26 [44] Fig. (28). The b -

OH O MeO Me O O R 2 R 1
OH
O
MeO
Me
O O
R 2
R 1

132 R 1 = H, R 2 = Me

133 R 1 = Me,

R 2 = H

134 R 1 = Me,

R 2 = Me

OH O MeO Me OH O Me . heme
OH
O
MeO
Me
OH
O
Me
.
heme

or

135

O MeO .
O
MeO
.

OH

OH O Me MeO O O Me . heme
OH
O
Me
MeO
O O Me
.
heme

Me

136

heme

O MeO . OH OH O Me MeO O O Me . heme M e 136

heme

MeO . OH OH O Me MeO O O Me . heme M e 136 heme

O O Me

heme Me

137

Fig. (27). Formation of tertiary and secondary radicals by reacting heme with trioxane 132.

1820

Current Medicinal Chemistry, 2001, Vol. 8, No. 15

Charles W. Jefford

benzyl derivative 138 was about as twice as active as the parent trioxane 26. However, the b -phenyl derivative 139 was devoid of activity confirming that a carbon radical stabilized by conjugation, or hyperconjugation like the aforementioned tertiary radical, is not aggressive enough to kill the parasite.

MeO O O O R
MeO
O
O O
R
MeO O Me Me O O Me
MeO
O
Me
Me
O O
Me

26

R

= H

140

138

R

=

CH 2 Ph

139

R = Ph

 

Fig. (28). Active and inactive tricyclic trioxanes.

Obviously the two factors, electronic and steric, go hand in hand. Yet the ability of the peroxide bond to get into intimate contact with heme is of paramount importance. The methyl derivative 140, in which the underside of the peroxide bond is sterically blocked provides an apt illustration Fig. (28). It is totally inactive [96, 97].

The effect of steric hindrance on the availability or propensity of a carbon radical to accomplish its deadly task is patently seen on inspecting the in vitro activities of substituted cyclohexane-1,2,4-trioxanes and 1,2,4,5- tetroxanes both of which have the same modus operandi [98]. The spirocyclic trioxane 141 has an IC 50 against the W2 clone in vitro of 3.9, comparing well with artemisinin (14) and its value of 1.2 ng/ml Fig. (29). Simply placing four methyl substituents on the cyclohexane rings sharply diminishes the activity of 142, its IC 50 rising to 94.0 ng/ml.

1,2,4,5-Tetroxanes have long been known to possess antimalarial activity, but much less so than their trioxane counterparts [99-101]. The cyclohexane derivative 143 is 4-5 times less active than 141 showing an IC 50 of 20 ng/ml Fig. (29). Loading the tetroxane with two pairs of geminal

O O O Ph
O
O O
Ph

141

Ph

Me

Me

Me Me O Me O O Ph
Me
Me
O
Me
O
O
Ph

Ph

142 heme Me Me O . Ph Me O O Ph heme
142
heme
Me
Me
O
.
Ph
Me
O
O
Ph
heme

145

Fig. (29). Sterically encumbered trioxanes and tetroxanes.

methyl groups on the cyclohexane ring raises the IC 50 of 144 to 255 ng/ml, wiping out activity. It can be concluded that steric encumbrance at the C3 position on the cyclohexane ring relative to the spirocyclic carbon atom somehow interferes with parasiticidal action. As in the case of the methylated tricyclic trioxanes, two hypotheses are possible. First, the bulky gem-dimethyl groups could simply prevent the peroxide bond from getting close to heme. Second, if heme manages to get near enough to effectelectronictransfer, the resulting neopentyl type radicals, e.g. 145 and 146, would be unreactive and therefore useless for killing parasites.

Substitutions on certain parts of the artemisinin skeleton can also profoundly perturb its activity. As previously mentioned, substitutions in the lactone ring, either as ester or ether derivatives of the epimeric lactols, are sufficiently remote from the peroxide linkage as to have little effect on docking with heme. In fact, such derivatives, like the lactols themselves, are often more active than the lactonic parent, simply for electronic reasons. However, substituents at the a -position to the lactone lie closer to the peroxide linkage. Changing them alters the activity in a systematic and, in certain cases, a dramatic way [102]. Replacing the b -methyl group in artemisinin (14) by ethyl or propyl leads to IC 50 values against the W2 clone for 147 and 148 that are roughly 12 times smaller than that of 14, in other words, the compounds are more active Fig. (30). However, substitution by a much longer aliphatic chain, as evidenced by 149, annihilates activity completely. Presumably, because the chain is long enough and sufficiently mobile to reach and cover the peroxide bond preventing approach to heme.

Surprisingly, in vitro tests carried out on epiartemisinin 151 revealed IC 50 values only 1.5 -2.0 times bigger than that of 14 [102, 103] Fig. (30). In contrast, the IC 50 of the bromo derivative 150 is 8 times that of than 14. The isostere of 150, the gem-dimethyl derivative 152, is completely inert. The expectation is that all three a -substituted derivatives would erect the same kind of steric barrier to an incoming molecule of heme. A reason for the low IC 50 values of 151

O O O O 143
O O
O O
143

Me

Me

Me Me O O Me Me O O Me Me 144
Me
Me
O
O Me
Me
O O
Me
Me
144
Me Me O . O Me Me O O heme Me Me
Me
Me
O
.
O
Me
Me
O
O
heme
Me
Me

146

Me

heme

Me

Implications for the Mode of Action

Current Medicinal Chemistry, 2001, Vol. 8, No. 15

1821

might be due to isomerization. Under the conditions of incubating P. falciparum during the test, 151, the less stable epimer, or at least some of it, could have reverted to the more active b -epimer 14.

O O O 2 R O O R 1
O
O
O
2
R
O
O
R 1

Me

Me

14

R 1 =

Me, R 2 = H

147

R 1 =

Et, R 2 = H

148

R 1 =

Pr, R 2 = H

149

R 1 = (CH 2 ) 13 Me, R 2 = H

150

R 1 =

Me, R 2 =Br

151

R 1 =

H, R 2 = Me

152

R 1 = R 2 = Me

Fig. (30). Some derivatives of artemisinin.

A recent X-ray study of 151 reveals that the a -methyl substituent lies in van der Waals contact with the proximal oxygen atom of the peroxide bond [104]. Evidently, the peroxide is hindered. The consequences of this hindrance are shown up by the in vivo activity. Against Plasmodium berghei N, the chloroquine-sensitive line, the ED50 and ED90 values of 151 are about seven times greater than those

of 14. Against P. yoelii ssp. NS, the resistant line, the ED50 and ED90 values are 4-6 times bigger. Clearly, the a - epimer 151 is 4-7 times less effective than its b -epimer 14. The persistence of some activity may be due to some epimerization to 14 under the test conditions. Comparison of the X-ray data of 14 and 151 reveals that the two molecules are superimposable, except for the lactone ring in 151 which is slightly distorted to alleviate congestion between the a - methyl group and the peroxide bond. Thus the difference in activity arises solely from the orientation of the methyl substituent on the lactone ring.

The diminished activity of 151 and its absence by 152 could be ascribed to poor docking on heme Fig. (31). Thus, the complexes 154 and 155 owing to obstruction by the a - methyl substituent either do not form or are not intimate enough for an electron to jump from the 3d orbital of iron to the anti-bonding orbital of the O-O sigma bond. Consequently, neither oxy nor successive carbon radicals arise from 154 and 155 like they do from 153. An alternative is that docking in 154 and 155 is close, but skewed obliging the iron atom of heme to selectively bind to the distal oxygen atom of the peroxide bond. As a result, electron transfer occurs leading solely to the secondary carbon radicals 158 and 159. The tight complex formed with artemisinin (153) would be expected to form mostly, if not all, the primary radical 156. The relative parasiticidal power of the primary and secondary radicals (e.g. 156 vs. 157) is not known, but the selective capture of the primary radical by Mn(II)TPP (v. supra) argues in favor of the former.

O O O R 2 Me R 1 O O Me Me Me N N
O
O
O
R 2
Me
R 1
O
O
Me
Me
Me
N
N
Fe
N N
Me
Me

HO 2 C HO 2 C

153

154 =

155

R 1 = Me, R 2

R 1 = H, R 2

R 1 = R 2

=

=

Me

H

Me

O O Me O O . Me O Me Me Me N Fe N N
O
O
Me
O
O
.
Me
O
Me
Me
Me
N Fe
N
N N
Me
Me

HO 2 C

HO 2 C

O O O 2 R Me R 1 OH O . Me Me Me Fe
O
O
O
2
R
Me
R 1
OH
O
.
Me
Me
Me
Fe
N
N
N
N
Me
Me

HO 2 C

HO 2 C

156 157 R 1 = Me, R 2 = H 158 R 1 = H,
156
157
R 1 =
Me, R 2
=
H
158 R 1 =
H, R 2
=
Me
2
159
R
1 = R
=
Me
PP
Me
Me
O
O
Me
O
HO 2 C
N
+ N
O
Fe
PP
Me
OH
+ HO 2 C
N N
Me
Me
Me
hemozoin
160
161

Fig. (31). Formation of radicals from artemisinin, epiartemisinin and congeners by reaction with heme.

1822

Current Medicinal Chemistry, 2001, Vol. 8, No. 15

Charles W. Jefford

Me Me O O
Me
Me
O
O

H

OH

Me

Me

OH

13

Me Me Me O OH H OH
Me
Me
Me
O
OH
H
OH

164

+

Fig. (32). Mode of action of yingzhaosu A.

heme

H OH 164 + Fig. (32) . Mode of action of yingzhaosu A. heme Me Me
Me Me O . O
Me
Me
O
.
O

hemin

H

OH

Me

OH

Me

163

excision

A. heme Me Me O . O hemin H OH Me OH Me 163 excision .
. PP + hemin O Me Me OH hemin
.
PP
+
hemin
O
Me
Me
OH
hemin

165

166

hemozoin

How Artemisinin and Peroxidic Antimalarials Kill the Parasite

How the parasite is actually killed is also not known. It must be emphasized that FeCl 2 .4H 2 O in MeCN is not the same as heme inside the red blood cell. Both entities display the same redox properties towards artemisinin. The first acts as a catalyst as already mentioned, but intraerythrocytic heme acts as a reagent when a parasite is present. Alkylation by a reactive hemin-radical e.g. 156 undoubtedly takes place

The cis-fused cyclopentene-trioxanes, exemplified by the most potent candidate 53, like cis-decalin easily undergo conformational inversion. They therefore are able to make a close fit with the surface of heme. The resulting complex from 53 then unravels to the hemin derivative of the primary radical 74, which then proceeds to alkylate parasite protein. The motor for antimalarial activity is the thermodynamic stability which comes from the formation of a carbonyl group at its simplest, or better one that is conjugated. Such a structural feature is found in yingzhaosu A (13) and C (162)

Me Me heme O O OH Me
Me
Me
heme
O
O
OH
Me

Tol-p

162

Me Me . Me O OH O O Me hemin Tol-p Tol-p 167 168
Me
Me
.
Me
O
OH
O
O
Me
hemin
Tol-p
Tol-p
167
168
Me Me Me Me . O OH OH O + PP hemin hemin 169 170
Me
Me
Me
Me
.
O OH
OH
O
+
PP
hemin
hemin
169
170

Fig. (33). Mode of action of yingzhaosu C.

inside the food vacuole of the parasite Fig. (31). Addition to an unsaturated center in the parasite protein (PP) would afford a new radical, which could then eject an electron yielding a cation followed by its deprotonation. Lastly, protonation of the resulting PP-artemisinin-hemin adduct 156 releases the alkylated protein 160 from its hemin appendage (161) which subsequently polymerizes to hemozoin. Experimentally, it was found that on treatment with artemisinin trophozoites of P. falciparum still produce hemozoin, while with chloroquine less is produced [105]. In any event, heme(FeII) is first oxidized to hemin(FeIII), which then polymerizes. How this happens is controversial, but it may well be a purely chemical process [106]. In summary, in a sort of quid pro quo, a toxic C-centered radical replaces toxic heme to kill the parasite.

and may contribute to their potency [68] Fig. (32). Complexation of heme with 13 gives the hemin oxy radical 163. Excision of the a ,b -unsaturated ketone 164 releases the cyclohexyl radical 165 which then alkylates parasite protein finally producing 166 and eventually hemozoin. Yingzhaosu C (162) behaves the same way Fig. (33). The hemin-oxy radical 167 cleaves to the acetophenone 168 and the ethyl radical 169. Alkylation gives the hemin-PP adduct 170 which finally ejects hemin as before.

Confirmation of the validity of the foregoing schemes has been obtained by treating arteflene(16) with FeCl 2 .4H2O in MeCN [107]. Evidence for cleavage of the ferric oxy radical 171 to the cyclohexyl radical 172 and the enone 173 was obtained by capture of the former with 5,5-dimethyl-1- pyrroline N-oxide and the identification of its adduct by EPR

Me C O O O Fe 2+
Me
C
O
O
O
Fe 2+

6 H 3 (CF 3 ) 2 -2,4

Me

Me . O O
Me
.
O O

O

Fe 2+

C 6 H 3 (CF 3 ) 2 -2,4

Me

. O
.
O

O

Me

Fe 2+

16

171

172

+

Fig. (34). Ferrous ion-induced cleavage of arteflene.

C 6 H 3 (CF 3 ) 2 -2,4 O Me
C 6 H 3 (CF 3 ) 2 -2,4
O
Me

173

Implications for the Mode of Action

spectroscopy [108] Fig. (34). The potency of arteflene (16) is proof that secondary C-centered radicals are reactive enough after all to be effective schizonticides. Nevertheless, the fact that the p-fluorophenyl-trioxane 53 is even more potent points to the efficacy of a primary radical (Table 1).

Evidence that artemisinin on treatment with a Lewis acid or benzylamine opens to a hydroperoxide has been suggested as a basis of parasiticidal action [109, 110]. It will be interesting to see if such reactions occur under physiological conditions and how biomolecules react with the purported electrophilic oxygenating species derived from the hydroperoxide. In general, hydroperoxides that are unable to cleave to a carbon radical only manifest weak antimalarial activity [29].

Molecular Considerations in Designing New Peroxidic Antimalarials

The preceding mechanistic schemes clearly depend on the optimal concurrence of several molecular properties in order to produce the ultimate antimalarial. In other words, the parasiticidal action of potent peroxides like 14 and 53 depends on the efficient operation of a sequence of chemical events, namely, docking with heme, electron transfer, formation of an oxy radical, scission to an unencumbered C- centered radical powered by the formation of a carbonyl group or something similar, and finally, the death of the parasite by alkylation. Many trioxanes and peroxides are going to fulfill the above criteria. In designing new peroxidic antimalarials, any one of the above desiderata needs to be examined to see if the candidate will fit the bill.

Many spatial arrangements are possible between heme and artemisinin (14) and it does not necessarily follow that the one usually drawn is the right one. However, computer- assisted molecular modeling of various docking arrangements of hemin(FeIII) (serving as a model for heme) and 14 using the Sybil program showed that in the most stable configuration the peroxide bond and the carbonyl oxygen atom interact closely with hemin iron [111]. The a - face of artemisinin is close to heme putting the interatomic distance between 2.6 and 2.8Å from the peroxide oxygen atoms to the iron. A similar procedure with deoxyartemisinin (23) and hemin favored close binding on the opposite or b -face.

A QSAR study using Comparative Molecular Field Analysis of 11-alkyl derivatives of artemisinin as well as some tricyclic trioxanes similar to D-seco-artemisinin gave a good correlation between calculated and observed activities [102]. From the relative contributions of steric and electrostatic terms the former had the greatest bearing on activity. The latter finding accords well with docking on heme as the crucial first step for effective drug action.

In a 3D-QSAR study using a pharmacophore search method (CATALYST) two hydrophobic features and hydrogen bonding were identified as the hypothesis or pharmacophore responsible for activity [112]. By taking a training set of trioxanes having known in vitro and in vivo activities the best arrangement of the hypothesis consonant