Why Artemisinin and Peroxides Antimalarials

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*

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 Plasmodiumfalciparum, has opened a new era in the chemotherapy of malaria. In vitro and in vivotests with synthetic structurally simpler trioxanes clearly demonstrate that much of theskeleton 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 highantimalarial activity can be displayed by molecules which do not resemble the geometryof 13 and 14 at all. The possible mode of action of 13 , 14 , and synthetic peroxides is examined. They are believedto kill intraerythrocytic Plasmodium by interacting with the heme discarded by proteolysis of ingestedhemoglobin. Complexation of heme with the peroxide bond followed by electron transfer generates an oxyradical that evolves to the ultimate parasiticidal agent. Experiments with ferrous reagents indicate that activeperoxides 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. Thereview covers our own and other contributions to this timely topic and evaluates the different mechanismsproposed for the mode of action of peroxidic antimalarials.

INTRODUCTION chronic infections can lead to kidney failure, hyperactivemalarial splenomegaly and Burkitt’s carcinoma. Mostmalaria is caused by P. vivax and is essentially benign, butPlasmodiun falciparum is virulent. It is the most dangerousbecause the level of parasitemia is much higher than for theother three species. Also more than 80% of the hemoglobinwithin the cell gets degraded to heme by the parasite.Moreover, the parasitized red blood cells in the latematuration stage lose their platelet-like form and no longerfreely circulate in the blood. They become knobbly and sticktogether blocking the microvasculature in vital organs suchas the brain; with coma and death being the usual sequel. Intropical Africa alone falciparum malaria claims each year thelives of 1.5-3 million children most of whom are under fiveyears of age.

Background to the Disease

Malaria is an age-old disease and one of the commonestcauses of illness in the world. It is estimated that about 2.5billion people living in malarious zones are at risk and that300-500 million clinical cases of malaria occur each year [1].The disease is transmitted in a two-stage process by the biteof an infected female anopheline mosquito which can transferto the human host four species of parasite, Plasmodiummalariae, P. ovale, P. vivax and P. falciparum [2]. Theparasites first invade the liver and then as merozoitespenetrate erythrocytes where they proliferate and eventuallyburst the parasitized cells releasing about 25 daughtermerozoites which then invade more erythrocytes so initiatinga new infectious cycle. Some merozoites evolve into maleand female gametes which undergo fertilization after beingdrawn into the gut of a second mosquito when it bites theinfected host. The resulting sporozoites are then injected viathe salivary glands into a fresh host through another bitethereby maintaining transmission.

Treatment and Prevention of Malaria

The prevention and treatment of malaria today constitutesan acute challenge for modern medicine and public healthmanagement because many of the traditional quinoline-baseddrugs are becoming increasingly ineffective in certain parts ofthe world owing to multi-drug resistance [3]. In order to putthe problem in perspective a short overview of thedevelopment of traditional antimalarials is presented.

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

Jesuit missionaries in Peru around 1630 discovered thatthe bark of the cinchona tree allayed fever. A few years laterexportation of the bark to Europe at Rome’s behest led to itsinclusion in the pharmacopoeia as a cure for fever [4]. In1820 the active principle was found in to be the alkaloidquinine (1). This finding biased subsequent drugdevelopment in the years prior to 1939 towards chemicallyrelated remedies such as chloroquine (2), amodiaquine (3),

*Address correspondence to this author at the Department of OrganicChemistry, University of Geneva, CH-1211 Geneva, Switzerland; Ph.:+41-22-7763316; Fax: +41-22-7763601;e-mail: [email protected]

0929-8673/01 $28.00+.00 © 2001 Bentham Science Publishers Ltd.

Dr. Mansoor Alam

1804 Current Medicinal Chemistry, 2001, Vol. 8, No. 15 Charles W. Jefford

N

MeO

HN

N

OMe

ClNEt2

Me

N

MeEt2N

NH

Cl N N

HHO N

MeO

Cl

NH

HO

Et2N

H

MeEt2N

NH

1 2 3

4 5Fig. (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 thesequinoline derivatives was not known at the time.Consequently, the design of antimalarial drugs in the early

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

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

HN

NH

NH

CHMe 2

Cl

NHHN H2N NH2

EtCl

N

N

NH2SHN

N

NMeO

MeO

NH2SH2N

O

O

O

O

6 7 98Fig. (2). Some traditional inhibitors of dihydrofolate reductase and synthetase.

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

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

Disadvantages of Traditional Nitrogen ContainingDrugs

In any event, the risk of severe cutaneous reaction(Stevens-Johnson syndrome) by Fansidar rules out itsprophylactic use against falciparum malaria [11].Furthermore, Fansidar is inefficacious against P. vivax andcarries a significant risk of mortality. In an endeavor to fillthe niche left by progressively obsolescent chloroquine,

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

OH

Cl

O

O CF3 Cl

ClNBu

OH

Bu

N CF3

CF3

NHO

11 1210

H

Fig. (3). Some successor antimalarials.

Implications for the Mode of Action Current Medicinal Chemistry, 2001, Vol. 8, No. 15 1805

recourse was recently made to antique drugs such asatovaquone (11) and proguanil (6), combined together asMalarone, and a derivatized phenanthrene isosterehalofantrine (12) [12].

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

Apart from the diminished effectiveness of theaforementioned nitrogen heterocycles due to resistance by P.falciparum, there are certain personal disadvantages and risksfor the patient or user. Chloroquine is limited in itsgeographical use, only working in the Middle East, Mexicoand Central America. Mefloquine is expensive, 100 timesmores so than chloroquine, and has resulted in seizures andpsychiatric disorders. Halofantrine is equally expensive,unsuitable for prophylaxis, and has led to cases ofcardiotoxicity. Even quinine is never totally effective, and itstoxic side effects deter its usage [13].

Semi-synthetic Derivatives

In the case of yingzhaosu A (13), a synthetic route to thenatural product based on (-)-carvone (15) as starting material[30] was adapted for preparing in some 10 or so stepsessentially the surrogate molecules arteflene or Ro 42-1611(16) and Ro 41-3823 (17) as pure enantiomers in addition toother analogues [31] Fig. (5). In comparison, derivatives ofartemisinin were much easier to come by, merely entailingthe initial reduction of the freely available and crystallinenatural product to the epimeric α and β-lactols (18) whichwere then converted to esters and ethers by standardprocedures Fig. (6). Scores of semi-synthetic derivatives of18 were produced of which the most important are β-arteether(19), β-artemether (20), sodium β-artelinate (21) and α-artesunic acid (22). All have antimalarial activitiescommensurate with that of the parent 14 [32, 33].

THE ADVENT OF PEROXIDIC ANTIMALARIALS

Yingzhaosu and Artemisinin

Against this disheartening backdrop of increasinglyunsatisfactory N-heterocyclic drugs coupled with the risingincidence of the deadly falciparum malaria, the advent of twonon-nitrogenous lead compounds was not only timely, butsignaled a new era of antimalarial chemotherapy [14, 15]. Itwas discovered in 1979 that two sesquiterpene peroxidesobtained from Chinese medicinal plants, yingzhaosu A (13)and qinghaosu or artemisinin (14), possessed powerfulantimalarial activity [16] Fig. (4). Yingzhaosu A (13), atypical 1,2-dioxane, occurs as a decomposition productarising in the stored roots of a sparsely growing vine,Artabotrys uncinatus (Lam.) Merr. [17]. Artemisinin (14) isa complex tetracyclic 1,2,4-trioxane found as the activeprinciple of Artemisia annua Linn., a wild shrub of wide-spread habitat [18-20].

Despite many successful syntheses, those presentlydevised for 13 and 14 are impractical either in terms oflength or cost. For example, the surrogate arteflene has notbeen further promoted as a commercial pharmaceuticalproduct, mainly for reasons of cost. Even the extra couple ofsteps required to convert 14 into 19-22 probably add toomuch to the price of the products for their intended use inthird world markets. The idea of using the lead or even afirst generation product as the actual commercial entity isshortsighted. The usual proceeding for creating a new drug isfirst to identify the pharmacophore of the providentiallyprovided natural product by stripping away the extraneousbits. Once the essential structural elements are laid bare, thenext step is to elucidate the mode of action. With suchinformation in hand, it should be possible to design morepotent, cheaper, synthetically accessible, and morebiologically suitable antimalarials, and to gain, into thebargain, mechanistic insights that might reveal othertherapeutic applications.

O

Me

Me

OMe

O

OO

Me

OH

Me

Me

Me

OH

6

14

11

4

3

O O

13

A

B C

D

Fig. (4). Yingzhaosu A and artemisinin.

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

O

MeMe O

Me

Me

OO (CH2)10MeO

O

Me

MeO

CF3

CF3

and

10 or so steps

15 16 17

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


O

O

Me

Me

OMe

HO

EtOMe

O

Me

Me

O

O O

O

Me

Me

OMe

O

O

O

COOH

O

MeO

Me

Me

O

O

O

Me

Me

OMe

MeO

C6H4CO2Na

19

20

O

21

O

22

O O O

14

18

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

LOOKING FOR THE PHARMACOPHORE The 1,2,4-trioxane ring per se is or rather was arelatively unknown chemical entity. Before 1957 none wasreported in the literature. Subsequently and especially sincethe discovery of artemisinin, many methods for theirsynthesis were devised [36]. As a result, numerous trioxanesof simplified, but varied structure were prepared and screenedfor in vitro activity against P. falciparum using theDesjardins test [37, 38]. In this test concentrations of thedrug (expressed in ng/ml) which inhibit growth of theparasite by 50 and 90% are estimated (IC50 and IC90).

Tricyclic Trioxanes

On inspecting the artemisinin skeleton several questionsarise. How many and which of the four rings (A,B,C, and D)are necessary for high activity? Is the boat conformation ofthe trioxane ring really necessary? Would a peroxide do aswell? Is chirality important? What sort of peripheralsubstituents are required to bolster activity? It should bementioned straightaway that the peroxide linkage in 14 is acritical 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 itoffers a site for hydrolytic degradation and also constitutes aninherent therapeutic weakness as attested by the 8-fold greateractivity of deoxoartemisinin (24) [35].

The synthesis and testing of many simpler tricyclictrioxanes reveal that certain rings in 14 are redundant. Thehigh activity of the dimethyl derivative 25 shows that ring Dcan be dispensed with altogether or rather that the spirocyclicpentane ring serves in its stead Fig. (8). Clearly, the lactonefunction in ring B is superfluous. Further evidence that the Bring is unnecessary is attested by the high activity of theendo-methoxy trioxanes 26 and 27. The pair of exo-methoxyepimers 28 and 29 performs less well, some five-fold. Inboth instances, the phenyl group at the bridgehead booststhe activity over the methyl derivative. The peroxide 30 isdevoid of activity; so there are limits to what extent thebasic sesquiterpene skeleton can be modified [39-41].

OMe

O

Me

Me

O

O O

O

Me

Me

OMe

O

23 24

Several other active, tricyclic trioxanes e.g. 31-33, havebeen synthesized, tested, and confirm that the full tetracyclicFig. (7). Deoxyartemisinin and deoxoartemisinin.

O O

MeO OR

OO

OO

Me

Me

O O

MeO OR

OH

OMeO Me

O

O O

PhCH2O OMe

O OMeO

OMe

OTs

O OMeO

OMe

OH

Ph

3025 2627

2829

R = MeR = Ph

R = Me

31

R = Ph

32 33

DA

CB

D

C

A A

D

A

C

Fig. (8). Some tricyclic trioxanes.


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

[41, 45-46]. However, switching the parent ring fromdihydronaphthalene to cyclohexene brought about a changein the right direction; the second set, 37-40, gave IC50values going from 729 to 104 ng /ml. The antimalarialactivity, poor to start with, improved progressively onattaching spirocyclic rings at the C3 position. A dramaticjump was achieved in the case of the cyclopentenehomologues 41-44; their IC50 values against the W2 cloneranged from 5.31 to 0.49 ng/ml, demonstratingunequivocally that the carbocyclic framework of artemisininis simply not required for activity. It is worth mentioning byway of reference that IC50 values for artemisinin are usuallyabout 1-2 ng/ml, so the preceding values are indicative ofworthy contenders.

Bicyclic Trioxanes

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

Unlike artemisinin, the cis-fused bicyclic structures of 41-44 are flexible, permitting the trioxane ring to undergoinversion between two chair conformations. Clearly, the cis-fused, almost planar cyclopentene and the spirocyclopentanerings are crucial features of the pharmacophore in these fullysynthetic analogues (v. infra). Another structural requirementwhich proved of supreme importance for attaining high

Ph

R2

R1

PhO O

O

R2

R1

PhO O

O

Ph

R2

R1O O

O

MeMe

34

R1 = H, R2 = (CH2)6C6H3Cl2-3,4

37R1 = R2 = Me R1 = H, R2 = CH2C6H4OMe-435

33 3

41

R1, R2 = (CH2)5

R1, R2 = (CH2)O(CH2)236

R1, R2 = (CH2)4

43

38

R1, R2 = (CH2)5R1, R2 = (CH2)4

39

R1, R2 = (CH2)O(CH2)2

R1 = R2 = Me

40

R1 = R2 = Me

42

44Fig. (9). Some bicyclic trioxanes.

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

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

H

C6H4Me

C6H4Me

O

OO

OH H

C6H4Me

C6H4Me

O

OO

OH

H

C6H4Me

C6H4Me

O

OO

O

H

C6H4F-p

C6H4F-p

O

OO

OR

H

C6H4F-pC6H4F-p

O

OO

N N

O

O

H

C6H4F-p

C6H4F-p

O

OO

HNN

O

O

48 R = CH2C6H4CO2Na

R = P(O)(ONa)2R = CO(CH2)2CO2Na

45 46 47

49 50

5152

5

6

5

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


Table 1. In vitro and In vivo Activity of Peroxides 16 and 17 and Trioxanes 44 and 53

Me(CH 2)10 MeOO

Me

OO

Me

O

O

Me

CF3

CF3

OO O

Ph

Ph

OO O

C6H4F-p

C6H4F-p

6.0-

1.81.2

1.81.3

1.21.0

2.31.9

75.0 25.0 ED50 p.o.

ED90 p.o.10.4 18.0 7.4

5.9 2.5 5.0 14.0

2.50.92.5

6.03.44.1 3.9

2.7ED50 s.cED90 s.c.

4.08.0

P. falciparum K1 strain, ng/mlED50

P. berghei,mg/kg/day x 4

31.127.6 9.51 5.57

ART CLQ MFQ

4.6 7.697.9

Controls

16 44 53

rac. rac.

17

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

9.5 and 5.6 against the chloroquine-resistant K1 strain of P.falciparum, values which are superior to those of 27.6 and31.1 ng/mls shown by 16 and 17 respectively (Table 1).The p-fluorophenyl trioxane 53 also performs 2-4 timesbetter than 16 against P. berghei both at the ED50 and ED90levels by oral administration. A brief description of the invivo test protocol is given later (see Table 2). The in vitroand in vivo results demonstrate that the yingzhaosu skeletondoes not have to be copied to confer high activity. Itdemonstrates as well that 53 is a potential competitor to 16.

The activity of the best candidate 44 is also sensitive tosubstitution in other parts of the ring. Replacing the phenylby tolyl or p-fluorophenyl substituents alters activityslightly. Substitution on the double bond, e.g. 45, andsubstitution at the C5 position in either the exo or endoconfiguration with neutral groups, e.g. 46-49, leads to EC50values of about 9-14 ng/ml, again for the W2 clone, whichare about 3-4 times less active than the parent cyclopent-5,6-enes Fig. (10). When ionic water soluble groups areappended at the C5 position, as illustrated by trioxanes 50-52, activity disappears entirely [48, 49]. The conclusion isthat the configuration at C5 does not impinge strongly onactivity, whereas the ionic nature of the substituent does.

Chirality and Configuration

As all the synthetic trioxanes discussed so far are racemicmixtures, only one enantiomer being depicted for the sake ofclarity, it might be asked if their IC50 values should behalved to double their potency to what it appears to be onthe assumption that only one of the enantiomers is the actualtherapeutic agent. However, it can be concluded withconfidence that in every case both enantiomers are equallyparasiticidal on the basis of the proof offered by the exo 5-hydroxy enantiomers 54 and 55 Fig. (11). Each turned out

As yingzhaosu A (13) is a fugitive species semi-syntheticderivatives have not been prepared. So arteflene (Ro 42-1611) (16) and Ro 41-3823 (17) have to serve as indices ofactivity for the natural product [50]. It is therefore worthnoting that the wholly synthetic cyclopentene-trioxanes 44and its p-fluorophenyl analogue 53 exhibit ED50 values of

OH

Ph

PhO

OO

O

OHOO

OPh

Ph

O

O

Ph

PhO

OO OOO

OPh

Ph

O

MeMe Me

O

CO

O O

O

MeMe Me

O

CO

54

SSR

R(+) (-)

R

R

S

S

SS

(-) (+)

55 56 57

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


to have the same activity. Even the diastereomericcamphanoate derivatives 56 and 57 have similar activities toeach other and also to 54 and 55, all compounds havingIC50 values lying between 1.5 and 2.4 ng/ml. In otherwords, they are all indistinguishable from artemisinin intheir potency [51].

Further confirmation is tellingly provided by the in vivoactivities of the pure enantiomers of the p-fluorophenylderivative of cyclopentene-trioxane 58 and 59 whencompared to that of the racemic mixture 53 (Table 2) [52]. Inthe Peters 4-day test against murine malaria, batches of miceare initially infected with chloroquine-sensitive P. berghei Nor chloroquine-resistant P. yoelii NS lines respectively anddosed each day for 4 days subcutaneously (sc) or orally (po)with the test antimalarial [53]. On the 5th day theparasitemia is read and the effective dose which suppresses50% and 90% estimated. It is immediately seen that theracemate 53, the (+) and (-)-enantiomers, 58 and 59, haveessentially identical ED50 values against the sensitive strainin both the sc and po modes of administration (Table 2).The same is true for the ED90 values. A similar alignment ofED50 and ED90 values is also evident for the resistant line. Itis also to be noted that the synthetic trioxanes (53, 58 and59) are superior to artemisinin in the resistant line.Moreover, 53 offers a bonus in being gametocytocidal andsporontocidal [54].

The foregoing results from the two sets ofenantiomerically pure synthetic trioxanes convey anunambiguous message. Configuration and chirality play norole in the mode of action. The trioxane ring in the syntheticbicyclic molecules have interconverting chair conformations,so a fixed boat conformation is not a requirement.

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

MODE OF ACTION

The above mentioned natural and synthetic trioxanes andperoxides are chemically very different from the traditionalaminoalkyl substituted quinoline remedies such as quinineand chloroquine. The former are neutral species whereas thelatter are bases. Both classes of drug are schizonticides actingon the parasite within the infected erythrocyte, but they doso by entirely different mechanisms. Curiously enough, thereceptor in both cases has been identified as heme. Sinceheme is achiral it is understandable that the configuration ofan enantiomerically pure antimalarial has no bearing onpotency.

In order to be effective, an antimalarial must first of alldiffuse into the red blood cell and interact with heme. It isnow known from a large body of evidence that artemisinin(14), its lactol derivatives 18-22 and peroxidic antimalarialskill the malarial parasite in the blood of the host by astepwise process of chemical induction Fig. (6). During thetrophozoïte stage of the intraerythrocytic cycle the parasitesinvade the red blood cells and ingest hemoglobin to provideamino acids for growth. After proteolysis the spent prostheticgroup, heme, because of its solubility and toxicity to theparasite, is eliminated immediately by oxidation andpolymerization 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

(racemic)RR + SS

( )

5.6 10.0 7.6 4.5

2.5 6.0 6.0 2.5

ED50 ED90 ED90ED50

1.8 3.4

5.01.4

2.6 4.83.6 2.1

P. yoelii NSposc

1.1 3.12.81.5

P. berghei N

ED50

sc

po

1.8 3.23.62.1

53

3.11.8

ED90

scED50

P. berghei N2.4 56scP. yoelii NS

65 170

290 128

ED50 ED90 ED90ED50

2.3 0.9

C6H4F-p

C6H4F-pO O

O

Chloroquine

ED90

5.8 10.0

Quinine

(+)

Artemisinin

58 59

_O

O OC6H4F-p

C6H4F-p

mg/kg/day x 4

mg/kg/day x 4

R

R

SS


NN

N

NFe

HO2CHO2C

Me

Me

Me

Me

NFe

Me

Me

Me

Me

HO2CHO2C

N

NN

Ar

ArO

O

O O

OAr

Ar

O

O

60 61

53 62

6364

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

chelation and its toxic effect potentiated so that the parasiteis killed [56-58]. During the mortal event chloroquineremains chemically unchanged neither reacting with norkilling the parasite. Instead, it lets heme do the job.Treatment of the host with an antimalarial peroxide alsointerrupts the aforementioned detoxification process. But itdoes it in a different way from the quinoline-type drugs. Itclosely coordinates with heme and then reacts with it toproduce a lethal agent.

The nature of the latter, and how it forms took some timeto be delineated. Experiments performed with hemin andartemisinin confirmed their interaction to form an unknownadduct after generating an unidentified oxy radical [59-62].Artemisinin was thought to alkylate heme and parasiteproteins, but no structures were proposed [63]. It was alsodiscovered that the administration of radiolabeled β-arteether,

dihydroartemisinin, and arteflene to P. falciparum-infectederythrocytes resulted in the transfer of label to six malarialproteins [64]. It was therefore concluded that in someundefined manner, not necessarily involved with parasitedeath, reaction with specific malarial proteins had occurred.Nonetheless, signs were seen that the parasiticidal processsomehow involved transient radicals. Gradually, by devisingmodel experiments with simpler peroxides and 1,2,4-trioxanes, the nature of the lethal agent and the mechanisticdetails of the mode of action became clearer and morecomplete.

In recent years numerous model experiments have beenperformed and even today some of the results andinterpretations presented as the final word are open toquestion as they are at variance with the canons ofcontemporary organic chemistry.

O

HOAr

ArAr

ArO

O

O

53

MeCN, 22o, 2h

66%

67 R = H

65

2+

73

74

.

O

Ar

ArOO

O

O

OO

Ar

Ar

Ar

ArOO

FeCl2.4H2O (0.35 equiv)

R = Cl R = OH 66 8%

2%

3+

._

75

_

O

Ar

ArO_

76

+

3+

2+

O

Ar

ArOO

_

77

O

R(CH2)4

O

Ar = C6H4F-p

+Cl, H2O_

Fe

Fe

Fe

Fe

H

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


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, mightbehave as oxygen atom transfer agents. The idea was fosteredby the fact that deoxyartemisinin (23) was the mainmetabolite and that heme might behave like a P450-typemonooxygenase [65, 66]. It was postulated that activetrioxanes would first transfer an oxygen atom to heme (60) toform the corresponding iron-oxene intermediate (61) whichin turn would destroy a neighboring parasite bymonooxygenating its protein Fig. (12). Heme might beexpected to convert 14 into 23 during the parasiticidal event.Similarly, 53 would give the dioxolane 62. The idea wastested by using ferrous chloride tetrahydrate in acetonitrile asa model for heme. As a simple chemical model for theparasite cyclohexene (63) was chosen as it should be easilyepoxidized to 64 by the ferryl equivalent of 61. Actualtreatment of 53 and 14 with ferrous chloride caused rapidunraveling of the trioxane rings [67, 68] Fig. (13). Threeproducts were formed from 53; the cyclopentenyl esters of δ-chloropentanoic (65), δ-hydroxypentanoic (66) and pentanoicacids (67) in yields of 66, 8 and 2% respectively [62]. Theracemic diphenyl cyclopentene-trioxane 44 under the sameconditions gave analogous results. Repetition of bothexperiments in the presence of cyclohexene (63) led tobasically the same result. Neither dioxolane 62 nor epoxide64 was formed.

In the case of artemisinin (14), only isomerizationoccurred [68]. Exposure to FeCl2 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) weredetected. β-Artemether (19) gave a similar result [68] Fig.(15). It reacted in 5 min. isomerizing to the correspondingfuran-acetate 70, the hydroxy-deoxyartemsinin derivative 71,and an epimeric mixture of formyl diketones 72 in yields of32, 23, and 16%. Running the reaction in cyclohexeneimproved the yields of the same three products to 36, 30 and19%.

The Intermediacy of Carbon Radicals

The foregoing product compositions are stronglyindicative of the intermediacy of radicals in their formation.In fact, there are many precedents for the redox behavior offerrous ion towards cyclic peroxides which inducesdecomposition by forming radicals [69-72]. In the presentinstance, ferrous ion functions in the customary mannercomplexing with the O-O bond of 53 with concomitantsingle electron transfer Fig. (13). Scission of the resultingoxy radical 73 is driven by the formation of the ester functioncreating simultaneously the terminal pentanoate radical 74.Recuperation of the electron by ferric ion furnishes 75, whichprobably for reasons of entropy is unable to cyclize to thelactone 76. Instead, ambient chloride ion and to a lesserdegree water, attack 75 giving the chloro and hydroxyproducts 65 and 66. Overall, the conversion of 53 to 65 and66 can be considered as an isomerization with adjunction of

O-O

O

Me

Me

OMe

O O

O

OO

Me

Me

Me

O

O

OH

MeO

Me

Me

O

O

OMe

O

Me

Me

O

OOFe O

O

O

Me

Me

O MeO

Fe

14 68 69

cyclohexene (1.18 equiv.)

without cyclohexene

1-8%84%17%78%

.2+

2+.

2+

O O

O

Me

Me

O O

Me

O

Me

Me

O

OHOH

O

O

O

Me

Me

O

Me

OMe

O

H

Fe

25o, 5-15 min+

Fe

FeCl2.4H2O, MeCN

2+

H

2+ 2+

1,5 shift ..

2+

2+

OH

O

Me

Me

O

Me

O

2+

78

79

80

81 82 83

3

6

3

4

C-C scission

Fe

O=Fe

O=Fe

Fe

Fe

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


MeO

O

OO

Me

Me

Me

O

OH

MeO

Me

Me

O

O

MeO

O

Me

OCHO

Me

Me

O-O

O

Me

Me

OMe

MeO

+

70 7119 72

cyclohexene (1.18 equiv.)without cyclohexene

30%36%23%32%

25o, 5 min

19%

16%

MeOMe

O

Me

Me

O

OO

Fe

Me

Me

Me

OO

OMe

Me

Me

O

OO

MeO O

+

+MeO, CO, H

97

.2+

2+

98

.2+

MeOMe

O

Me

Me

O

OO

Fe

+

2+

2+ 2+

99 100

_

Fe

Fe

Fe Fe

FeCl2.4H2OMeCN

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

a molecule of hydrogen chloride or water. The origin of thepentanoate 67 can be attributed to the abstraction of ahydrogen 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. Indirectconfirmation of the radical 74 was obtained by carrying outthe same experiment with ferrous sulfate and cupric acetate inmethanol. The terminal olefin 77 was the exclusive product.As soon as 74 forms it is oxidized by cupric ion to thecorresponding masked primary cation 75 which promptlyeliminates a proton.

In just the same way as before ferrous ion complexes withthe peroxide bond of artemisinin (14) Fig. (14). Althoughnot specified in the preceding example, the ferrous ion aftersingle electron transfer forms a covalent bond with theoxygen anion of the sundered peroxide bond giving a pair ofequilibrating ferric monodentate oxygen radicals 78 and 79which are formed in different proportions. They evolvedifferently too. The main course is followed by 78 whichcleaves to the pendent primary ethyl radical 80; the drivingforce being the acquisition of thermodynamic stability byformation of the acetate group. Expulsion of the contiguousferric ion as ferrous ion and union of the two radical centerscreates the tetrahydrofuran 68.

The evolution of the minor oxy radical 79 was originallypuzzling 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 bythe excision of O=Fe2+ [76]. The latter, if not dispersed ordestroyed, could then react again with 82 to afford theepoxide 83. Alternatively, 83 could be formed directly byloss of ferrous ion from 81. Finally, opening of the epoxide83 by internal nucleophilic attack by the tertiary hydroxylgroup accounts for the formation of the minor product 69[77]. The intermediacy of O=Fe2+ may have been inventedto account for deoxyartemisinin (23), the human metaboliteof 14, which is formed in addition to 68 and 69 whenartemisinin was exposed to ferrous bromide in THF (v.infra). Although C-centered radicals like 78 and 79 had beenpreviously invoked [75] as possible agents responsible forthe high antimalarial activity of the tricyclic tosylate (32),the later predilection for O=Fe2+ as the toxic agent may havebeen influenced by the large body of research carried out withP450-monooxygenases.

The 1,5 H Shift

Despite their plausibility, some of the foregoing stepsseemed unsatisfactory and deserve comment. The 1,5hydrogen transfer, although favored over 1,3 and 1,4transfers, must meet certain geometric criteria for it to occur[78]. It appeared from an examination of the calculated andX-ray structures of 14 taken as a model for the Fe3+-boundcomplex that the interatomic distance between H-C(3) andO-C(6) in 79 is 2.478-2.803 Å [68]; a distance greater than

2+

.2+2+

.

O

Me

O

Me

Me

O

OFe

OFe

OH

O

H

O

Me

Me

OMe

O

84 85

78 69

Fe

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


the critical distance of 2.1 Å above which migration isthought 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 stillexceeded 2.1 Å. As there was no clear-cut preference for 1,5over 1,3-H shift, the latter was favored for plotting a pathfrom 78 to 69 Fig. (16). Consequently, a 1,3-H shiftfollowed by a 1,2 shift of a hydroxyl group (78→ 84→ 85)were proposed to give the tertiary radical which then wassupposed to close to 69 by loss of ferrous ion.

Unfortunately, the preceding path is the wrong one asattested by the subsequent isolation of epoxide 83 and thetrapping of the secondary radical 81 (v. infra). The findingsof a recent study using density functional theory alsoprovided a mechanistic corrective [80]. Taking 6,7,8-trioxybicyclo[3.2.2]nonane (86) as the model for 14, formaladdition of a hydrogen atom affords the pair of oxy radicals87 and 89 Fig. (17). Calculation confirmed that theinteratomic distance between the oxygen radical and thecontiguously oriented 1,5 disposed hydrogen atom in theground state of 87 is 2.340 Å, a distance definitely greaterthan the critical value of 2.1 Å. However, the activationenergy for 1,5 H-transfer to give the secondary carbon radical

stable than its acetal precursor 89 by 12.2 kcal/mol. Thesecalculations of course strictly apply to the artificial oxyradicals 87 and 89, but nonetheless they are relevant to the1,5 H shift 79→ 81 and the C-C scission 78→ 80. Theconclusion is ineluctable. Converting an oxy radical to a C-centered radical by shifting a H-atom or cleaving a C-C bondis going to be easy. More importantly, the C-centeredradicals 80 and 81 derived from artemisinin, once formed,will be unable to regress to their oxy radical predecessorsbecause the energy of activation in the back reaction is nowfar higher than it was in the forward reaction. In other words,these C-centered radicals are kinetic products and theirformation is irreversible.

A further indication that ester formation is the drivingforce for producing an active primary C-centered wasprovided by calculations on the 1-methoxycyclopentyl-1-oxyl radical (91) and its scission product the δ-radical (92)[52]. Their geometries were optimized by semi-empiricalunrestricted Hartree-Fock calculations according to the PM3method Fig. (18). The respective heats of formation, -59.5and -76.6 kcal/mol, confirm that the conversion 91→ 92 isstrongly exothermic by 17.1 kcal/mol. A precedent for the

OO

O

O OH

O

O OH

HO.

O O

HO

O O

HO

.

.

86 87 88

89

H

90

1,5 shift

C-C scission

.

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

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

Carbon Radicals as Kinetic Products

The evolution of the acetal-type radical 89 was similarlyfavored. The activation energy for its cleavage to the formylprimary 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 byadding a hydrogen atom to artemisinin serves as a model forboth ferric derivatives 78 and 79 Fig. (19). The heats offormation for 93 and its rearranged products the primary andsecondary radicals 94 and 95 were found to be -199.4,-216.3, and 219.3 kcal/mol respectively [68]. In otherwords, the act of creating the primary and secondary radicalsreleases 16.9 and 19.9 kcal/mol. Thus, both the 1,5 shiftand C-C scission are strongly exothermic processes. Byextrapolation, the rearrangements 78→ 80 and 79→ 81

H O

O

H

H H

H H

H H

HH

HH H

OH

HH

O

HH

H

H

H

H91 92

..

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


959493

+

O

MeO

O

OOH OHO

OH

O O MeO

MeHO

OO

Me

O

Me

Me

Me

Me

Me

. . .

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

should be exothermic to the same degree and thereforeirreversible. These conclusions, as it will be seen later, havespecial relevance to the mode of action of peroxidicantimalarials.

Is O=Fe2+ an Intermediate?

No direct evidence has been reported for the existence of82, while that in support of O=Fe2+ is circumstantial. Theexperiments, the rationalization of which led to the proposalof such exotic species, were conducted by subjectingartemisinin (14) to ferrous bromide in THF alone or in thepresence of various easily oxidizable or aromatizable addendsas possible mechanistic witnesses [27, 76]. Under theseconditions, 68 and 69 were formed as before in a similarratio, but in diminished yields of 29 and 10%, the balance ofmaterial being deoxyartemisinin (23) (59%). The proportionof 23 went up on adding progressively more 1,4-cyclohexadiene. Hexamethyl Dewar benzene (HMDB) alsoboosted production of 23, becoming partially aromatized tohexamethylbenzene in the process. Consequently, it wasinferred that 23 arose by protonation and intramolecularclosure of the putative cyclic enol ether 82, increasinglyfurnished by loss of O=Fe2+ from 81 Fig. (14). Asupplementary avenue to 23 was assumed to issue from 81by abstraction of hydrogen from 1,4-cyclohexadiene.

However, 23 could have arisen by a less exotic andmechanistically more likely route. The reduction ofartemisinin (14) or perhaps, more appropriately, the ferricoxy radicals 78 and 79 by abstraction of hydrogen from THFor added 1,4-cyclohexadiene without a hydrogenationcatalyst (none is needed) to give the dihydroxy derivative 96followed by dehydration to 23 offers a reasonable explanationFig. (20). Ferrous ion or a transient radical, rather thanO=Fe2+, may well have catalyzed the aromatization ofHMDB. Other addends such as methyl phenyl sulfide andtetralin, the oxygenation of which was presented as proof forO=Fe2+, may have abstracted oxygen directly from 14 orcould have been oxidized adventitiously. The recent

observation that large amounts of diol are formed whensimple bicyclic endoperoxides are treated with FeBr2/THFattests to its reductive nature [82]. Finally, it is difficult toexplain why O=Fe2+, a non-selective oxidant, if reallypresent, which has no trouble in epoxidizing the enol ether82, fails to react with admixed 1,4-cyclohexadiene to give itsepoxide.

Lastly, it should be mentioned that the simple extrusionand transfer of an oxygen atom from a peroxide to a ferrouscation might be mechanistically difficult. In order to be aneffective oxidant, the high valent iron-oxene species mayneed to be perferryl, the species formed in the P450 enzymecatalytic cycle, rather than ferryl [83]. If this be the case, thesecond step after coordination with ferrous ion, the rupture ofthe FeO-R bond to produce the perferryl species O=Fe3+

requires the departure of R as an anion, not a radical, whichis more costly in energy and less likely.

In view of the inertness of cyclohexene in the FeCl2-induced reactions with 53 and 14 together with the non-formation of deoxyartemisinin (23) or the dioxolane (62), theoriginal proposal that only C-centered radicals are involvedis undoubtedly correct and does not need to be modified tofit a P450 mono-oxygenase mechanism in spite of itsattractiveness [84]. The FeCl2-induced reaction of β-artemether (19) with and without cyclohexene follows thesame course and can be similarly interpreted Fig. (15).Deoxy-β-artemether is not formed. The furan-acetate 70 andhydroxy-deoxyartemisinin 71 arise in the standard way viacleavage and rearrangement of the pair of ferric oxy radicals97 and 98. The third product the mixture of diketones 72must have arisen by the formal loss of a molecule of methylformate. How this happens is explicable in terms of theelimination of ferrous ion in a counterclockwise or clockwiseradical fragmentation of 97 or 98. The resultingmethoxymethyl formate 99 then disintegrates by loss of aproton, carbon monoxide, and methoxide ion, giving thepenultimate diketo-aldehyde 100, which finally isomerizes to72.

The final piece in the mechanistic jigsaw puzzle was theisolation of the unstable epoxide 83, albeit in a tiny amount(1%), from the reaction mixture obtained by treating 14 withone equivalent of ferrous sulfate in aqueous acetonitrile. Thisvaluable piece of information confirms the sequence 79→81→ 83→ 69 [85, 86]. However, it must be noted that thekey experiment of actually isomerizing 83 to 69 appears notto have been done or at least reported. Proof for a secondarycarbon radical, presumably 81, was secured by its capturewith 2-methyl-2-nitrosopropane and the identification of theresulting nitroso radical by its ESR spectrum. Why the

OHHO

O

Me

Me

O

Me

O H2O

96

237814 or

Fig. (20). Reduction of artemisinin or its oxy radical 78 to thediol 96 and deoxyartemisinin 23 .


MnNN

N

N

Ph

Ph

Ph

Ph

OMe

O

Me

Me

O

OO

MnNN

N

N

Ph

Ph

Ph

Ph

O MeO

Me

Me

O

OO

MnNN

N

N

Ph

Ph

Ph

Ph

O MeO

Me

Me

O

OHO

NN

N

N

Ph

Ph

Ph

Ph

O MeO

Me

Me

O

OHO

HH

MnNN

N

N

Ph

Ph

Ph

Ph

.

+

O MeO

Me

Me

O

OHO

+

.

101 102 103

104 105

+.H

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

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

By recourse to a hydrophobic model which parallels thebiological event, primary carbon radicals formed fromartemisinin (14) could be trapped [87]. Instead of heme,which is notoriously unstable, managanoustetraphenylporhyrin (Mn(II)TPP), generated in situ by theinteraction of Mn(III)TPPPOAc with tetrabutylammoniumborohydride, was allowed to react with 14 Fig. (21). Singleelectron transfer occurred in the usual way by breaking the O-O bond. The resulting manganese(III) derivative 101 cleavedto the primary radical 102. Thereafter, intramolecularaddition to one of the nearby pyrrole rings affords the pyrroleradical 103, then the cation 104 by internal transfer of anelectron. Reduction by borohydride to the dihydropyrroleand demetallation afforded the covalent adduct 105. Byapplying the same procedure, the primary radicals from β-artemether (19) and the cyclopentene-trioxane 53 weresimilarly produced and captured by the pyrrole ring [88, 89].

It is reasonable to assume that in the biological contexttoo the decomposition of artemisinin (14) and β-artemether

(19) proceeds via primary and secondary carbon radicals.Metabolic studies with 14 and β-arteether confirm theformation of products like 70 and 71 as well asdeoxyartemisin (23) and its ethoxy derivative [90]. Theorigin of the deoxy compounds could be due to enzymaticdeoxygenation which has nothing to do with theparasiticidal event. For example, an in vitro study of themetabolism of β-arteether in rat liver cytosol revealed thatdeoxy-β-artether was formed directly under the influence of anNADH-dependent cytosolic enzyme [91]. As a chemicalequivalent of reduction by a Zn-containing NADHdehydrogenase zinc dissolving in acetic acid was taken as anappropriate reagent [92]. Adding an equivalent of Zn powderto a solution of 14 in AcOH with stirring at roomtemperature gave after a few hours a quantitative yield ofdeoxyartemisinin (23) [68]. Under the same conditions 19gave deoxy-β-artemether (108) as the sole product in 68%yield Fig. (22). These results indicate that deoxygenationinvolves a two-electron reduction, which is a characteristic ofzinc, but not of ferrous ion. First, Zn donates two electronsto the O-O bond of 19 to form the bidentate intermediate106. The latter thanks to the oxophilic nature of zincrearranges to 107. Finally, deoxyartemether (108) is formed

O-O

O

Me

Me

OMe

MeO

O O

O

Me

Me

OMe

MeOZn

O

Zn

O

Me

Me

OMe

MeO

O

19 106 107

_O

O

Me

Me

OMe

MeO

108

+

Zn=OZn

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


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 FeBr2, THF 15 min 29 10 59 0 [76]

2 FeBr2, THF, CHD 40% 15 min 17 4 71 0 [76]

3 FeCl2.4H2O, MeCN 5 min 77 11 0 0 [76]

4 FeCl2.4H2O, MeCN 5 min 78 17 0 0 [68]

5 FeCl2.4H2O, MeCN, CH 5 min 84 8 0 0 [68]

6 FeCl2, imidazole, MeCN 5 min 78 16 6 0 [109]

7 Hemin, PhCH2SH, THF 15-40 min 63 5.5 1.0 0 [76]

8 FeSO4, H2O, MeCN hours 25 78 0 1-2 [86]

by extrusion of ZnO which dissolves in AcOH givingZn(OAc)2.

Comments on the Proposed Unified MechanisticFramework

Having in hand sufficient proof for the intermediacy of theepoxide 83, the primary and secondary radicals 80 and 81together with their formation as kinetic products, and anabsence of proof for the cyclic enol ether 82 and the ferrylspecies O=Fe2+, all the pieces are in place for defining theultimate mechanism for the ferrous ion-cleavage ofartemisinin and its congeners. A recent attempt, whileambitious and somewhat audacious, falls short of this goal[86]. A so-called unified mechanistic framework wasformulated on the basis of an imperfect understanding of thedata and some of the principles governing organic reactions.The data taken for consideration, although disparate, werequite limited (Table 3). With such a small sample a reliableinterpretation is already compromised. It is immediatelyseen that deoxyartemisinin (23) is only produced in THFand also to a very minor degree when imidazole was present(entries 1, 2, 6, and 7). As mentioned earlier, it seems very

likely that 23, arising by reduction, stems from a reactionwhich is separate from that of the catalyzed isomerization tothe furan-acetate 68 and the hydroxypyran 69. Consequently,a doubt exists about the validity of any mechanism that ispartly based on 23. A scheme to be valid, and indeed to beunified, can only take into account those products that sharea common mechanism.

The reaction speeds (they cannot be called rates), namely,the time required to complete the reaction, are very roughestimates (Table 3). All that can be said is that FeSO4 (entry8) is much slower than the rest, which are more or lessequally rapid in their action. Its slowness differentiates itfrom the other reagents by permitting other products to beidentified, notably the epoxide 83 and one of itsrearrangement products (109). As the formation of 109 wasonly alluded to, a word on how it arises is appropriate. Theisomerization of 83 to hydroxypyran 69 was correctlydetailed Fig. (23). Protonation of 83 to 110 followed byopening of the epoxide transfers positive charge to the acetalfunction as indicated by the species 111. Annihilation ofcharge on the oxonium ion by internal attack of the hydroxylgroup then gives 69. An alternative course is to open theprotonated epoxide 110 to form a five-membered ring. Attack

O

Me

O

Me

Me

O

OH

O

O

Me

O

Me

Me

O

OH

O H

O

MeO

Me

Me

O

OH

OH

O

Me

O

Me

Me

O

O

O H

O

Me

O

Me

Me

O

O

O H

83 110+

111

110+

O

Me

OR

Me

Me

O

O

O

R = H

+

112

R = Ac109

113

H

69

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


by the hydroxyl group on the least substituted terminus ofthe epoxide entity leads to the furan 112. Deprotonation andcleavage gives 109, one of the observed decompositionproducts which was subsequently identified as its acetate. Infact, this dichotomy of epoxide opening is a characteristicfeature in the rearrangement of artemisinins and depends onthe nature of the adjacent ring atoms (v. infra).

The main mechanism proposed incorporates as its corethe pair of equilibrating oxy radicals and 78 and 79(produced from 14) which are assumed to be in equilibriumwith their derived carbon radicals 80 and 81 Fig. (24). Itshould be remarked at the outset that the subsidiaryequilibrating oxy radicals 114 and 115 shown as arisingfrom 78 and the conversion of 114 to 116 are purelyhypothetical in the case of artemisinin as no products formedfrom them were observed. They are meant to serve asreference structures for reactions of derivatives. The coreequilibrium is supposed to be displaced to favor differentamounts of the products 68, 69, and 23 depending on thekind of ferrous reagent and solvent employed. Two factorswere invoked to account for the product composition. Thebetter described is the solvent factor. The basic idea is thatthe preferential breakage of one over the other of the twobonds in Fe-O-R as in 80 and 81 is susceptible to the natureof the solvent. It is suggested that the Fe-O bond is strongerin THF than in MeCN, therefore favoring homolysis of theO-R bond, whereas the reverse is true in aqueous MeCN.This particular solvent effect was used to explain why 23 isformed as the major product in THF, but not observed inMeCN (Table 3, entry 1). Such an explanation is difficult toaccept since radical reactions are generally insensitive tosolvent polarity [93].

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

bond. It is argued, without substantiation, that FeSO4,being less active than FeBr2 (Table 3, entries 8 and 1) slowsdown "the steps of" (the rate of conversion of?) 81 to 69 andof 80 to 68, but aq. MeCN "works the opposite way"(speeds them up?). Put another way, it can be supposed thatthis means that the ratio of the equilibrating species 81 to 80is about the same (3:1) when produced by either FeSO4 orFeBr2 in their respective solvents. Decomposition of 80 justgives 68 while 81 bifurcates to 23 and 69 in THF, but goescompletely to 69 in MeCN. When FeCl2.4H2O in MeCN isused as reagent (entry 4), no 23 is formed as the solvent doesnot facilitate Fe-OR bond cleavage. The ratio of 68 to 69now changes to 4.6:1. Apparently in this case the effects ofsolvent and reagent do not cancel out which means that theequilibrium shifts away from 81 towards 80 because as "the1,5 shift is not so fast" 79 gives 81 less quickly. The majorand minor radicals 80 and 81 then react giving 68 and 69 inthe aforementioned ratio.

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

The above mechanism was also deemed to be applicableto certain artemisinin derivatives. To simplify discussion,just two are considered here. β-Artemether (19) and its β-benzyloxy analogue on treatment with FeSO4 in aqueousMeCN gave derivatives analogous to 68 and 69 in ratios of 1to 1.2 and 1.8 to 1 respectively. The switch in ratios wasattributed to slower 1,5 shifts due to conformational changescaused by the "larger" benzyloxy substituent. In reality the

OMe

O

Me

Me

O

OOFe

2+.

2+

1,5 shift

O

Me

O

Me

Me

O

OFe O

2+78

O

Me

O

Me

Me

O

OFe O

scission

2+

.

2+

.114

115

O

OH

MeO

Me

Me

O

O

OMe

O

Me

Me

O

OH

OH

O

O

O

Me

Me

O

MeO

Fe

69

2+

2+

.81

83

O

O

OO

Me

Me

Me

O

OO

O

Me

Me

O MeO

Fe

2+

68

2+

80

O

Me

O

Me

Me

O

OO

.

116

O O

O

Me

Me

O

Me

OFe

OH

O

Me

Me

O

Me

O

23

O

MeO

Me

Me

O

O

2+.

79

6

822+

Fe

O=Fe

C-C

FeFe

Fe

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


difference in steric compression on the rigid tetracyclicskeleton between such remote substituents must benegligible.

Arguments have been mustered to provide purportedlycorrect mechanisms for the reactions of certain carbaartemisinins [94]. The action of FeBr2/THF ondeoxoartemisinin (24) produced the completely unraveledformyl diketone (117) and the deoxodeoxyartemisinin (118)in yields of 79 and 8% Fig. (25). The first step is the settingup of the pair of equilibrating oxy radicals 119 and 120.Thereafter, their evolution was ascribed to the differentialeffect of THF. Cyclization 119→ 122 was retarded, allowingcleavage 120→ 123, which is favored, to take over. Closureof the hydroxyl group onto the double bond in 123 wouldafford 118 in the postulated way. Fragmentation of 119 bycircular movement of four electrons brings about cleavage to117. It was not made clear why so little of the deoxygenatedproduct was formed compared with the case of artemisinin. Asimpler explanation is that 120 fragments just as well as 119which necessarily bypasses the potential excision of O=Fe2+,while deoxygenation to 118 is probably the consequence ofan independent reduction process arising from 119 and 120through the diol 121.

The dideoxoartemisinin (124) seemed to presentdifficulties of interpretation even though the result wassimple enough Fig. (26). With the same reagents as beforejust a single product was obtained, the hydroxy furan 125.The mechanism proposed [86] starts by adjunction of ferrousion giving the pair of ferric oxy radicals 126 and 127. Thistime there is no avenue of cleavage open to 126 becausethermodynamic stabilization cannot accrue by creation of acarbon-carbon double bond. The reaction proceeds entirely

from 127 which undergoes 1,5 H shift to 128. Even thoughit is in THF, a solvent alleged to favor excision of O=Fe2+,128 fails inexplicably, if the unified mechanism is to bebelieved, to react in this sense and loses Fe2+ insteadforming the epoxide 129. The next step proposed is far-fetched as it entails opening of the epoxide with FeBr2 tofurnish 130. In order to get the groups in the right positions,the two ligands are now obliged to exchange with each othergiving 131. The goal, the hydroxy furan 125, is finallyreached by nucleophilic displacement of bromide ion by theoxide substituent. Of course, this tortuous route is notfollowed at all, because there is a rational shortcut.Protonation of the epoxide 129 will engender backside attackby the hydroxyl group to form the furan ring 125 directly.This is the expected closure. Closure to the six-memberedring only occurs when positive charge is formally located atthe methylated terminus by an acetal function, e.g. 83→110→ 111→ 69 Fig. (23).

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

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

O-O

O

Me

Me

OMe

Me

O

Me

Me

O

OO

Fe

OO

O

Me

Me

O

Me

Fe

OO

O

Me

Me

O

Me

OHHO

O

Me

Me

OMe

FeBr2, THF

O

Me

Me

OMe

O

11724

2+

2+

25o, 5 min

.2+

2+

.

120119

118

O

OO

Me

Me

Me

O

6

2+

OH

O

Me

Me

O

Me

+

122 123

121

FeFe Fe H2O

Fig. (25). Ferrous ion-induced rearrangement of deoxoartemisinin 24 .


O-O

O

Me

Me

Me

O

Me

Me

Me

O OH

Me

Me

Me

O

OO

Fe

OO

O

Me

Me

Me

Fe

OHO

O

Me

Me

Me

Fe

O

O

O

Me

Me

Me

124

Fe

OH

O

Me

Me

Me

25o, 5 min

Br

OFeBr

2+

.2+

O

Me

Me

Me

2+

.

Br

128

OH

126

FeBrO

127

2+.

129

125

130 131

FeBr2, THF

125

H

129

+

FeBr2FeBr2

H

Fig. (26). Rearrangement of dideoxoartemisinin by FeBr2. Mechanistic avenues.

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

Peroxides Generating Hindered and SubstitutedCarbon Radicals have Poor Antimalarial Activity

It is now clear that artemisinin gives with ferrousreagents and presumably with heme, two types of radical,primary and secondary. An obvious question to ask is whatis the relative parasiticidal power of the two? Is one moreactive than the other? Are other types of carbon radicalequally as active? Another question concerns theirsusceptibility to steric hindrance.

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

artemisinin in vitro, having an IC50 value of 4.5 ng/mlagainst the W2 clone. The α-methyl and dimethylderivatives 133 and 134 were inactive. Two conclusions canbe drawn. The first is that 133 and 134 are unable to accessheme failing to elicit electron transfer, whereas 132 sitsnicely on top of heme, generating one or both of the tertiaryand secondary radicals 135 and 136 with perhaps the latterbeing the more effective at killing the parasite. The second isthat coordination with heme takes place with all threetrioxanes, but as 133 cannot undergo 1,5 shift only thesecondary radical 136 forms. Similarly, the only option opento 134 is cleavage to the unreactive tertiary radical 137.Therefore, it appears that hindrance to coordination withheme is the determining factor for antimalarial activity inthis instance. Nevertheless, the electronic nature of the alkylradical is important too.

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

O OMeO O

Me

OH

R2

R1heme

heme

OHO

MeO OMe

OH

Me

heme

O OMeO

O MeOH

Me

hemeMe

O OMeO

O MeOH

Me

heme132 R1 = H, R2 = Me133 R1 = Me, R2 = H134 R1 = Me, R2 = Me

or.

135 136

.

.137

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


benzyl derivative 138 was about as twice as active as theparent trioxane 26. However, the β-phenyl derivative 139was devoid of activity confirming that a carbon radicalstabilized by conjugation, or hyperconjugation like theaforementioned tertiary radical, is not aggressive enough tokill the parasite.

O O

MeO OMe

26138

R = H

O O

MeO OMe

140

Me

R = CH2Ph

R

139 R = Ph

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

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

The effect of steric hindrance on the availability orpropensity of a carbon radical to accomplish its deadly taskis patently seen on inspecting the in vitro activities ofsubstituted 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 IC50 against theW2 clone in vitro of 3.9, comparing well with artemisinin(14) and its value of 1.2 ng/ml Fig. (29). Simply placingfour methyl substituents on the cyclohexane rings sharplydiminishes the activity of 142, its IC50 rising to 94.0 ng/ml.

1,2,4,5-Tetroxanes have long been known to possessantimalarial activity, but much less so than their trioxanecounterparts [99-101]. The cyclohexane derivative 143 is 4-5times less active than 141 showing an IC50 of 20 ng/ml Fig.(29). Loading the tetroxane with two pairs of geminal

methyl groups on the cyclohexane ring raises the IC50 of 144to 255 ng/ml, wiping out activity. It can be concluded thatsteric encumbrance at the C3 position on the cyclohexanering relative to the spirocyclic carbon atom somehowinterferes with parasiticidal action. As in the case of themethylated tricyclic trioxanes, two hypotheses are possible.First, the bulky gem-dimethyl groups could simply preventthe peroxide bond from getting close to heme. Second, ifheme manages to get near enough to effect electronic transfer,the resulting neopentyl type radicals, e.g. 145 and 146,would be unreactive and therefore useless for killingparasites.

Substitutions on certain parts of the artemisinin skeletoncan also profoundly perturb its activity. As previouslymentioned, substitutions in the lactone ring, either as esteror ether derivatives of the epimeric lactols, are sufficientlyremote from the peroxide linkage as to have little effect ondocking with heme. In fact, such derivatives, like the lactolsthemselves, are often more active than the lactonic parent,simply for electronic reasons. However, substituents at theα-position to the lactone lie closer to the peroxide linkage.Changing them alters the activity in a systematic and, incertain cases, a dramatic way [102]. Replacing the β-methylgroup in artemisinin (14) by ethyl or propyl leads to IC50values against the W2 clone for 147 and 148 that are roughly12 times smaller than that of 14, in other words, thecompounds are more active Fig. (30). However, substitutionby a much longer aliphatic chain, as evidenced by 149,annihilates activity completely. Presumably, because thechain is long enough and sufficiently mobile to reach andcover the peroxide bond preventing approach to heme.

Surprisingly, in vitro tests carried out on epiartemisinin151 revealed IC50 values only 1.5 -2.0 times bigger thanthat of 14 [102, 103] Fig. (30). In contrast, the IC50 of thebromo derivative 150 is 8 times that of than 14. The isostereof 150, the gem-dimethyl derivative 152, is completely inert.The expectation is that all three α-substituted derivativeswould erect the same kind of steric barrier to an incomingmolecule of heme. A reason for the low IC50 values of 151

OO

OPh

Ph

Me

Me

MeMe

OO

O

Ph

Ph

O

OO

O

OO

OOMe

Me

MeMe

Me

Me

MeMe

heme heme

OO

O

Ph

Ph

Me

Me

MeMe

heme

OO

OOMe

Me

MeMe

Me

Me

MeMeheme

141 142 144143

145

.

146

.

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


might be due to isomerization. Under the conditions ofincubating P. falciparum during the test, 151, the less stableepimer, or at least some of it, could have reverted to themore active β-epimer 14.

O

O

R1

Me

OMe

O

147

R2

O

R1 = R2 = Me R1 = H, R2 = Me

R1 = Pr, R2 = HR1 = (CH2)13Me, R2 = H

148

149

150

R1 = Et, R2 = H

R1 = Me, R2 =Br151

152

14 R1 = Me, R2 = H

Fig. (30). Some derivatives of artemisinin.

A recent X-ray study of 151 reveals that the α-methylsubstituent lies in van der Waals contact with the proximaloxygen atom of the peroxide bond [104]. Evidently, theperoxide is hindered. The consequences of this hindrance areshown up by the in vivo activity. Against Plasmodiumberghei N, the chloroquine-sensitive line, the ED50 andED90 values of 151 are about seven times greater than those

of 14. Against P. yoelii ssp. NS, the resistant line, the ED50and ED90 values are 4-6 times bigger. Clearly, the α−epimer 151 is 4-7 times less effective than its β-epimer 14.The persistence of some activity may be due to someepimerization to 14 under the test conditions. Comparison ofthe X-ray data of 14 and 151 reveals that the two moleculesare superimposable, except for the lactone ring in 151 whichis slightly distorted to alleviate congestion between the α-methyl group and the peroxide bond. Thus the difference inactivity arises solely from the orientation of the methylsubstituent on the lactone ring.

The diminished activity of 151 and its absence by 152could be ascribed to poor docking on heme Fig. (31). Thus,the complexes 154 and 155 owing to obstruction by the α-methyl substituent either do not form or are not intimateenough for an electron to jump from the 3d orbital of iron tothe anti-bonding orbital of the O-O sigma bond.Consequently, neither oxy nor successive carbon radicalsarise from 154 and 155 like they do from 153. An alternativeis that docking in 154 and 155 is close, but skewed obligingthe iron atom of heme to selectively bind to the distaloxygen atom of the peroxide bond. As a result, electrontransfer occurs leading solely to the secondary carbon radicals158 and 159. The tight complex formed with artemisinin(153) would be expected to form mostly, if not all, theprimary radical 156. The relative parasiticidal power of theprimary and secondary radicals (e.g. 156 vs. 157) is notknown, but the selective capture of the primary radical byMn(II)TPP (v. supra) argues in favor of the former.

N

N

N

NFe

HO2C

HO2C

Me

Me

Me

Me

O

MeO

Me

R2

O

OOR1

N

N

N

N

FeHO2C

HO2C

Me

Me

Me

Me

O MeO

Me

O

OOMe

N

N

N

N

FeHO2C

HO2C

Me

Me

Me

Me

MeO

Me

R2

OOHR1

N

N

N

NFe

HO2C

HO2C

Me

Me

Me

Me

O MeO

Me

O

OOHMe PP

153

161

+

155

160

.

R1 = R2 = Me

.

156 157 R1 = Me, R2 = HR1 = H, R2 = MeR1 = R2 = Me159

158R1 = H, R2 = Me154

R1 = Me, R2 = H

OO

+

hemozoin

PP

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


Me

HO OH

MeMe

OH

H

OO

OH

Me

Me

Me

OH

Me

O

hemin

Me

OH

Me

Me

Me

OHOO

hemin H

Me

MePP

OHhemin

.

excision

+

heme

hemozoin

+

13 163

164 165 166

.

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

How Artemisinin and Peroxidic Antimalarials Kill theParasite

How the parasite is actually killed is also not known. Itmust be emphasized that FeCl2.4H2O in MeCN is not thesame as heme inside the red blood cell. Both entities displaythe same redox properties towards artemisinin. The first actsas a catalyst as already mentioned, but intraerythrocyticheme acts as a reagent when a parasite is present. Alkylationby a reactive hemin-radical e.g. 156 undoubtedly takes place

The cis-fused cyclopentene-trioxanes, exemplified by themost potent candidate 53, like cis-decalin easily undergoconformational inversion. They therefore are able to make aclose fit with the surface of heme. The resulting complexfrom 53 then unravels to the hemin derivative of the primaryradical 74, which then proceeds to alkylate parasite protein.The motor for antimalarial activity is the thermodynamicstability which comes from the formation of a carbonyl groupat its simplest, or better one that is conjugated. Such astructural feature is found in yingzhaosu A (13) and C (162)

.+Me

Tol-p

MeMe

OHO O Ohemin

OOH

MeMe

Tol-pMe

Me

Tol-p

MeMe

OHOO

hemin

162 167 168 169

hemeMe

Me

OHO

hemin

170

PP

.

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

inside the food vacuole of the parasite Fig. (31). Addition toan unsaturated center in the parasite protein (PP) wouldafford a new radical, which could then eject an electronyielding a cation followed by its deprotonation. Lastly,protonation of the resulting PP-artemisinin-hemin adduct156 releases the alkylated protein 160 from its heminappendage (161) which subsequently polymerizes tohemozoin. Experimentally, it was found that on treatmentwith artemisinin trophozoites of P. falciparum still producehemozoin, while with chloroquine less is produced [105]. Inany 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]. Insummary, in a sort of quid pro quo, a toxic C-centeredradical 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 radical163. Excision of the α,β-unsaturated ketone 164 releases thecyclohexyl radical 165 which then alkylates parasite proteinfinally producing 166 and eventually hemozoin. YingzhaosuC (162) behaves the same way Fig. (33). The hemin-oxyradical 167 cleaves to the acetophenone 168 and the ethylradical 169. Alkylation gives the hemin-PP adduct 170which finally ejects hemin as before.

Confirmation of the validity of the foregoing schemes hasbeen obtained by treating arteflene (16) with FeCl2.4H2O inMeCN [107]. Evidence for cleavage of the ferric oxy radical171 to the cyclohexyl radical 172 and the enone 173 wasobtained by capture of the former with 5,5-dimethyl-1-pyrroline N-oxide and the identification of its adduct by EPR

C6H3(CF3)2 -2,4

OO

Me

MeO

C6H3(CF3 )2-2,4

OO

Me

MeO

16

+

171

C6H3(CF3)2-2,4

OO

MeMeO

..

172 173

Fe2+ Fe2 + Fe2+

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


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

Evidence that artemisinin on treatment with a Lewis acidor benzylamine opens to a hydroperoxide has been suggestedas a basis of parasiticidal action [109, 110]. It will beinteresting to see if such reactions occur under physiologicalconditions and how biomolecules react with the purportedelectrophilic oxygenating species derived from thehydroperoxide. In general, hydroperoxides that are unable tocleave to a carbon radical only manifest weak antimalarialactivity [29].

Molecular Considerations in Designing New PeroxidicAntimalarials

The preceding mechanistic schemes clearly depend on theoptimal concurrence of several molecular properties in orderto produce the ultimate antimalarial. In other words, theparasiticidal action of potent peroxides like 14 and 53depends on the efficient operation of a sequence of chemicalevents, namely, docking with heme, electron transfer,formation of an oxy radical, scission to an unencumbered C-centered radical powered by the formation of a carbonylgroup or something similar, and finally, the death of theparasite by alkylation. Many trioxanes and peroxides aregoing to fulfill the above criteria. In designing new peroxidicantimalarials, any one of the above desiderata needs to beexamined to see if the candidate will fit the bill.

Many spatial arrangements are possible between hemeand artemisinin (14) and it does not necessarily follow thatthe one usually drawn is the right one. However, computer-assisted molecular modeling of various dockingarrangements of hemin(FeIII) (serving as a model for heme)and 14 using the Sybil program showed that in the moststable configuration the peroxide bond and the carbonyloxygen atom interact closely with hemin iron [111]. The α-face of artemisinin is close to heme putting the interatomicdistance between 2.6 and 2.8Å from the peroxide oxygenatoms to the iron. A similar procedure withdeoxyartemisinin (23) and hemin favored close binding onthe opposite or β-face.

A QSAR study using Comparative Molecular FieldAnalysis of 11-alkyl derivatives of artemisinin as well assome tricyclic trioxanes similar to D-seco-artemisinin gave agood correlation between calculated and observed activities[102]. From the relative contributions of steric andelectrostatic terms the former had the greatest bearing onactivity. The latter finding accords well with docking onheme as the crucial first step for effective drug action.

In a 3D-QSAR study using a pharmacophore searchmethod (CATALYST) two hydrophobic features andhydrogen bonding were identified as the hypothesis orpharmacophore responsible for activity [112]. By taking atraining set of trioxanes having known in vitro and in vivoactivities the best arrangement of the hypothesis consonant

with the highest activity was found. Typically, the highestantimalarial activity is shown by those cis-fused trioxaneswhich map well with the hypothesis (41-44 and 53) as theyare able to adjust their conformations at little cost in energy.An interesting result was the predicted inertness of the cis-fused cyclohexene trioxanes (37-40), the reason being theirinability to adopt a low enough energy conformation thatoverlaps with the computed ideal pharmacophore. It was alsofound that 53 docks closely with heme putting the iron atomclose to the peroxide bond.

Attempts have been made to correlate computedmolecular electrostatic potentials (MEPS) with antimalarialactivity with little success [113]. A problem is the difficultyof comparing the MEPS of molecules that are quite differentin shape. A technique, which paradoxically facilitatescomparison, is to reduce the dimensionality of the MEPs byrecourse to Kohonen Neural Network transforms [114]. Tovisually improve the characteristic features of a giventransform, each is surround by eight replicas. The firstindications are that such 3 x 3 tilings of active trioxanes,such as artemisinin, give continuous strips of charge,whereas inactive peroxides give broken strips. Morecalculations on a bigger sample base need to be undertakento confirm the aforementioned early conclusions.

CONCLUSION AND PROSPECTS

It is seen from the preceding discussion that the evidencefor the involvement of carbon radicals produced from aperoxidic precursor by heme is completely established.Subsequently, the killing of the parasite within its foodvacuole by alkylation is a logical and convincing sequel.However, the identity and characterization of the alkylatedparasite protein needs to be elucidated to fully confirm thereality of this crucial step. At the same time, suggestionsthat the death of the parasite may occur by oxygen atomtransfer or by the action of an oxy electrophilic speciesshould be clarified. As alkylation and oxygenation arechemically different they should be easily distinguishable.

The finding that artemether and arteether are neurotoxic athigh dose in animal models is probably due to alkylation byradicals, namely, by the same mechanism that works forkilling the parasite [115]. Similarly, the iron-triggeredalkylation mechanism which characterizes antimalarialtrioxanes and peroxides indicates that they would find use asantitumor agents as well [116].

There is no doubt that the conventional antimalarialremedies based on quinoline derivatives have outlived theirusefulness. In view of the increase of multi-drug-resistantfalciparum malaria, new, effective drugs are urgently needed.Experience has shown that reformulations of old drugs arenot going to provide an adequate response to the growingmenace. A new antimalarial must be as simple in structureas aspirin or paracetamol, taken as a tablet by mouth, and ascheap and safe to manufacture on the ton scale. The synthetictrioxane 53 is a prime candidate, fitting the aforementionedcriteria. Nonetheless, the design of other effective simpleperoxides, bearing neutral water soluble groups such ashydroxyl substituents, is now entirely feasible in the light of


the knowledge accumulated from mechanistic studies and thesystematic preparation and testing of many trioxanes andperoxides of varied structure. For example, easily accessiblealkyl and hydroxyalkyl analogues of 1-methylcyclopropylhydroperoxide, as well as other variations on the basicstructure, offer promise and should be prepared and tested.

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