CHAPTER 2

BACKGROUND OF THE STUDY AND PROJECT AIMS

2.1 Background of the study

The first generation derivatives of ART are some successful drugs that have been

found effective against multi-drug resistant P. falciparum malaria. These drugs are

used to treat uncomplicated and complicated (P. falciparum and P. vivax) cases of

resistant malaria. They represent a novel class of antimalarial drugs and their

combination therapy is considered first-line treatment against resistant malaria. The

semi-synthetic derivatives of ART were developed in order to improve solubility and

pharmacokinetics of the parent, naturally occurring ART molecule. They are usually

administered by oral or parenteral route. Artemether and arteether are more potent than

ART, but have shorter biological half-lives, and may produce fatal toxicities such as

hematopoetic, cardiac and CNS toxicities. The sodium salt of artesunic acid (water-

soluble derivative of ART) is capable of rapidly reducing parasitemia in severe

complicated malaria (e.g. cerebral malaria). But, because of the high recrudescence

rates (or disease recurrence due to poor pharmacokinetics), sodium artesunate is

usually given in combination with slowly eliminating potent drugs from different

antimalarial classes like CQ, MQ, AQ, SP and pyronaridine. Artemether is usually

given in combination with lumefantrine, and DHA is administered in combination

with piperaquine. The combination therapy of ARTs increases their efficiency,

particularly in terms of overcoming the development of resistance. Sodium artelinate,

65
the sodium salt of artelinic acid, is sometimes used in place of artesunate to overcome

the hydrolytic instability experienced with the later. In comparison to oil soluble

analogues (artemether and arteether), sodium artelinate is not only more stable in

aqueous solution but also has a much longer biological half-life (1.5-3.0 h) with less

CNS toxicity. The water soluble ART derivatives have, therefore, rapid onset of action

which makes them especially effective against severe malaria. At the same time, rapid

disappearance from the blood may be a key reason behind their slow development of

resistance against malaria parasites. The problems associated with ART derivatives

(pharmacokinetic and toxicity issues) prompted investigations into the development of

newer semi-synthetic derivatives of ART and synthetic endoperoxides as potent

antimalarials with better physiochemical, pharmacological and toxicological

properties1-3.

2.1.1 Development of newer generation peroxide antimalarials

Hundreds of natural and synthetic peroxide compounds were developed from the

first generation derivatives of ART. The second generation peroxide compounds

possess good clinical efficacy against drug-resistant strains of P. falciparum, and also

have better metabolic stability than first generation analogues. The second generation

drugs include C(10) aryloxy derivatives (a-d) of artelinic acid, deoxyartemisinin (e)

and its derivatives, C(9)-substituted compounds (f-h), and C(10) heterocyclic

derivatives (i, j)4 (Figure 2.1).

66
 H H H

O O O
H H H
O O O
O O O
H H H
O O O
R

O R
R

R= CH(p-NO2 C6H4)CO2Et, a R= CH3, e R= furan-2-yl, i

R= CHNH2CH2CO2Et, b R= (CH2)3,CH3, f R= indole-2-yl, j
R= CH(p-CF3C6H4), c R= (CH2)3,C6H5, g
R= CF3, d R= (CH2)3, p-ClC6H5, h

Figure 2.1: Some second generation derivatives of ART

Newer semi-synthetic and synthetic endoperoxides (a-p) are being developed

(Figure 2.2). C(10)-ether derivatives (g, h) and C(10)-alkylamino analogues (i-o) of

ART are also included in this category. Some novel candidates are arteflene (b),

artemisinic acid (c), artemisitene (d), SM905 (e) arterolane (OZ277, f), artemeside (i),

artemisone (BAY449585, l), RW1777 (n), and artefenomel (OZ439, p)5,6. These drugs

are highly effective against P. falciparum resistant severe malaria like cerebral

malaria. Arteflene is a stable, safe and effective drug, but it shows a certain degree of

recrudescence rate. It is a synthetic derivative obtained from a natural precursor

Yingzhaosu A, a. The suppressive activity of arteflene is comparable to CQ, but

superior to artesunate. Artemisone is 10 times more potent than artesunate 7.

67
 O
H
O H
O
O
O O
O
O
O H
H
OH HO O

OH
O
O
F3C CF3
a b c d

H
H
O O
O
O H
O
O O NH O
O
H O NH2 H
O
O
OH
H O
O N R
R=CH2CH2C6H4C6H5, g
e f
R= COC6H4C6H5, h

H
H O O
O
O H
O O O
O O
O
H H
O
O
N N
N
X= NH, i R= CH2-Ph, m p
R= CONH-Ph, n O
X= O, j N
X X= S, k R= pyrimidin-2-yl, o
X= SO2, l R

Figure 2.2: Some newer synthetic peroxide compounds

Arterolane (OZ277) is a synthetic trioxolane (ozonide) analogue of ART. It is

orally active and rapidly acting blood schizonticide against all blood stages of P.

falciparum without any effect on liver stages and acts by inhibition of Pf ATP6, a

sarcoplasmic endoplasmic reticulum calcium ATPase encoded by P. falciparum. It has

been found effective against CQ-resistant strains of P. falciparum. Arterolane was

tested in combination with piperaquine in areas of ART resistance (under TRAC II).

68
The discovery of OZZ277 was continued with the development of OZ439

(artefenomel) which is a newer generation synthetic trioxolane antimalarial compound.

In vitro data on the early ring stages confirmed that OZ439 is active on ART-resistant

strains of P. falciparum. It also showed good efficacy against resistant malaria when

tested in combination with piperaquine. Another interesting compound is BAY449585

(artemisone) which cures malaria in Aotus monkeys infected with P. falciparum with a

single dose at 10 mg/kg body weight when combined with mefloquine (5 mg/kg).

Artemisone also showed resistant preventing activity against P. falciparum when used

in combination with piperaquine5-7.

Newer endoperoxide drugs also include several 11-azaartemisinins and fluorinated

analogues (Figure 2.3). Aza dvts. (a-f) are more stable anactive than ART. Fluorinated

analogues have good oral bioavailability (due to increased lipophilicity) and high

metabolic stability. Compound g is a fluorinated derivative of DHA and compounds,

h-j are the 10-carba analogues of ART4,8.

O O O O
O O O O
O O O O
H H H H
N O O R O
R
O R1 R2
CF3
F F
R= H, a R1= H, R2= CF3, g, i j
R= NH2, b R1= OH, R2= CF3, h
R= OH, c
R= CH2CH2OH, d
R= CH2CHO, e
R= CH2-(pyridin-2-yl), f

Figure 2.3: Newer aza and fluorinated endoperoxides

69
2.1.2 Developmeent of 1,2,4-Trioxane-based and related peroxide antimalarials

 1,2,4-Trioxane-based antimalarials

Among the 1,2,4-trioxane derivatives (a-l) depicted below (Figure 2.4), the 2-

adamantyl derivatives (j-l) have been reported to be the most active compounds. These

compounds reduce parasitaemia to an appreciable extent both in vitro and in vivo4,9.

O R R H
H
H
O O H
O H
O O
R O O
O O H3CO
OCH3
c R= alkyl
a R=H/aryl/heteroaryl b R= alkyl OH

O O O O O O OH
O NHR

R O R O R O R'

f
d R= aryl e R= aryl
R=aryl, R'= H/CH3
Cl '

O O OH O O
CCO2C2H5 R O R2
R O R O
CH2CO2C2H5 R'
O2S O O R1
g R= aryl h R= aryl, R'= H/CH3 i
R=aryl, R1= R2=alkyl

O O O O O O

HO
R O O O
R
R
k R= CH3/
j R= cycloalkyl/aryl cyclopropyl/n-Pr l R= aryl

Figure 2.4: Some 1,2,4-trioxane-based antimalarials

 1,2,4-Trioxolane-based antimalarials

The derivatives of 1,2,4-trioxolane, the ozonides, (a-l) comprises another important

class of organic peroxides. They exhibit excellent activity against malaria parasite like

70
structurally similar 1,2,4-trioxanes. Their activities are comparable to artesunate and

artemether, both in vivo and in vitro, but with improved pharmacokinetic (oral

bioavailability, half-life etc.) properties4,10,11 (Figure 2.5).

O O O O O O

NH2 NH2
O O N
O NH2
O
b c
a
O O
O O O O
O O
O N
O O N S
OEt O
d CO2H e f
O O
O O
O O
O
O O NH NH2
OH
O
g h i

O O N H2N
O O
O O
N
O NH O
O N
O
N
j k l
N

Figure 2.5: Some 1,2,4-trioxolane-based antimalarials

 1,2,4,5-Tetraoxane-based antimalarials

Derivatives of 3,6-Substituted 1,2,4,5-tetraoxacyclohexane are known as 1,2,4,5-

tetraoxanes. A group of 3,6-dispiro-1,2,4,5-tetraoxanes (a-i) (Figure 2.6) have been

reported to exhibit pronounced antimalarial activity against both CQ-sensitive and -

resistant strains of P. falciparum. The compounds (e-i) with adamantly substituents

possess superior antimalarial effectiveness as compared to other compounds.

Tetraoxone h (RKA182) and tetraoxane i (TDDE209) are the next-generation potent

antimalarial compounds which are under clinical phase of development. The role of

71
adamantyl substituent is that it stabilizes the structure and improves the antimalarial

activity. Some derivatives (e) that contain polar sulphonamide groups at one end and a

highly lipophilic adamantly group at the other end also possess potent antimalarial

activity4,12-19.

O O O O O
O O

O O O O
O O R R
R
O
a R=NHCH2CH2N(CH3)2 b R= OH/NH2/NHPr/NHCH2Ph c

O O
O O O O O O

O O O O O O
R

R= OCH3/NH2/NHCH2/CH2N(CH3)2 f
e
d

O O O
O O O R
O O
N O O
N S R
O O R
O O
O
H N N N
R=Et/Pr/Cyclopropyl N f O
R= h
e R=
O
N O g
N F
i

Figure 2.6: Some 1,2,3,4-tetraoxane-based antimalarial agents

2.1.3 Hybrid peroxide antimalarials

To overcome the problem of drug resistance, combination therapy has been used

for over years, but with limited success, and therefore, the concept of hybrid molecules

has recently been introduced for the development of new drugs for treating resistant

malaria. In hybrid molecules, two pharmacophores are covalently fused together to a

single molecular entity (hybrid molecule) that exhibit dual modes of action which

might be beneficial in the treatment of resistant malaria because of their high

antimalarial efficacy and resistant preventing action20-25. In hybrid endoperoxides, also

known as chimeric peroxides, the quinoline and peroxide moieties are the contributing

72
pharmacophoric groups. Such hybrid analogues are attributed to have the property of

resistant preventing action with some important advantages like improved

pharmacokinetic properties over traditional drugs such as QN, CQ, MQ, ART and also

newer trioxanes, ozonides and tetraoxanes4,26. Endoperoxide-based hybrid compounds

therefore, represent an attractive alternative therapeutic approach to ACTs.

Some ART endoperoxide-based hybrid compounds are ART-QN (a), ART-MQ (b),

ART-9-aminoacridine (c), ART-vinylsulfones (d) and ART-4-aminoquinolines (e)

(Figure 2.7). They possess higher activity than ART and QN or MQ or CQ4,27,28.

H H
O O
H H
O O
O O
O O
O O CF3
O
O O
N O N
H
HO O

O
N CF3
CF3

a b

H H H
O
H
O
H
O R3 O H
O
O O O S O O
O
n=2/3/4/5
O O R2
H
O N O N O Cl
N H H
HN nN H R1 N N
O O
H n
Cl e N
c n=2/3 d

Figure 2.7: ART-based hybrids

Based on the ‘covalent bitherapy’ approach, hybrids of 1,2,4-trioxane and

aminoquinoline (AQ), named as trioxaquines have been developed. A trioxaquine

molecule is constituted as follows: Quinoline-Linker-Trioxane. The 1,2,4-trioxane

73
entity of trioxaquine is responsible for the activity of ART, whereas, the

aminoquinoline entity is necessary for the accumulation (pharmacokinetic property) of

the drug within the parasite. The antimalarial activities of trioxaquines (a-h) depicted

in Figure 2.8 are significantly higher than the activity of each of the individual

fragment, indicating a synergistic action of the trioxaquine and aminoquinoline

components4,29-34.

Ph

O O
Cl N H Ph
H O
O N O
CH3 H O
O HN HN nN
O N H
HN HN H O O CH3
H n= 2/3/4
b Cl N c
a
H
N
O Ar
O O
O
O O HN
O O
HN nN O
HN nN H
H
Cl N
n=2/3/4 n=2/3/4, O O
Cl N Ar= phenyl/biphenyl
Cl N f R1
e O O
d Ph
R
H O
O N
Ph HN H O O
O O O
O O
HN nN Fe
H Cl N
n=2
R1=R2=R3=alkyl
Cl N g R2
h i

Figure 2.8: Trioxquines and other trioxane-based hybrids

Compounds a, c-g are 1,2,4-trioxaquine-4-aminoquinoline hybrids, b is a hybrid of

1,2,4-trioxaquine-8-aminoquinoline and compound h is a 1,2,4-trioxaquine which is a

ferroquine-trioxane hybrid (Trioxaferroquine, SSR97193). This hybrid compound is

under phase II of clinical development that showed antiparasitic efficacy against P.

falciparum in blood stage malaria infection with well defined safety and tolerability.

74
Some of these hybrid analogues show excellent oral effectiveness, but some molecules

suffer from limitations such as poor stability and poor solubility. Trioxaquines are

regarded as first generation endoperoxide-based hybrids4,33. Recently, novel coumarin-

trioxane hybrids (i) have been reported to possess good antimalarial activity both in

vitro and in vivo against CQ-sensitive strain of P. falciparum35.

In recent literature, second-generation hybrids of endoperoxide, called

trioxalaquines have been reported. Trioxalaquine hybrids are adamantylated

derivatives of 4- aminoquinoline (a, b, e) or 8- aminoquinoline (c) or aminoacridine

(d) moiety and simply nonadmantylated vinylsulfonylurea conjugates (f) of 1,2,4-

trioxolane1,27,36 (Figure 2.9).

Cl

Cl
O O H O O
N
O n N N N
n=2/3 O
N
Cl
a b
Cl
O O H O O
N
N N N
O n O
N
n=2/3
O O
c d
HN
NH O O R2 O
H O
O O N S
O X
R R= R3
O O R1
Cl
X= CONH
e
f

Figure 2.9: Trioxalaquines and trioxolane-vinylsulfone hybrids

In trioxalaquines, the quinoline moiety, mostly 4- aminoquinoline is conjugates

with a 1,2,4-trioxolane moiety through a functional linker (Trioxalaquines =

75
Quinoline-Linker-Trioxolane). These hybrid analogues exhibit potent antimalarial

activity including gametocytes.

The 1,2,4,5-tetraoxane-based hybrids (Figure 2.10) include tertraoxane-4-

aminoquinoline known as tetraoxaquines, (a, b) tetraoxane-vinylsulfone analogues (c)

and adamantylated tetraoxane-vinylsulfone analogues (d). They are as active as ART

or MQ against the P. falciparum strain and possess higher activity than CQ against

CQ-resistant strains27,36.

O HN NH
O O O O
R=
O O R O O R
Cl
a b

O O R2 O O R2 O
H O O H O
N S N S
X X
O O R3 O O R3
O R1 O R1

X= CONH X= CONH/ CH2CONH/ NH/ CH2NH

c d

Figure 2.10: Tetraoxaquines and related hybrid analogues

2.2 Rationale of investigation

2.2.1 Key problem and research gaps

During the past few decades, the emergence of multi-drug resistant strains of P.

falciparum has become an increasingly serious concern in malaria control and

prevention worldwide. This has limited the use of currently available drugs, especially

those in the aminoquinoline group (chloroquine, amodiaquine, mefloquine) including

non-quinolines like antifolates, atovaquone and antibiotics, and the novel ART

derivatives, in the treatment of resistant malaria6,7. WHO has recommended ACTs for

the treatment of multi-drug resistant malaria caused by P. falciparum. However, some

76
ACTs (for example, artemether + lumefantrine, artesunate + mefloquine,

dihydroartemisinin + piperaquine) have been reported to develop resistance against P.

falciparum in South-East Asia including Western Cambodia, Vietnam, Thailand,

Eastern Myanmar and Northern Cambodia37,38. Besides resistance problem, bio-

pharmaceutical (unsatisfactory pharmacokinetics due to poor lipid/water partitioning

behavior, poor water solubility/drug absorption, inadequate bioavailability and short

half-life), chemical (limited availability, purity and cost), toxicity (hematopoetic,

cardiac and CNS), and treatment (non-compliance with repeated regimens and

recrudescence/disease recurrence) issues limited the therapeutic potential of semi-

synthetic derivatives of ART2,4,36. Because of the above challenging issues, the clinical

effectiveness of ACTs used in the treatment of resistant malaria has been seriously

impaired. Therefore, there is an urgent need to develop novel and affordable

antimalarial agents as alternative to ART-based drugs or ACTs that would be effective

against resistant P. falciparum malaria.

2.2.2 Design considerations and research hypothesis

According to WHO, ACTs are considered as the frontline treatment of multidrug-

resistant P. falciparum malaria7. The clinical importance of ACTs is attributed to be

mainly due to the high antimalarial efficacy of ART-derived drugs (artemether,

artesunate, dihydroartemisinin). The second non-artemisinin component (mefloquine,

amodiaquine, lumefantrine) is added to combination regimens to circumvent the

problem of drug resistance37,38. Owing to excellent antiparasitic action of ART-based

drugs, research into synthetic endoperoxides (1,2,4-trioxanes, 1,2,4-trioxolanes,

1,2,4,5-tetraoxanes and their hybrid analogues) based on the endoperoxide scaffold of

77
ART have gained significant interest, in recent years, for the development of

antimalarial drugs that are structurally simpler, synthetically accessible and at the

same time potent and cost-effective. Pharmacodynamic study suggests that the

endoperoxide component (i.e., 1,2,4-trioxane ring) is the key pharmacophore of ART

and its semi-synthetic analogues5. Although the 1,2,4-trioxane ring system is the

critical pharmacophoric moiety of ART, its presence alone is insufficient to bring

about desired antimalarial efficacy as most synthetic 1,2,4-trioxanes are less active

than ART and DHA40.

Several design strategies have already been initiated by MMV (Medicines for

Malaria Venture) program for the development of effective, safe and affordable

synthetic peroxides as potent antimalarial drugs. As a results many endoperoxide-

based antimalarial compounds have been developed that possess better

pharmacokinetics and antimalarial properties than ART-derived drugs. Some

endoperoxide-based molecules that are currently under clinical phase of development

include arterolane (OZ277), artefenomel (OZ439) and artemisone (BAY449585),

Tetraoxanes (TDDE209, RKA182), and trioxaferroquine (SSR97193)41. In view of

above considerations, developing newer series of 1,2,4-trioxane derivatives is a

rationale need for investigating novel peroxide-based antimalarial compounds as an

effective and alternative approach for the treatment of resistant malaria. It would thus

offer simple peroxide molecules based on single drug conjugate structure that may be

used in single drug conjugate therapy rather than hybrid drug analogues or drug

combinations with high antimalarial efficacy against resistant malaria.

78
2.3 Project aims

Based on background information obtained from recent literature reports, our study

was aimed at developing 1,2,4-trioxane derivatives as novel antimalarial agents

against resistant P. falciparum malaria. The objectives of the project are summarized

below:

 Design, modeling and in silico screening of newer series of 1,2,4-trioxane

derivatives

 Synthesis and characterization of 1,2,4-trioxane derivatives

 Antimalarial screening (in vitro and in vivo) of synthesized trioxane derivatives

 Study of structure-activity relationship (SAR) quantitative structure-activity

relationship (QSAR), docking and mode of action of potent 1,2,4-trioxane

derivatives for antimalarial activity

2.4 Expected outcome

The 1,2,4-trioxane derivatives proposed to be developed are expected to be potent

antimalarial agents against resistant P. falciparum malaria. The novelty of potent

trioxane-based antimalarial compounds active against resistant malaria may be

patentable. Novel trioxane molecules may not only be an alternative, but effective

approach in the treatment of malaria with resistant preventing action and ready

availability at affordable cost. Trioxane compounds may represent as lead molecules

for further modification to more potent antimalarial agents.

79
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