Identification of a 1,2,4,5-tetraoxane antimalarial drug-development candidate (RKA 182) with superior properties to the semisynthetic artemisinins

Article abstract of DOI:10.1002/anie.201001026

(Figure Presented) Fighting drug resistence: From a library of over 150 1,2,4,5-tetraoxanes, the candidate RKA182 was selected for preclinical development as an antimalarial agent. RKA182 has outstanding in vitro activity against resistant strains of P. falciparum and retains this level of activity against southeast asian isolates that failed artemisinin-based combination therapy.

Full text of DOI:10.1002/anie.201001026

Angewandte
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DOI: 10.1002/anie.201001026
Antimalarial Agents
Identification of a 1,2,4,5-Tetraoxane Antimalarial Drug-Development
Candidate (RKA182) with Superior Properties to the Semisynthetic
Artemisinins
Paul M. OꢀNeill,* Richard K. Amewu, Gemma L. Nixon, Fatima Bousejra ElGarah,
Mathirut Mungthin, James Chadwick, Alison E. Shone, Livia Vivas, Hollie Lander,
Victoria Barton, Sant Muangnoicharoen, Patrick G. Bray, Jill Davies, B. Kevin Park,
Sergio Wittlin, Reto Brun, Michael Preschel, Kesheng Zhang, and Stephen A. Ward
Artemisinin (1) is an extract of the Chinese wormwood
Artemisia annua and has been used since ancient times to
treat malaria.[1] Today, semisynthetic derivatives artesunate
(2) and artemether (3) are used clinically in drug combina-
tions (ACT; artemisinin-based combination therapy).[2] How-
ever, first-generation analogues (e.g. 2 and 3) have a limited
availability,[3] high cost,[4] and poor oral bioavailability
(Scheme 1a).[5] In addition to these drawbacks there have
been recent reports of high failure rates associated with ACTs
suggesting the possibility of clinical artemisinin resistance
along the Thai–Cambodian border.[6] In the light of these
observations there is an urgent need to develop alternative
endoperoxide-based therapies.[7]
The crucial structural functionality within artemisinin and
synthetic 1,2,4-trioxanes[8] is the endoperoxide bridge.
Recently a series of molecules based on an ozonide structure
were developed from which the candidate OZ277[9] was
shown to have impressive antimalarial activity profiles in vitro
and in rodent models of malaria. However, the recent
Scheme 1. a) Artemisinin and its semisynthetic analogues. b) Compar-
ison of tetraoxanes with trioxolane-based antimalarials.
[*] Prof. P. M. O’Neill, Dr. R. K. Amewu, F. Bousejra ElGarah,
Dr. J. Chadwick, Dr. V. Barton
Department of Chemistry, University of Liverpool
development of OZ277 has been hampered as this molecule
Liverpool, L69 7ZD (UK)
was found to be unstable in the plasma of malaria patients
E-mail: pmoneill@liverpool.ac.uk
during a phase II dose-ranging study.[10a,b]
Prof. P. M. O’Neill, Dr. J. Chadwick, Prof. B. K. Park
MRC Centre for Drug Safety Science, Department of Pharmacology
University of Liverpool, Liverpool (UK)
In our hands, studies of endoperoxide stability have shown
that 1,2,4,5-tetraoxanes[11a] (e.g. 6) have significantly higher
stability[11b] than their 1,2,4-trioxolane (4) or 1,2,4-trioxane
M. Mungthin
Department of Parasitology
counterparts.[11b] To exemplify further the chemical and
Phramongkutklao College of Medicine
Bangkok (Thailand)
biological differences between these two heterocycles it has
been noted that the simple dispiro-1,2,4-trioxolane 4 is
antimalarially inactive and unstable whereas the close chemi-
cally stable tetraoxane analogue 6 expresses antimalarial
activity in the nanomolar range (IC50 = 25 nm) (Scheme 1b).
For good levels of antimalarial activity in the ozonide series,
fusion of the 1,2,4-trioxolane ring system to an adamantane
Dr. G. L. Nixon, Dr. A. E. Shone, Dr. S. Muangnoicharoen,
Dr. P. G. Bray, J. Davies, Prof. S. A. Ward
Liverpool School of Tropical Medicine, Liverpool (UK)
Dr. S. Wittlin, Prof. R. Brun
Swiss Tropical and Public Health Institute, Parasite Chemotherapy
Basel (Switzerland)
core (see for example 5, Scheme 1) was found to be
Dr. L. Vivas, H. Lander
Department of Infectious and Tropical Disease
essential.[9] Given the foregoing observations we reasoned
that similar substitution of the cyclohexyl group in antimala-
rially active analogue 6 would generate a new series of
molecules with improved stability profiles and improved oral
and pharmacokinetic profiles through optimization of the side
chain in the generic structure 7 (R = polar water-solubilizing
London School of Hygiene and Tropical Medicine, London (UK)
Dr. M. Preschel, Dr. K. Zhang
Carbogen AMCIS, Hunzenschwil (Switzerland)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201001026.
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group). Identification of the optimal, metabolically stable
hit series 1 this template was selected over series 2 for lead
optimization which ultimately led to the synthesis, profiling,
and selection of RKA182 (15; vide infra) as the development
candidate.
polar side chain to counterbalance the lipophilicity of the
adamantane functional group was the primary focus of the
medicinal chemistry optimization.
In order to candidate-select a 1,2,4,5-tetraoxane we set a
rigorous target product profile from the onset of our
medicinal chemistry optimization, and for the candidate
selection of RKA182 (15) over 150 novel 1,2,4,5-tetraoxanes
were synthesized and screened from two independent hit
series (Scheme 2). After extensive in vitro, in vivo, and
DMPK (drug metabolism and pharmacokinetics) studies on
The synthesis of lead tetraoxanes (hit series 1) is depicted
in Scheme 3. The synthetic route is high-yielding, comprises
only five steps, and is divergent in the last step making
expedient parallel synthesis possible (see the Supporting
Information).[12a] In vitro and in vivo data of the most potent
compounds synthesized in hit series 1 can be seen in Table 1.
These molecules exhibit an IC50 of less than 6 nm against both
chloroquine-sensitive 3D7 and chloroquine-resistant K1
strains of P. falciparum with the lowest IC50 being 0.8 nm
(Table S1 in the Supporting Information).[14] Tetraoxane
analogues also have ED50/ED90 values of less than 3.5/
9.5 mgkgÀ1, with the lowest values being ED50/ED90 = 0.99/
Scheme 2. Tetraoxane candidate development:[13] SAR trends of novel
1,2,4,5-tetraoxanes and candidate selection of RKA182.
Scheme 3. Synthesis of polar tetraoxane derivatives 12–18.
Table 1: In vitro and in vivo[15] data for tetraoxanes 12–18 and pharmocokinetic parameters after a single intravenous (1 mgkgÀ1) and single oral
(10 mgkgÀ1) administration in the rat.
Compound
IC50 [nm]
% Inhibition
30 mgkgÀ1[a]
ED50
ED90
10 mgkgÀ1 (po)
1/2 [h]
F[b] [%]
[mgkgÀ1
]
[mgkgÀ1
]
t
3D7
1.4Æ0.1
K1
0.9Æ0.2
12
13
14
15
100
100
99.7
100
100
100
100
100
100
100
100
3.47
3.18
3.02
1.82
1.33
2.23
0.99
1.60
3.96
3.80
2
5.40
3.88
9.25
8.38
4.18
5.12
1.41
2.91
11.72
12.24
4.5
1.64Æ0.34
5.89Æ1.12
1.72Æ0.76
3.53Æ1.12
2.38Æ0.90
NC
1.08Æ0.33
1.23Æ0.45
NC
9
11
9
5.2Æ0.5
2.5Æ0.2
4.9Æ1.21
0.8Æ0.2
6.0Æ1.2
1.2Æ1.0
1.2Æ0.8
1.8Æ0.6
7.8Æ0.9
12.5Æ5.6
0.8Æ0.3
2.8Æ1.2
1.9Æ1.9
1.1Æ0.8
1.5Æ1.0
0.9Æ0.7
0.9Æ0.4
1.6Æ0.8
3.2Æ2.3
24
15 (tosylate salt)
16
17
18
artesunate
artemether
chloroquine[c]
38 (42)[d]
36
9
23
NC
1.4[c]
–
ND
–
250.0Æ20.2
[a] Parasitemia was determined by microscopic examination of Giemsa stained blood films taken on day 4. Microscopic counts of blood films from
each mouse were processed using spreadsheet (Microsoft Corp.) and expressed as percentage of inhibition from the arithmetic mean parasitemias of
each group in relation to the untreated group. [b] Indicates F (oral bioavailability in rat) calculated using AUC0–t and actual doses (see the Supporting
Information). [c] Data taken from Vennerstrom et al.[9] NC=not calculable; ND=not determined. [d] Oral bioavailability (8 mgkgÀ1 oral/1 mgkgÀ1 iv)
in mouse (average of two experiments).
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1.41 mgkgÀ1 (tetraoxane 17) when tested in mice infected
with the P. berghei ANKA parasite. The activity of 17, in
terms of ED50 and ED90 in the four-day test,[15] surpasses that
of any synthetic endoperoxide reported in the literature to
date after oral administration. Piperidinyl piperazine-func-
tionalized tetraoxane 15, when formulated as a tosylate salt,
has outstanding in vitro activity (< 1 nm) and in vivo activity
with an ED50/ED90 of 1.33/4.18 mgkgÀ1 which is superior to
artemether and artesunate and comparable to artemisone, the
leading next-generation semisynthetic artemisinin which is
currently undergoing phase II clinical trials.[16] In studies
conducted on mouse survival, mice treated at 3 ꢀ 10 mgkgÀ1
per day with 15 survived 22 days in comparison with only
9 days for artesunate (see the Supporting Information).
Pharmacokinetic parameters and oral bioavailability were
determined after intravenous (1 mgkgÀ1) and oral adminis-
tration (10 mgkgÀ1) in Sprague–Dawley rats (Table 1). The
most significant observation from this study is that tetraox-
anes 15, 16, and 18 have oral bioavailabilities of greater than
20% (Tables S2 and S3 in the Supporting Information).
Taking this information together with the antimalarial activity
data it was clear that although the activity of 12 and 17 in the
four-day test was very good, the poor bioavailability of 17ꢁs
and 12ꢁs complicated in vivo metabolic profile meant that
these molecules were not considered further. Compounds 13
and 18 were ruled out at this point due to relatively poor
bioavailability for the former and a very short half-life
predicted for the latter tetraoxane. Although 16 was more
potent in the mouse survival studies this tetraoxane was also
ruled out due to lower maximum tolerated dose (MTD) in
rats (100 mgkgÀ1 for 16 versus 400 mgkgÀ1 for 15) and lower
overall exposure (AUC) than 15 in rat pharmacokinetic
studies. Based on the outstanding in vitro and in vivo
antimalarial activity and pharmacokinetic profile 15 was
selected as the lead candidate. Formulation work on 15
delivered the compound as a ditosylate salt which had
improved oral bioavailability of 38% in the rat (42% in the
mouse; Table 1 and Table S4 in the Supporting Infomration).
Having selected 15 as the drug candidate a scalable,
industrial synthesis was sought. The industrial synthesis
(Carbogen AMCIS) of 15 comprises only four steps (due to
the synthesis of 8 in one step from direct hydrogenation of
phenol ester 19) involving a single chromatography step and
has a projected low cost of goods (Scheme 4). The key step in
the scale-up of this potentially hazardous chemistry involved
the in situ generation of the keto ester 10 without isolation of
the gem-dihydroperoxide 9 followed by hydrolysis (of the
ester function) to provide the acid 11 in acceptable yields on a
1.2 kg scale. The application of the Dussault procedure
(employing Re2O7 as a mild Lewis acid)[12b] for the key
tetraoxane ring-forming step was also crucial to the scale-up
of this chemistry.
Scheme 4. Industrial synthesis of tetraoxane 15 (RKA182).
Figure 1. IC50 data against Cambodia–Thai patient isolates for 15
compared with other antimalarials. Blue: mefloquine; red: artesunate;
green: artemisinin; purple: RKA182.
the true test of activity versus artemisinin-resistant strains can
only be assessed in a dynamic human model of malaria due to
lack of stable inherent resistance phenotype in vitro.) To
assess the speed of action a single oral dose of 30 mgkgÀ1 15
was administered to mice infected with 4 ꢀ 106 P. berghei
ANKA infected red cells two days postinfection (Figure 2).
Parasitemias rapidly decreased to undetectable levels 24 h
after treatment with 15, whereas treatment with the same
dose of artesunate (ASN) reduced parasitemias up to 5% 8 h
posttreatment increasing rapidly thereafter.
To demonstrate the remarkable stability of tetraoxane 15
in both noninfected and infected red blood cells, in vitro
studies were carried out to assess the percentage recovery of
drug at set time points in noninfected and infected blood
(Figure 3). It can be seen from this data that OZ277 rapidly
degrades in infected blood cells giving no recovery of drug
after only 35 min. In sharp contrast, RKA182 (15) shows 79%
recovery after 4 h in infected blood. In vivo pharmacokinetic
analysis was also performed to demonstrate the impact of
As a part of our preclinical development 15 was also
tested against eleven different southeast asian isolates from
patients who had failed ACT combination chemotherapy
(Figure 1), and RKA182 (15) clearly demonstrated superi-
ority over mefloquine, artesunate, and artemisinin with all
measured IC50 values below 5 nm. (Whilst the five- to tenfold
increase in potency versus artemisinins for 15 is reassuring,
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Figure 2. Speed of action of 15 (RKA182) following a single oral dose
of 30 mgkgÀ1. Red: no drug; green: ASN 30 mgkgÀ1; blue: RKA182
30 mgkgÀ1
.
Scheme 5. TEMPO spin-trapping of C-radical intermediates generated
following 12–FeII activation. R=N(CH2CH2)2NCH2iPr.
Figure 3. a) Stability of 15 in noninfected and infected red blood cells
in comparison with OZ277; blue diamonds: RKA182 noninfected
RBC; red squares: RKA182 2% infected RBC; light gray ꢀ: OZ277
noninfected RBC; green triangles: OZ277 1% infected RBC. b) Phar-
macokinetic parameters in infected versus noninfected mice.
infection on the exposure profile of 15 and the data (Fig-
ure 3b) clearly demonstrates equivalent exposure (AUC) in
both infected and noninfected mice. Taken together, these
data suggests 15 is more stable than OZ277 in which malaria
infection was associated with a significant reduction in drug
plasma concentrations in phase II trials.[17]
Scheme 6. Heme alkylation by tetraoxane 12.
In order to characterize the potential mediators of the
antimalarial activity of RKA182 we performed mechanistic
studies with iron(II) bromide in THF in the presence of the
spin-trapping agent 2,2,6,6-tetramethyl-1-piperidine-1-oxyl
(TEMPO). From these studies, we were able to intercept
both the primary and secondary carbon centered radicals to
produce two TEMPO adducts A and B (Scheme 5).
The behavior of the tetraoxanes reported here is distinct
from 1,2,4-trioxolanes since only the secondary carbon
centered radical species has been characterized from
OZ277 and other 1,2,4-trioxolanes.[9] Since heme alkylation
is believed to play a vital role in the mechanism of action of
endoperoxide antimalarials[17] (Scheme 6) we examined the
reactivity of 12 with ferrous heme. LC–MS analysis confirmed
an m/z 782.3 Da for three adducts (maximum absorption of
the Soret band at 430 nm) that result from the covalent
bonding of the tetraoxane-derived secondary carbon centered
radical and the heme porphyrin. This process may play an
important role in the molecular mechanism of action of these
derivatives.
In conclusion, we have identified the first water-soluble
1,2,4,5-tetraoxane drug candidate that has outstanding anti-
malarial activity, stability, low toxicity (see the Supporting
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K. McIntosh, M. Padmanilayam, J. Santo Tomas, C. Scheurer, B.
Scorneaux, Y. Tang, H. Urwyler, S. Wittlin, W. N. Charman,
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Information), and ADME properties (absorption, distribu-
tion, metabolism, and excretion) that overcome most of the
problems encountered previously with the synthetic and
semisynthetic antimalarial endoperoxide drugs that have
progressed into preclinical development. This work firmly
establishes the potential of the tetraoxane pharmacophore to
provide the next generation of synthetic drugs for deployment
in the control and eradication of malaria as a component of
combination chemotherapy.
[10] a) P. Olliaro, T. N. C. Wells, Clin. Pharmacol. Ther. 2009, 85,
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[11] For details of amide and amine 1,2,4,5-tetraoxacyclohexanes and
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Received: February 18, 2010
Revised: April 7, 2010
Published online: July 13, 2010
Keywords: antimalarial agents · drug development ·
.
endoperoxides · medicinal chemistry · tetraoxanes
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