Endoperoxide carbonyl falcipain 2/3 inhibitor hybrids: Toward combination chemotherapy of malaria through a single chemical entity

Article abstract of DOI:10.1021/jm1009567

We extend our approach of combination chemotherapy through a single prodrug entity (O'Neill et al. Angew. Chem., Int. Ed. 2004, 43, 4193) by using a 1,2,4-trioxolane as a protease inhibitor carbonyl-masking group. These molecules are designed to target the malaria parasite through two independent mechanisms of action: iron(II) decomposition releases the carbonyl protease inhibitor and potentially cytotoxic C-radical species in tandem. Using a proposed target "heme", we also demonstrate heme alkylation/carbonyl inhibitor release and quantitatively measure endoperoxide turnover in parasitized red blood cells.

Full text of DOI:10.1021/jm1009567

8202 J. Med. Chem. 2010, 53, 8202–8206
DOI: 10.1021/jm1009567
Endoperoxide Carbonyl Falcipain 2/3 Inhibitor Hybrids: Toward Combination
Chemotherapy of Malaria through a Single Chemical Entity
Peter Gibbons,† Edite Verissimo,‡ Nuna C. Araujo,‡ Victoria Barton,† Gemma L. Nixon,§ Richard K. Amewu,† James Chadwick,†,
Paul A. Stocks,§ Giancarlo A. Biagini,§ Abhishek Srivastava,§ Philip J. Rosenthal,^ Jiri Gut,^ Rita C. Guedes,# Rui Moreira,#
Raman Sharma,†,§ Neil Berry,† M. Lurdes S. Cristiano,‡ Alison E. Shone,§ Stephen A. Ward,§ and Paul M. O’Neill*,†,
†Department of Chemistry, University of Liverpool, Liverpool, L69 7ZD, U.K., ‡Department of Chemistry, Biochemistry and Pharmacy, and
CCMAR, University of the Algarve, Campus de Gambelas, Faro, 8005-039, Portugal, §Liverpool School of Tropical Medicine, Pembroke Place,
Liverpool, L3 5QA, U.K., MRC Centre for Drug Safety Science, Department of Pharmacology, University of Liverpool, Liverpool,
L69 3GE, U.K., ^Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California 94143-0811,
United States , and #iMed.UL, Faculty of Pharmacy, University of Lisbon, Av Prof. Gama Pinto, 1649-003, Portugal
Received July 27, 2010
We extend our approach of combination chemotherapy through a single prodrug entity (O’Neill et al.
Angew. Chem., Int. Ed. 2004, 43, 4193) by using a 1,2,4-trioxolane as a protease inhibitor carbonyl-
masking group. These molecules are designed to target the malaria parasite through two independent
mechanisms of action: iron(II) decomposition releases the carbonyl protease inhibitor and potentially
cytotoxic C-radical species in tandem. Using a proposed target “heme”, we also demonstrate heme
alkylation/carbonyl inhibitor release and quantitatively measure endoperoxide turnover in parasitized
red blood cells.
Introduction
degrade by a designed cascade to produce in a concomitant
fashion a potentially toxic carbon radical (2a) and a chalcone
molecule (2b), an entity capable of inhibiting the malaria
trophozoite cysteine protease falcipain 2.7
As a tractable solution to Plasmodium falciparum resistance
development,1 we have developed a novel protease inhibitor
peroxide hybrid strategy that encapsulates and refines the
concept of combination chemotherapy. Our approach is based
on the ability of Fe(II) or heme to selectively cleave the peroxide
bridge of the endoperoxide class of drug, a process that appears
to be restricted to the malaria parasite. We have designed
synthetic routes to produce antimalarial drug hybrids that,
as a function of heme (or ferrous iron dependent) peroxide
cleavage, will liberate not only free radicals (proposed arte-
misinin type of action)2 but also a second antimalarial drug
with an independent mechanism of action.
Although the specific mechanism of action of the endoper-
oxide antimalarials remains controversial,3-5 we have based
our hybrid strategy on previous findings using biomimetic
Fe(II) chemistry and electron paramagneticresonance(EPRa)
spectroscopy. We have provided evidence that the Roche anti-
malarial arteflene releases a potentially toxic carbon centered
radical in tandem with an electrophilic enone.6 On the basis of
a mechanistic understanding of this process, we developed
chemistry for the preparation of a new series of endoperoxide
cysteine protease inhibitor (ECPI (1a)) prodrugs, compounds
that have the capacitytoselectively delivertwo toxic entitiesto
the malaria parasite. Specifically, we demonstrated that under
ferrous mediated conditions, our prototype antimalarials (1a)
Strategy
The selective parasite reductive cleavage of the endoper-
oxide prodrug (e.g., 1a) to an enone system and a secondary
C-centered radical might suggest that this approach is rela-
tively limited in the type of inhibitor that can be embedded
within the prodrug. On the contrary, mechanistic studies by
Vennerstrom on the ferrous mediated decompositions of the
1,2,4-trioxolane pharmacophore have revealed that this hetero-
cycle also readily degrades to carbonyl species in tandem with
free radical production (Figure 2A).8 With this knowledge
and as a paradigm for a potentially generic approach to com-
bination chemotherapy, we have focused on peroxides incor-
porating novel peptidic cysteine protease (falcipain 2/3) inhib-
itors, compounds with antimalarial effects at the level of the
parasite digestive vacuole. Falcipain 2 has been selected as
a target, since we have a good working knowledge of the
biochemistry and structure-activity relationships (SAR) for
carbonyl-based inhibitors of this key protease enzyme.9
A key consideration for this strategy is the interaction of the
two components combined within the single chemical entity.
Combining artemisinin with a chalcone derivative indepen-
dently as an artemisinincombination therapy (ACT) resultsin
synergistic or additive interactions suggesting that endoper-
oxides and chalcones can be used together effectively.10 Thus,
the prodrug approach previously explored (Figure 1) has
significant advantages over the use of either drug in combination
or alone, since it allows selective drug delivery (i.e., metabol-
ically stable) to the parasite food vacuole reducing toxicity to
*To whom correspondence should be addressed. Phone: 0151 794
3553. Fax: þ44 (0)151 794 3588. E-mail: pmoneill@liverpool.ac.uk.
a Abbreviations: ALLN, calpain inhibitor I, N-acetyl-Leu-Leu-Norleu-al;
ECPI, endoperoxide cysteine protease inhibitor; ACT, artemisinin
combination therapy; SAR, structure-activity relationship; CP, cysteine
protease;EPR, electron paramagnetic resonance; MRM, multiple reaction
monitoring.
r
pubs.acs.org/jmc
Published on Web 10/27/2010
2010 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8203
ketones areconsiderably morestablethan their aldehydecounter-
parts, and for this reason we also included the cysteine protease
(CP) inhibitors 3c-f as targets (Figure 2).11
Docking studies with the falcipain 2 crystal structure (PDB code
12
2OUL, 2.20 A) were performed for the aldehyde and ketone
˚
based inhibitors, and docking poses are presented in Figure 3.
Each ligand was energy-minimized and then subjected to 10 000
docking runs. The top 10 ranking solutions (i.e., those with the
highest fitness score13) were visually analyzed for (i) the distance
between the potentially reactive carbon atoms (i.e., ketone/alde-
hyde carbonyl and CH2OR) and the sulfur atom of the catalytic
Cys42 residue, (ii) hydrogen bond interactions in the proposed
oxy anion hole formed by the residues Gln36, His174, and Cys42,
and (iii) hydrophobic interactions between the ligand and non-
polar regions of the enzyme surface. Energy minimized structures
place the carbonyl warhead of the inhibitor in the vicinity of
˚
Cys42 residue (<4 A) and the gold scores for 3c-f encouraged
ketone inhibitor synthesis.
Figure 1. Designed ferrous mediated degradation of chalcone prodrug.
Peptides 3c-g were prepared in good overall yields using pre-
viously reported standard peptide coupling techniques. The syn-
thesis proceeded with hydrolysis of dipeptides 5a and 5c to acids
7a and 7b followed by Weinreb amide formation and subsequent
Grignard addition chemistry to deliver 3c-f (Scheme 1B).14
Following optimization studies the target prodrugs (4a-f) werepro-
duced by Griesbaum co-ozonolysis using O-methyl 2-adamantanone
oxime/O3 and the prodrugs were purified by HPLC as an insepa-
rable mixture of 1:1 diastereomers.15
Discussion
Table 1 lists enzyme inhibitory data of all compounds prepared
against recombinant falcipains 2 and 3 and the antimalarial
activity against 3D7 malaria parasite strain.16 It is immedi-
ately clear that the aldehydes 3a and 3b are potent and selective
inhibitors of falcipain 2 with low nanomolar activity. In the
keto series only R-phenoxy analogue 3g expresses nanomolar
activity against falcipains 2 and 3. As noted previously, 3a and
3b also express low nanomolar antimalarial activity versus the
3D7 strain of Plasmodium falciparum; apart from 3g, alkoxy
ketone inhibitors had low activity against both enzymes
(2-21 uM). This is surprising given the fact that (a) these
molecules contain the optimalresiduesLeu andhomophe/Phe
within the structure of the inhibitor and (b) the docking
studies performed with FP2 suggested a good enzyme fit for
this series. The modest activity for 3c-g suggests that the
reactivity of the R-alkoxy group, when compared with the
reactive aldehyde in 3a and 3b, is insufficient to achieve efficient
inactivation of Cys 42 through the usual 1,2-addition process.
The most potent endoperoxides from the series are the alde-
hyde prodrugs 4, 4a, and 4b and ketone prodrugs 4c and 4e.
Trioxolane 4b is active in the low nanomolar region (35 nM)
with 4a active at 55 nM versus the 3D7 strain of Plasmodium
falciparum. The activities of the parent prodrugs 4a and 4b
were significantly less against the recombinant enzyme
(>400 nM). Since there is more than a 400-fold difference
between the inhibitory potency for aldehyde 3a and endoper-
oxide 4a versus falcipain 2, the fact that these molecules express
similar activity versus cultured parasites (27 nM versus 55 nM)
indicates that activation and inhibitor release are contributing
to the observed whole cell parasite growth inhibition for 4a.
That 4c-f express moderate antimalarial activity demon-
strates that molecules containing the privileged 1,2,4-trioxo-
lane heterocycle do not necessarily have potent antimalarial
properties. The fact that 4c-f are not as active as some spiro-
cyclohexyl systems may be again a stability issue (or more
likely, as suggested later, the alkoxy group could be hindering
Figure 2. (A) Fragmentation of endoperoxide to secondary carbon
centred radical species and protease inhibitor. (B) Carbonyl based
falcipain 2/3 inhibitors: Cbz=benzyloxycarbonyl; Mu=morpholine
urea.
the host cell and its dual mechanism of action should reduce
the chance of parasite resistance acquisition.
In line with the above discussion, in the case of peptidic
carbonyl based cysteine protease inhibitors (e.g., calpain
inhibitor I, N-acetyl-Leu-Leu-Norleu-al (ALLN)) we have
demonstrated by isobologram analysis that the interaction
with artemisinins and synthetic 1,2,4-trioxanes are additive
and not antagonistic. With this in mind, for the purposes
of comparison, two subsets of carbonyl-based inhibitor were
targeted; the carbonyl containing peptides (3a-g) and the
1,2,4-trioxolane prodrugs (4a-f) (Figure 2B).
Chemistry Experimental
The synthesis of target aldehyde inhibitors is presented in
Scheme 1A. Briefly, reduction of dipeptide esters 5a and 5b followed
bySwernoxidationofalcoholsof6aand 6bprovided aldehydes 3,
3a, and 3b. In studies by Veber on potent low nanomolar peptidic
cathepsin K inhibitors it was demonstrated that alkoxymethyl

8204 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Gibbons et al.
Figure 3. Docking poses of aldehydes 3a (A), 3b (B), and 3g (C) in the active site of falcipain 2, obtained from PDB 2OUL. Figures were
prepared using Pymol.
Scheme 1. Synthesis of 1,2,4-Trioxolane Prodrugsa
a Part A: (a) CaCl2, NaBH4, MeOH, -5 ꢀC, 2 h; (b) (COCl)2, DMSO, Et3N, CH2Cl2, 0 ꢀC to room temp, 3 h; (c) O3, pentane/CH2Cl2 (4:1), carbonyl
protease inhibitor. Part B: (d) 2 N NaOH, MeOH, room temp, 16 h; (e) MeONHMe HCl, HOBt, EDAC, DMAP, DIEA, CH2Cl2, 0ꢀC, 3 h; (f) Mg,
3
HgCl2, ROCH2Cl, THF, -25 ꢀC, 1.5 h.
Table 1. Enzyme Inhibition (Falcipain 2/3) and Antimalarial Activity
of Inhibitors and ECPI Prodrugs
Scheme 2. Degradation of 1,2,4-Trioxolane Aldehyde Model
IC50 (nM ( SD)
IC50 (nM)
Plasmodium
falciparum 3D7
recombinant
falcipain 2
recombinant
falcipain 3
analogue
3a
3b
1.4 ( 0.2
16.3 ( 0.9
198.9 ( 1.06
214.6 ( 11.3
5597 ( 1563.7
8613 ( 913.5
853 ( 149
21600 ( 198
222 ( 77.8
947 ( 133
332 ( 18.1
<19780 ( 3903
44155 ( 7332
<24215 ( 5239
>25000
27.1
9.3
3c
3d
7149.5 ( 245
1966 ( 254
2048.5 ( 14.8
1961 ( 247
437.7 ( 47.7
425.3 ( 34.5
510.23 ( 25.2
<11500 ( 452
1200 ( 1230
<8668 ( 501
<8583 ( 794
NA
>10000
>10000
>10000
>10000
310
3e
3f
3g
4a
4b
55.5
35.3
4c
4d
582
2564
572
4e
4f
artemisinin
ND
12.5
NA
the approach of reducing iron to the endoperoxide-bridge in
this trioxolane).
the benzylic C-radical intermediate produced itself by a 1,5-H-
shift of the initially formed oxyl centered radical as shown in
Scheme 2.
With this result in hand we turned to peptide aldehyde
prodrug analogue 3. Scheme 3 provides data on the conver-
sion of selected trioxolane 4 in the presence of ferrous bromide
and with heme. For 4, using FeBr2, we were able to character-
ize a secondary carbon centered radical spin-trapped adduct
Initial mechanistic studies conducted with trioxolane model
compound 4g (IC50=100 nM vs 3D7) indicated the feasi-
bility of our approach, since this aldehyde prodrug was
efficiently turned over to the requisite aldehyde as shown in
Scheme 2 in addition to two distinct carbon centered radical
species. The expected adduct 10a was obtained in 20% yield in
combination with 10d. We propose that 10d is derived from

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8205
Figure 4. Representative chromatograms of 3 and 4 obtained after the analysis of 20 μL aliquot of 100 nM incubation of 4 with 3D7 malaria
parasites. Ion current intensities and multiple reaction monitoring (MRM) transitions are shown in each chromatogram.
Scheme 3. Ferrous Mediated Fragmentation of Prodrug 4 (R=Cbz-Leu) and Spin Trapping with 4-Oxo TEMPOa
a Reactions were performed at 35 ꢀC under nitrogen. Solvent for FeBr2 is ACN/DCM (1:1). Solvent for heme is ACN/H2O (3:1).
9a (quantitative yield) along with lactone 9b (see mechanism
involving a single electron transfer (SET)) from ferric iron and
oxidation of the radical to a carbocation with subsequent ring
closure. Treatment of trioxolane 4 with Fe(II) in the absence
of 4-oxo TEMPO provided a 96% yield of the aldehyde along
with 30% of the lactone 9b. The corresponding reaction with
heme (generated from hemin chloride and sodium dithionite)
also led to cleavage of the prodrug to the aldehyde in 54%
yield in tandem with heme alkylated adduct 9c which was
characterized by LC/MS.
In terms of carbonyl release the reactivity of the corre-
sponding R-alkoxy ketone inhibitor 4e was much slower with
62% turnover depending on the iron source used. Itwas consi-
dered that the alkoxy group could be hindering the approach
of reducing iron to the endoperoxide bridge in this prodrug.
In order to obtain definitive proof of inhibitor release within
the living malaria parasite two experiments were performed in
late ring/early trophozoites. Parasiteswereexposedto1μMor
100 nM prodrug 4. After this time, the media were removed
via centrifugation (2000g ꢀ 5 min), and red blood cells were
lysed by the addition of 5 mL of acetonitrile/methanol/water
(40:40:20). The lysed material was triturated through a pipet
and the lysis solution separated by centrifugation (3000g ꢀ
5 min). The lysis solution was then analyzed via LC/MS/MS
(Supporting Information). As a control, nonparasitised red
blood cells were treated in exactly the same manner. Figure 4
shows the representative chromatograms of 3 (Figure 4A) and
4 (Figure 4B) obtained after the analysis of 20 μL aliquot of
100 nM incubation of 4(incubationof20μL aliquot of 100 nM)
with chloroquine-sensitive Plasmodium falciparum (3D7) para-
sitized red blood cells for 4 h. It was found that after 4 h of
incubation 4 was converted to 54 nM 3 (54% yield) with only
minor amounts of the trioxolane remaining (1.5 nM detected
by LC/MS/MS). Notably, in uninfected erythrocytes, turn-
over to the aldehyde 3 was considerably lower.
Conclusion
We have completed the synthesis and assessment of car-
bonyl based protease inhibitors and their endoperoxide hybrids.
Studies on the ferrous mediated decomposition of these molec-
ules have definitively proven that these systems degrade by
carbonyl inhibitor formation in tandem with C-radical for-
mation. In studies with a viable parasitic target we have demon-
strated efficient heme alkylation with concomitant carbonyl
inhibitor release. Furthermore, using LC/MS/MS, we have
described for the first time a quantitative intraparasitic esti-
mation of endoperoxide turnover in 3D7 parasites following
a 4 h exposure to trophozoites. One potential caveat to this

8206 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Gibbons et al.
approach with the 1,2,4-trioxolane heterocycle is the capacity
of this system to undergo Hock cleavage under mildly acidic
conditions (see Supporting Information Figure S4). While our
in vitro studies with iron(II) species definitively prove C-radical
formation in tandem with carbonyl release, we cannot rule out
that this process is occurring within the acidic digestive
vacuole of Plasmodium falciparum. A Hock mediated release
of the inhibitor, while ensuring efficient delivery, would not
provide the desired dual targeting effect. This competing
degradation pathway could explain, in part, the lower than
expected antimalarial activity for 4a and 4b. Further work
will focus on the optimization of the endoperoxide carbonyl
masking group in terms of tuning the reactivity to achieve
selective and controlled drug release within the parasite target
while maintaining appropriate pharmacokinetic properties.
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Supporting Information Available: Experimental details. This
material is available free of charge via the Internet at http://
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Note Added after ASAP Publication. The version of this
paper that was published ASAP October 27, 2010, was missing
author Nuna C. Araujo from the author list. The revised
version was published October 29, 2010.
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