Enantioselective Synthesis and in Vivo Evaluation of Regioisomeric Analogues of the Antimalarial Arterolane

Article abstract of DOI:10.1021/acs.jmedchem.7b00699

We describe the first systematic study of antimalarial 1,2,4-trioxolanes bearing a substitution pattern regioisomeric to that of arterolane. Conformational analysis suggested that trans-3″-substituted trioxolanes would exhibit Fe(II) reactivity and antipa

Full text of DOI:10.1021/acs.jmedchem.7b00699

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Enantioselective Synthesis and in Vivo Evaluation of Regioisomeric
Analogues of the Antimalarial Arterolane
Brian R. Blank,† Jiri Gut,‡ Philip J. Rosenthal,‡ and Adam R. Renslo*,†
†Department of Pharmaceutical Chemistry and ‡Department of Medicine, University of California San Francisco, 1700 Fourth Street,
San Francisco, California 94158, United States
S
* Supporting Information
ABSTRACT: We describe the first systematic study of antimalarial 1,2,4-trioxolanes bearing a substitution pattern regioisomeric
to that of arterolane. Conformational analysis suggested that trans-3″-substituted trioxolanes would exhibit Fe(II) reactivity and
antiparasitic activity similar to that achieved with canonical cis-4″ substitution. The chiral 3″ analogues were prepared as single
stereoisomers and evaluated alongside their 4″ congeners against cultured malaria parasites and in a murine malaria model. As
predicted, the trans-3″ analogues exhibited in vitro antiplasmodial activity remarkably similar to that of their cis-4″ comparators.
In contrast, efficacy in the Plasmodium berghei mouse model differed dramatically for some of the congeneric pairs. The best of
the novel 3″ analogues (e.g., 12i) outperformed arterolane itself, producing cures in mice after a single oral exposure. Overall, this
study suggests new avenues for modulating Fe(II) reactivity and the pharmacokinetic and pharmacodynamic properties of 1,2,4-
trioxolane antimalarials.
bore a peroxide bond in a sterically hindered environment. Most
notably, Vennerstrom and co-workers identified the 1,2,4-
trioxolane-based pharmacophore that produced both arterolane3
(OZ277, 2a) and artefenomel4 (OZ439, 3) (Figure 1). Clinical
development of arterolane by Ranbaxy led to its approval in India
and some African countries as a combination with piperaquine.
Artefenomel was subsequently selected for clinical development
INTRODUCTION
■
Despite recent progress in the control and treatment of malaria,
this devastating disease still affects millions around the world and
was estimated by the World Health Organization to have caused
429,000 deaths in 2015. The blood stage of infection is
responsible for symptomatic disease, which is characterized by
cyclical rounds of asexual replication of Plasmodium spp.
parasites within host erythrocytes. To support its rapid
proliferation in this stage of infection, the parasite depends on
the catabolism of host hemoglobin as a source of amino acids.
However, the consequent production of toxic free ferrous iron
heme during this process represents an Achilles heel that is
targeted in different ways by multiple classes of approved
antimalarials, including quinolines and artemisinins.
The sesquiterpene artemisinin (qinghaosu, 1) and its
analogues dihydroartemisinin (DHA) and artesunate are
employed in artemisinin-based combination therapies (ACT),
the current WHO recommended front line therapy for
uncomplicated malaria caused by Plasmodium falciparum, the
most virulent human malaria parasite. Artemisinins exhibit a
novel pharmacology that requires initial activation of a hindered
endoperoxide bond by reduced iron sources in the parasite. The
iron-dependence and likely involvement of carbon-centered
radical intermediates in artemisinin pharmacology were first
revealed in the early 1990s in elegant mechanistic work involving
synthetic artemisinin derivatives.1,2 Soon, more synthetically
accessible chemotypes were identified that, like the artemisinins,
Figure 1. Structures of antimalarial endoperoxides.
Received: May 11, 2017
© XXXX American Chemical Society
A
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on the basis of its much superior Fe(II) stability and in vivo PK/
PD properties5 and is currently in Phase 2 clinical trials.6
and cis-4″ analogues, we furthermore demonstrate that trans-3″
analogues can exhibit in vivo PK/PD properties distinct from cis-
4″ comparators. Finally, we describe an efficient and stereo-
controlled synthesis of trans-3″-substituted 1,2,4-trioxolane
analogues that will enable further lead optimization studies of
this chemotype.
Recent chemoproteomic studies7−9 from two different groups
have confirmed the long-hypothesized pleiotropic action of the
artemisinins and demonstrated7 a remarkable concordance in the
proteins covalently targeted by artemisinins and synthetic 1,2,4-
trioxolanes. Hence, artemisinin and 1,2,4-trioxolane-derived
affinity probes were shown to irreversibly label numerous
proteins in the parasite, including those involved in energy
acquisition, antioxidant response, and protein and DNA
synthesis. Yet despite having many potential targets in the
parasite, resistance to artemisinins has emerged in Southeast Asia
and is now a significant clinical problem, especially with
concomitant resistance to partner drugs.10 The resistant
phenotype results from mutations in the propeller domain of
the PfKelch13 (K13) protein, which shares homology with the
mammalian Keap1 protein.11 These mutations appear to confer
an enhanced stress response and a prolonged ring stage that
allows K13 mutant parasites to better sustain the potent but
short-lived insult conferred by artemisinins.12
RESULTS AND DISCUSSION
■
Design and Synthesis. The Fe(II) reactivity and resulting
antimalarial effects of 2a and 3 can be rationalized in terms of the
conformational dynamics of the cyclohexane ring. In ground
state conformer I (Scheme 1), the axial endoperoxide lies in the
Scheme 1. Conformational Effects on Fe(II) Reactivity in
Antimalarial 1,2,4-Trioxolanes
A number of groups have now explored whether K13
mutations confer resistance also to antimalarial 1,2,4-trioxolanes,
employing in vitro assays that focus on the kinetics of ring-stage
killing. Thus, Tilley and co-workers13 studied killing kinetics of
DHA, 2a, and 3 against a clinical K13 mutant strain and a
genetically matched K13 revertant strain bearing the wild-type
K13 allele. For all three agents, the time to reduce early ring
viability by 50% (t∧50) was more than doubled in the K13 mutant
strain compared to that in the revertant parasites. Thus, DHA
and 2 exhibited t∧50 values of ∼3 and ∼1 h for the K13 mutant
and revertant strains, respectively, whereas 3 exhibited slower
killing with t∧50 values of ∼5 and ∼2 h. Using a ring-stage survival
assay and a larger panel of K13 mutants, Fidock and co-workers14
found that DHA and 2a were uniformly less effective against the
K13 strains when compared to revertant or wild-type controls. In
contrast, compound 3 showed cross-resistance to only one of the
K13 mutants examined (I543T). Finally, Wittlin and co-
workers15 studied clinical (2a, 3) as well as additional preclinical
trioxolane analogues against a K13 clinical isolate, finding that
the synthetic trioxolanes were superior to DHA in a ring stage
survival assay. Although the findings of these three studies are in
some cases at odds, they are in general agreement that
structurally distinct endoperoxide antimalarials can exhibit
divergent effects on K13 mutant rings and that the superior in
vivo exposure profile of 3 in humans predicts for efficacy in
patients with K13 mutant parasites. Early data6 from the clinical
investigation of 3 suggests this is indeed the case.
The findings summarized above provide essential guidance for
the optimization of endoperoxide antimalarials in the current
environment of K13-mediated resistance. New agents should
ideally confer rapid killing of wild-type and mutant K13 parasites
while retaining a prolonged in vivo exposure profile that ideally
enables single-dose therapy.9,13 Among currently available
agents, 3 meets this pharmacokinetic (PK) target, whereas 2a
and current artemisinins do not due to shorter durations of
exposure in vivo. The identification of novel 1,2,4-trioxolanes
that combine rapid ring-stage killing kinetics with superior
pharmacokinetics will be expedited by new approaches for the
control of endoperoxide reduction/activation by Fe(II). Here,
we provide data suggesting that trans-3″-substitution of the
cyclohexane ring, like canonical cis-4″ substitution, modulates
Fe(II) reactivity of 1,2,4-trioxolanes in a pharmacologically
relevant regime in vivo. Comparing congeneric sets of trans-3″
concave face of an aliphatic surface with approach to the σ*
orbital of the O−O bond effectively shielded by the four proximal
axial hydrogen atoms of the adamantane and cyclohexane rings
(Scheme 1). It is the peroxide-equatorial conformer II that
exposes the O−O bond for inner-sphere coordination with
Fe(II) and single-electron transfer leading to bond scission.16
Consistent with this interpretation is the highly regioselective
nature of peroxide cleavage with reaction at the oxygen atom
opposite the adamantane moiety predominating (O1, Scheme
1).16 The crucial role of cyclohexane ring conformation on Fe(II)
reactivity allows the latter to be finely tuned by varying the nature
and stereochemistry of the 4″ substituent (i.e., R4 in Scheme 1).16
The optimal balance of Fe(II) reactivity and antiplasmodial
effects was ultimately achieved in analogues bearing cis-R4
substitution that stabilizes unreactive conformer I. A focus on
cis-R4 substitution during lead optimization efforts ultimately
produced both of the clinical candidates from this class (2a and
3). Moreover, the improved stability of 34 as compared to 2a
toward endogenous Fe(II) sources can be understood as arising
from more severe 1,3-diaxial interactions in conformer II of
compound 3 due to the bulkier aryl R4 side chain. Importantly, 3
still retains sufficient reactivity with free Fe(II) heme in parasites
to confer a potent antimalarial effect.
The ability to predict Fe(II) reactivity on conformational
grounds is very appealing from a design perspective and may
prove essential in finding the right balance of in vivo stability
toward endogenous Fe(II) sources (principally labile iron) and
reactivity with Fe(II) heme necessary to target both susceptible
and resistant parasites. We considered that trans-3″ substitution,
nearly17 unexplored previously, should modulate conformational
dynamics in an analogous fashion as canonical cis-4″ substitution
(R3 vs R4, Scheme 1). In the case of trans-R3 analogues, however,
B
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a
1,3-diaxial interactions in conformer II would involve both O and
H atoms as opposed to only H atoms in cis-R4 analogues.
Depending on the nature of the R3/R4 side chain then, distinct
conformational dynamics of the cyclohexane ring might be
expected, and this would confer distinct in vitro and in vivo
activities. Other important properties such as solubility,
metabolism, and clearance were also expected to be affected by
the switch to trans-R3 substitution, which eliminates the internal
symmetry present in 2a and 3, producing a distinct topology.
The arterolane (2a) scaffold was selected for initial studies
because a late-stage amide coupling reaction would allow ready
access to the matched R3/R4 analogue pairs. As comparators, we
selected several R4-side chain derivatives described in patent
applications18 and reported to possess reasonable in vivo efficacy
with oral dosing. Thus, 2a and nine additional R4-side chain
analogues (2b−j) were synthesized19 from acid intermediate 417
and commercially available amines (Scheme 2). We then turned
our attention to construction of the analogous R3-side chain
variants.
Scheme 3. Enantiocontrolled Synthesis of Ketone 7
a
Reagents and conditions: (a) dimethyl malonate, (R)-ALB (1 mol
%), t-BuOK (0.9 equiv relative to ALB), 4 Å MS, THF, rt, 68 h, 87%;
(b) NaOH, H2O/THF (11:1), 0 °C, 2 h; (c) DMSO, 160 °C, 4 h,
85% over two steps.
in nearly quantitative yield. Hydrolysis of ester 10 with NaOH
then furnished the desired carboxylic acid 11 in excellent yield.
From 11, the desired 3″ amides 12a−k were prepared in 68−
95% yields via formation of the mixed anhydride (ethyl
chloroformate and Et3N at −10 °C) and subsequent reaction
with primary or secondary amines (Scheme 4). We were able to
confirm that the Griesbaum co-ozonolysis of 7 and 9 had
proceeded with good diastereoselectivity (91:9 dr) by the
preparation of analogue 12k in which trans and cis diastereomers
could be readily distinguished by NMR.20
Scheme 2. Synthesis of Arterolane and Related cis-4″
a
Analogues from Key Intermediate 4
a
In Vitro Evaluation of Congener Pairs. The effect of R3
substitution on antiplasmodial activity was evaluated by
comparing the regioisomeric analogues 2a−j and 12a−j (Chart
1). New trans-R3 analogues exhibited potent, low nM activities
against the chloroquine-resistant W2 strain of P. falciparum with
activity comparable to that of cis-R4 comparators. Moreover,
structure−activity trends tracked remarkably closely in the
regioisomeric scaffolds consistent with our hypothesis that trans-
R3 substitution should confer similar conformational constraints
and Fe(II) reactivity as cis-R4 substitution. In both scaffolds,
piperidine amides (12e and 2e) were the least potent,
approximately 10- to 20-fold weaker than piperazine analogues
12g and 2g. The most significant difference between regioisomer
pairs was observed for morpholine amides 12f and 2f, where 3″
analogue 12f was 3-fold more potent than that of 2f. Overall,
these findings confirmed that trans-R3 substitution modulates
1,2,4-trioxolane reactivity in a pharmacologically relevant range,
producing effects on cultured parasites that are comparable to
those of canonical cis-R4 substitution.
Reagents and conditions: (a) ethyl chloroformate, Et3N, CH2Cl2,
−10 °C, 75 min; R(R′)NH, CH2Cl2, −10 °C to rt, 2−24 h, 71−95%.
Reaction conditions adapted from ref 19.
Unlike in analogues 2a−j, which possess internal symmetry
and are achiral, R3 substitution leads to four possible stereo-
isomers. Because trans-R3 stereochemistry was desired based on
our conformational analysis, this left the trans-(R,R) and trans-
(S,S) enantiomers as potential candidates for evaluation. We
arbitrarily selected the trans-(R,R) stereoisomers for this initial
study. Previously, we found20 that the Griesbaum co-ozonolysis
reaction of 3-substituted cyclohexanones proceeds with high
(∼90:10 dr) intrinsic diastereoselectivity and favors the desired
trans stereoisomer. Preparing the desired trans-(R,R) stereo-
isomer then required starting from an appropriate nonracemic 3-
cyclohexanone. Toward this end, cyclohexanone 5 was prepared
in 87% yield via catalytic asymmetric Michael addition of
dimethyl malonate to 2-cyclohexen-1-one (Scheme 3).21−23
Notably, this very practical synthesis of 5 has been performed
previously on kilogram scales in a premanufacturing setting.23
We confirmed the high enantiomeric purity of 5 (95% ee) by
conversion to ketal 6,24,25 the two diastereomers of which could
be readily distinguished by 13C NMR spectroscopy.
A Krapcho decarboxylation was next considered as a means to
produce the desired ester 726 from 5. However, for the possibility
of epimerization via retro-Michael/Michael addition of dimethyl
malonate to be avoided, a two-step procedure was utilized
instead.26 Thus, hydrolysis of 5 with NaOH at 0 °C afforded the
mono-carboxylate intermediate, which was immediately heated
to 160 °C to facilitate decarboxylation. This afforded 7 in 85%
yield over two steps. Conversion of 7 to ketal 8 confirmed that
the hydrolysis/decarboxylation sequence had proceeded without
erosion of enantiomeric purity (94% ee for 7). Griesbaum co-
ozonolysis of 7 with two equivalents of oxime 9 at 0 °C using our
optimized conditions20 afforded 1,2,4-trioxolane intermediate 10
A second hypothesis regarding trans-R3 substitution was that
the resulting desymmetrized molecular topology would impact
the in vivo PK/PD properties of these analogues. Specifically, we
reasoned that the chiral trans-R3 analogues might interact
differently with CYP enzymes and drug transporters (e.g., P-gp).
To explore possible metabolic differences, we evaluated selected
analogues with mouse liver microsomes and derived intrinsic
clearance (CLint) values (Table 1). All the analogues evaluated
exhibited low or moderate CLint values that would predict
reasonable exposure in vivo. In four of the six analogue pairs
evaluated, it was the trans-R3 analogues that exhibited higher
CLint values with trans-R3 analogues 12a and 12c showing lower
intrinsic clearance than their cis-R4 comparators. Clearance
appeared to be CYP mediated in all cases, with the possible
exception of 2c, where chemical instability or non-CYP-mediated
clearance was implicated. The kinetic solubilities of arterolane
C
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a
Scheme 4. Stereocontrolled Synthesis of 1,2,4-Trioxolane Analogues 12a−k
a
Reagents and conditions: (a) 0.5 equiv of 7, O3, CCl4, 0 °C, 3 h, 95%; (b) NaOH, EtOH/H2O, 50 °C, 4 h, 95%; (c) ethyl chloroformate, Et3N,
CH2Cl2, −10 °C, 75 min; R(R′)NH, CH2Cl2, −10 °C to rt, 2−24 h, 68−95%.
a
Chart 1. In Vitro Activity of Trioxolanes 12a−j and 2a−j against W2 P. falciparum Parasites
a
In vitro activity of 12a−j and 2a−j against W2 P. falciparum parasites (EC50 SEM). Reported EC50 values are the means of three determinations
SEM
(2a) and its regioisomer 12a were evaluated and found to be
comparable. Overall, the in vitro ADME data suggested that
trans-R3 analogues were suitable for evaluation in animals and
that specific analogues might be differentiated from their
comparators in vivo.
pharmacodynamic response. Prior to evaluating additional trans-
R3 analogues, we sought to determine the 50% curative dose for
both 12a and 2a in this model and use this dose as a benchmark
for future studies. Thus, compounds 12a and 2a (both as free
bases) were administered to mice over 4 days at doses of 10, 6, 4,
or 1 mg kg−1 day−1, and survival and parasitemia were monitored
until 30 days postinfection. This study revealed a clear dose−
efficacy relationship and allowed us to estimate 50% curative
dose (PD50) values of ∼6 mg kg−1 day−1 for 12a and ∼4 mg kg−1
day−1 for 2a (Table 3). With the trans-R3 chemotype validated in
vivo and a relatively stringent dose/efficacy benchmark
established, we set out to more broadly evaluate the matched
pairs of regioisomeric analogues in animals.
The suppressive 4-day “Peters test”27 involving P. berghei
infected mice provides a convenient and cost-effective means to
assess the overall in vivo performance of preclinical antimalarials.
Thus, groups of five P. berghei infected female Swiss Webster
mice were treated via oral gavage with four daily (QD) doses of
either 2a (as the tosylate salt), regioisomeric comparator 12a
(both tosylate and free base), or chloroquine control (Table 2).
Mice were followed for 30 days and judged to have been cured of
the infection based on the lack of detectable parasitemia at the
end of the study. In this initial study, trans-R3 analogue 12a as
either tosylate salt or free base exhibited efficacy comparable to
arterolane tosylate, producing cures in all five animals.
As noted previously, the cis-R4 amide comparators were
selected in part based on in vivo efficacy data in patents and other
reports28 from the Vennerstrom group. Therefore, all nine cis-R4
analogues were expected to have good in vivo prospects, whereas
the in vivo performance of the trans-R3 comparators was less
certain. In the next study then, all 20 test compounds (2a−j and
This result indicated that 12a was orally absorbed and
achieved systemic exposure sufficient to produce a robust
D
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2a again showed good efficacy at these low doses with analogue
12a marginally more effective than 2a in this study (Table 4).
Table 1. In Vitro ADME Data for Selected Trioxolane
Analogues and Controls
T
T1/2 (min) no
solubility
c
1/2 a
b
Table 4. In Vivo Efficacy of Matched Analogue Pairs in P.
berghei-Infected Mice
cmpd
(min)
CLint
5.4
NADPH
(μM)
a
2a
128
169
stable
stable
stable
stable
70
433
429
b
compd
12a
dose (mg kg−1 day−1
)
mice cured (%)
12a
4.1
5.6
2b
124
4
6
4
6
4
6
4
6
4
6
4
6
4
6
4
6
4
6
4
6
4
6
4
6
4
6
4
6
4
6
4
6
4
6
4
6
4
6
4
6
30
60
100
20
80
0
12b
37.3
64.2
84.5
161
18.6
10.8
8.2
2c
2a
12c
136
2d
4.3
stable
stable
88.9
12b
2b
12d
64.2
48.1
25.5
277
10.8
14.4
27.2
2.5
0
2g
0
12g
stable
stable
stable
0
2i
12c
2c
100
100
0
12i
84.5
1.65
55.5
8.2
midazolam
diclofenac
amiodarone
testosterone
420
12.5
20
0
<3
12d
2d
315
0
a
b
Half-life when incubated with mouse liver microsomes. Clearance in
40
100
0
units of μL min−1 mg−1 of protein calculated as CLint = ln(2)*1000/
T1/2/protein concentration, where protein concentration is in mg/mL;
12e
2e
c
midazolam and diclofenac served as controls. Kinetic solubility in PBS
(pH 7.4) at a final DMSO concentraion of 5% as an average of three
determinations; amiodarone and testosterone served as controls.
0
0
0
12f
2f
0
Table 2. In Vivo Efficacy of Trioxolanes 12a and 2a and
Controls in P. berghei-Infected Mice Treated for 4 Days
0
a
0
b
treatment
2a
salt form
dose (mg kg−1 day−1
)
mice cured (%)
0
12g
2g
0
tosylate
tosylate
free base
13.6
13.6
9.5
100
100
100
80
0
20
100
100
0
12a
12a
chloroquine
vehicle treated
untreated
30
12h
2h
0
0
a
0
Beginning 1 h after infection, cohorts of five P. berghei-infected female
Swiss Webster mice were treated once a day for 4 days by oral gavage.
Mice were considered cured if there was no detectable parasitemia at
30 days postinfection.
0
b
12i
2i
100
80
100
100
60
100
80
80
40
0
Table 3. In Vivo Efficacy of 12a and 2a in P. berghei-Infected
Mice Following Four Daily Doses
a
12j
2j
a
b
treatment
dose (mg kg−1 day−1
)
mice cured (%)
12a
1
4
0
0
chloroquine
vehicle
6
60
100
0
a
10
1
Beginning 1 h after infection, cohorts of five P. berghei-infected female
2a
Swiss Webster mice were treated once a day for 4 days by oral gavage.
b
4
80
100
100
0
Mice were considered cured if there was no detectable parasitemia at
6
30 days postinfection.
10
vehicle
Among the other nine analogue pairs, four pairs failed to cure any
mice at either dosing level. These analogues included the
glycinamide (12b and 2b), piperidine (12e and 2e), morpholine
(12f and 2f), and N-methyl piperazine (12h and 2h) analogues.
Interestingly, whereas the piperidine and piperazine analogues
failed in these studies, the structurally very similar 4-amino-
piperidine derivatives 2i and 12i were among the most efficacious
analogues examined, curing all animals at the lowest dose of 4 mg
kg−1 day−1 and proving superior to 12a and 2a. This finding
untreated
0
a
Beginning 1 h after infection, cohorts of five P. berghei-infected female
Swiss Webster mice were treated once a day for 4 days by oral gavage
at the indicated daily dose. Mice were considered cured if there was
no detectable parasitemia at 30 days postinfection.
b
12a−j) were evaluated under 4 and 6 mg/kg daily dosing
protocols in groups of five Swiss Webster mice. Controls 12a and
E
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regarding 2i is consistent with a previous report28 demonstrating
the superiority of 2i over 2a in a similar murine model with 3 days
oral dosing at 3 mg/kg. Thus, for many of the analogue pairs, in
vivo efficacy seemed to track between the cis-R4 and trans-R3
comparators.
Table 6. In Vivo Efficacy of 12c and 12i in P. berghei-Infected
Mice with Less Frequent Dosing
a
b
compd
dose (mg/kg)
number of doses
mice cured (%)
12c
4
4
4
4
4
4
4
4
4
3
2
1
4
3
2
1
100
20
0
Of greatest interest were those analogue pairs for which in vivo
efficacy varied between the trans-R3 and cis-R4 comparators.
Most strikingly, cis-R4 analogues 2d and 2g were fully effective at
6 mg kg−1 day−1, whereas the analogous trans-R3 comparators
12d and 12g failed entirely or were minimally effective (Table 4).
In the case of the analogue pair 12c and 2c, however, it was the
novel trans-R3 analogue 12c that produced full cure at either
dose, whereas the cis-R4 derivative 2c was essentially ineffective.
Thus, regioisomeric trioxolane analogues can in some cases
exhibit starkly different efficacy in vivo despite having similar
intrinsic potency against cultured parasites. These results
confirmed our hopes that trans-R3 substitution might offer new
avenues for the optimization of PK/PD properties in preclinical
1,2,4-trioxolanes. In the case of analogue pairs 12c and 2c, 12d
and 2d, and 12g and 2g, CLint values from the mouse liver
microsome assay correctly predicted the superior analogue in
each pair (compare Tables 1 and 4). However, the magnitude of
differences in CLint values seems insufficient to fully explain the
differences observed, nor do the CLint values alone explain the
failure of 12b and 2b. It appears that additional in vivo PK/PD
studies will be required to fully understand the relative merits of
3″ and 4″ substitution within the context of specific side chain
types.
0
12i
100
60
0
0
a
Beginning 1 h after infection, cohorts of five P. berghei infected female
Swiss Webster mice were treated once a day by oral gavage for either
b
four, three, two, or 1 day as shown above. Mice were considered
cured if there was no detectable parasitemia at 30 days postinfection.
mouse survival was extended ∼3−6 days compared to that of
vehicle-treated control animals.
In a final in vivo study, we evaluated the efficacy of 12c and 12i
following a single dose of 40 or 80 mg/kg or two doses of 40 mg/
kg (Table 7). We included as a positive control in this study
Table 7. Efficacy of 12c, 12i, and Artefenomel in P. berghei-
a
Infected Mice Following a Single or Repeated Dose
b
compd
dose (mg/kg)
number of doses
mice cured (%)
3 (artefenomel)
40
80
40
40
80
40
40
80
1
1
2
1
1
2
1
1
100
100
100
0
Given that the novel trans-R3 analogues 12c and 12i
demonstrated complete cures at 4 mg kg−1 day−1, we next
explored whether even lower doses of these agents might be
effective in mice. Although 12c and 12i again cured all animals at
a 4 mg kg−1 day−1 dose, neither produced cures with reduced
doses of 2, 1, or 0.5 mg kg−1 day−1 (Table 5). We next asked
12c
12i
20
100
20
60
a
Beginning 1 h after infection, cohorts of five P. berghei-infected female
Table 5. In Vivo Efficacy of 12c and 12i in P. berghei-Infected
Mice at Various Dosing Levels
Swiss Webster mice were treated by oral gavage for either 1 or 2 days
at the indicated dose. Mice were considered cured if there was no
a
b
detectable parasitemia at 30 days postinfection.
b
compd
dose (mg kg−1 day−1
)
mice cured (%)
12c
0.5
1
0
0
artefenomel (3),4,29 which is known to be highly effective in these
models. Indeed, a single dose of 3 was remarkably effective,
curing all animals at both doses examined. Although the new
analogues 12c and 12i were completely effective with two doses
of 40 mg kg−1 day−1, a single dose of 40 mg/kg was only partially
effective for 12i and ineffective for 12c. Both compounds were
partially effective with a single 80 mg/kg dose and again 12i
proved superior to 12c (60 vs 20% cures). Thus, multiple PD
studies consistently revealed analogue 12i to be the most
promising of the novel trans-3″ analogues explored herein,
although it was inferior to artefenomel (3), which has
demonstrated4 single-dose cures at 20 mg/kg in similar models.
2
0
4
100
0
12i
0.5
1
0
2
0
4
100
a
Beginning 1 h after infection, cohorts of five P. berghei-infected female
Swiss Webster mice were treated once a day for 4 days by oral gavage.
Mice were considered cured if there was no detectable parasitemia at
b
30 days postinfection.
whether a full 4 days of treatment at 4 mg kg−1 day−1 was
necessary to achieve cures with 12c and 12i, cognizant that
current antimalarial target product profiles seek agents with
shortened dosing regimens. When 12c and 12i were
administered at 4 mg/kg for 4, 3, 2, or 1 day, a predictable
decline in efficacy with the number of doses was observed (Table
6). Thus, both compounds were again fully effective at four daily
doses of 4 mg/kg, and 12i was shown to be superior to 12c with
three daily doses of 4 mg/kg (60 vs 20% of animals cured for 12i
and 12c, respectively). Neither compound was curative with one
or two daily doses of 4 mg/kg, although under these regimens
CONCLUSIONS
■
The unusual Fe(II)-dependent pharmacology of antimalarial
1,2,4-trioxolanes necessitates an equally unusual approach to
their optimization, namely, one that recognizes and exploits the
strong connection between conformational dynamics, chemical
reactivity, and antiparasitic effects.16 Clinical experience with 2a
and 3 perfectly illustrates this point. Thus, early clinical studies of
2a revealed inferior drug exposure and half-life in malaria patients
when compared to healthy volunteers. This effect was traced to
reaction of 2a with endogenous Fe(II) sources in infected
F
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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
cyclodextrin in water, and 50% PEG400. There were five mice in each
test arm. Infections were monitored by daily microscopic evaluation of
Giemsa-stained blood smears starting on day 7. Parasitemia were
determined by counting the number of infected and uninfected
erythrocytes. Body weight was measured over the course of the
treatment. Mice were euthanized when parasitemia exceeded 50% or
when weight loss of more than 15% occurred. Animal survival and
morbidity were closely monitored for up to 30 days postinfection when
the experiment was terminated.
individuals. Nevertheless, by modifying the nature of the cis-4″
side chain, a superior drug candidate (3) with much improved
Fe(II) stability and in vivo PK properties was identified.4 Now,
an improved understanding13−15 of how emergent K13 mutant
parasites escape endoperoxide exposure provides rationale for
identifying new endoperoxides that more rapidly kill P.
falciparum K13 mutant ring forms while retaining stability
toward endogenous Fe(II) sources in the host. The result of just
such an effort focused on a tetraoxane chemotype has appeared
very recently.30
Here, we present the first systematic study comparing the
canonical cis-4″-substituted pharmacophore of 2a and 3 with
regioisomeric trans-3″-substituted analogues. On the basis of
their in vitro and in vivo properties, we conclude that the trans-3″
side chain modulates peroxide reactivity in a pharmacologically
relevant regime as hoped. In this preliminary study, we examined
just ten side chains employed previously in cis-4″ analogues. Even
with this limited survey, two novel analogues were identified that
exhibited in vivo properties superior to 2a in the P. berghei model,
and one that afforded single-dose cures at higher doses. The
ultimate potential of the new 3″-substituted chemotype will only
be revealed with the examination of a more diverse set of side
chains, and in particular, those designed to exploit steric and
conformational effects unique to this substitution pattern. Efforts
in this direction are well underway in our laboratories and will be
described in due course.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.7b00699.
Synthetic procedures and compound characterization for
new compounds (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: 415-514-9698; fax: 415-514-4507; e-mail: adam.
renslo@ucsf.edu.
■
ORCID
Adam R. Renslo: 0000-0002-1240-2846
Author Contributions
B.R.B., J.G., and A.R.R conceived experiments. B.R.B. devised
synthetic routes and synthesized compounds. B.R.B. and J.G.
acquired data. All authors analyzed data. A.R.R. supervised the
project. B.R.B. and A.R.R wrote the manuscript. All authors
reviewed, edited, and commented on the manuscript. All authors
have given approval to the final version of the manuscript.
EXPERIMENTAL SECTION
■
Known compounds were prepared according to literature procedures as
cited in the main text. The syntheses of new compounds 12a−k are
described in the Supporting Information. All compounds tested in
parasites or mice were judged to be of >95% purity as assessed using a
Waters Micromass ZQ 4000 equipped with Waters 2795 Separation
Module, Waters 2996 Photodiode Array Detector (254 nm), and Waters
2424 ELS detector. Separations were carried out with an XBridge BEH
C18, 3.5 μm, 4.6 × 20 mm column, at ambient temperature
(unregulated) using a mobile phase of water−methanol containing a
constant 0.10% formic acid.
Plasmodium falciparum EC50 Determinations. The growth
inhibition assay for P. falciparum was conducted as described
previously31 with minor modifications. Briefly, Plasmodium falciparum
strain W2 synchronized ring-stage parasites were cultured in human red
blood cells in 96-well flat bottom culture plates at 37 °C, adjusted to 1%
parasitemia and 2% hematocrit under an atmosphere of 3% O2, 5% CO2,
and 91% N2 in a final volume of 0.1 mL per well in RPMI-1640 media
supplemented with 0.5% Albumax, 2 mM L-glutamine, and 100 mM
hypoxanthine in the presence of various concentrations of inhibitors.
Tested compounds were serially diluted 1:3 in the range 10000−4.6 nM
(or 1000−0.006 nM for more potent analogues) with a maximum
DMSO concentration of 0.1%. Following 48 h of incubation, the cells
were fixed by adding 0.1 mL of 2% formaldehyde in phosphate buffered
saline, pH 7.4 (PBS). Parasite growth was evaluated by flow cytometry
on a FACsort (Becton Dickinson) equipped with AMS-1 loader (Cytek
Development) after staining with 1 nM of the DNA dye YOYO-1
(Molecular Probes) in 100 mM NH4Cl and 0.1% Triton x-100 in 0.8%
NaCl. Parasitemias were determined from dot plots (forward scatter vs
fluorescence) using CELLQUEST software (Becton Dickinson). EC50
values for growth inhibition were determined from plots of percentage
control parasitemia over inhibitor concentration using GraphPad Prism
software.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
A.R.R. acknowledges funding from the US National Institutes of
Health (R01 Grant AI105106). We thank Prof. J. Vennerstrom
for the generous gift of intermediate 4.
ABBREVIATIONS USED
■
DHA, dihydroartemisinin; ACT, artemisinins combination
therapy; WHO, World Health Organization; PK, pharmacoki-
netics; PD, pharmacodynamics; CLint, intrinsic clearance; MLM,
mouse liver microsome assay; QD, once-daily dosing
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