Tetraoxane-pyrimidine nitrile hybrids as dual stage antimalarials

Article abstract of DOI:10.1021/jm5004528

The use of artemisinin or other endoperoxides in combination with other drugs is a strategy to prevent development of resistant strains of Plasmodium parasites. Our previous work demonstrated that hybrid compounds, comprising endoperoxides and vinyl sulfones, were capable of high activity profiles comparable to artemisinin and chloroquine while acting through two distinct mechanisms of action: oxidative stress and falcipain inhibition. In this study, we adapted this approach to a novel class of falcipain inhibitors: peptidomimetic pyrimidine nitriles. Pyrimidine tetraoxane hybrids displayed potent nanomolar activity against three strains of Plasmodium falciparum and falcipain-2, combined with low cytotoxicity. In vivo, a decrease in parasitemia and an increase in survival of mice infected with Plasmodium berghei was observed when compared to control. All tested compounds combined good blood stage activity with significant effects on liver stage parasitemia, a most welcome feature for any new class of antimalarial drug.

Full text of DOI:10.1021/jm5004528

Article
pubs.acs.org/jmc
Tetraoxane−Pyrimidine Nitrile Hybrids as Dual Stage Antimalarials
†
§
†
‡
∥
‡
†
Rudi Oliveira, Rita C. Guedes, Patrícia Meireles, Ines
̂
S. Albuquerque, Lídia M. Gonca
̧
l v‡ es,
†,§
∥
Elisabete Pires, Maria Rosario Bronze, Jiri Gut, Philip J. Rosenthal, Miguel Prudencio,
́
̂
,†
,⊥
†
Rui Moreira,* Paul M. O’Neill,* and Francisca Lopes
†
Instituto de Investigaca
649-003 Lisboa, Portugal
̃
́
̧ o do Medicamento (iMed.ULisboa), Faculdade de Farmacia, Universidade de Lisboa, Av. Prof. Gama Pinto,
1
‡
Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa,
Portugal
§
ITQB-UNL, Av. da Repub
́
lica, Estaca
Department of Medicine, San Francisco General Hospital, University of California, San Francisco, San Francisco, California 94143,
United States
̃
́
̧ o Agronomica Nacional, 2780-157 Oeiras, Portugal
∥
⊥
Department of Chemistry, University of Liverpool, Liverpool, L69 3BX, U.K.
S Supporting Information
*
ABSTRACT: The use of artemisinin or other endoperoxides
in combination with other drugs is a strategy to prevent
development of resistant strains of Plasmodium parasites. Our
previous work demonstrated that hybrid compounds, compris-
ing endoperoxides and vinyl sulfones, were capable of high
activity profiles comparable to artemisinin and chloroquine
while acting through two distinct mechanisms of action:
oxidative stress and falcipain inhibition. In this study, we adapted this approach to a novel class of falcipain inhibitors:
peptidomimetic pyrimidine nitriles. Pyrimidine tetraoxane hybrids displayed potent nanomolar activity against three strains of
Plasmodium falciparum and falcipain-2, combined with low cytotoxicity. In vivo, a decrease in parasitemia and an increase in
survival of mice infected with Plasmodium berghei was observed when compared to control. All tested compounds combined
good blood stage activity with significant effects on liver stage parasitemia, a most welcome feature for any new class of
antimalarial drug.
INTRODUCTION
■
Although the number of deaths caused by Plasmodium
falciparum malaria has probably decreased in recent years, the
disease remains a huge problem in much of the developing
1
,2
world. Drug resistance is a major threat, and reports of
resistance to artemisinins are alarming, as we are now highly
dependent on the use of artemisinin-based combination
3
−5
therapies (ACTs) to treat falciparum malaria.
Semisynthetic
artemisinin derivatives (Figure 1) were a major breakthrough in
malaria chemotherapy because they produce a very rapid
therapeutic response against malaria parasites. ACT, the current
recommended treatment for uncomplicated falciparum malaria
in most of the world, includes five standard regimens, each
incorporating a rapid acting artemisinin derivative plus a slower
acting drug to kill parasites that may escape the rapid action of
the artemisinin and to limit selection of artemisinin-resistant
Figure 1. Structures of artemisinin, 1, its derivatives dihydroartemi-
sinin (DHA), artemether (AM), and artesunate (AS) and the
tetraoxane RKA182, 2.
7
debate, it is generally accepted that the resulting radicals of this
activation can alkylate several targets in the parasite, leading to
its death.
6
parasites. Artemisinins and other endoperoxides, such as
A key feature of hybrid drugs is the presence of different
mechanisms of action, against either a single target or different
tetraoxane 2 (Figure 1), are highly active against the asexual
erythrocytic stage of infection. These compounds are
reductively activated in the presence of high concentrations
of iron(II) accumulated inside the parasite food vacuole after
the digestion of large quantities of host hemoglobin. Although
the mechanism of action of artemisinins is still a matter of
8
targets. A recent publication associates artemisinin resistance
Received: March 22, 2014
©
XXXX American Chemical Society
A
dx.doi.org/10.1021/jm5004528 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
9
to a form of antioxidant defense system, so it is particularly
important that a distinct mechanisms of action is combined
with endoperoxide drugs in the treatment of highly adaptable
vivo using a murine model of malaria. The adamantane-based
tetraoxane moiety was selected throughout this study, as it is
known to provide adequate metabolic stability.
1
0
parasites. We recently reported that tetraoxane−peptide vinyl
sulfone hybrids, 3 (Figure 2), are rapidly activated by iron(II),
RESULTS AND DISCUSSION
■
Synthesis. Tetraoxane−pyrimidine nitrile hybrids were
synthesized as depicted in Schemes 1 and 2. The tetraoxane
a
Scheme 1. Synthesis of Tetraoxanes 6 and 7
a
Reagents and conditions: (a) (1) HCO H, H O 50%, ACN, rt, 45
2
2
2
min, (2) 2-adamantanone, Re O , DCM, rt, 3 h; (b) NaOH, MeOH/
2
7
H O (2:1), 80 °C, 1 h 30 s.
2
moiety was prepared by reacting the appropriate ketone 5 with
hydrogen peroxide 50% in acidic conditions to form a gem-
dihydroperoxide intermediate, which was then reacted with 2-
adamantanone in the presence of rhenium(VII) catalyst to give
Figure 2. General structures: tetraoxane−vinyl sulfone hybrids 3;
tetraoxane−pyrimidine nitrile hybrids 4.
2
0,21
6
a−b (Scheme 1).
The synthesis of the pyrimidine nitrile
19
releasing a potent irreversible inhibitor of falcipains inside the
moiety followed the procedure reported by Coteron et al.
́
11
parasitedigestive vacuole. Falcipain-2 and falcipain-3 are
cysteine proteases from P. falciparum involved in the digestion
of host hemoglobin in the digestive vacuole of the parasite,
Reductive amination of tert-butyl carbazate, 8, with the
appropriate aldehyde or ketone and subsequent nucleophilic
aromatic substitution reaction of the appropriate 2,4-dichlor-
opyrimidine with hydrazines 9a−c afforded the 5- and 6-
substituted 2-chloropyrimidines 10a−f (Scheme 2). Cyanation
of 10a−f using potassium cyanide gave the corresponding 2-
12
providing amino acids for its survival and development.
Although hybrid compounds 3 displayed weak to moderate
falcipain inhibitory activity, they were able to inhibit
hemoglobin digestion at low nanomolar concentrations,
consistent with the release of the parent peptide vinyl sulfone
triggered by iron(II).
19
cyanopyrimidines 11a−f. Boc removal with p-toluenesulfonic
acid and subsequent coupling with the acid chloride derived
from 7 yielded the final products 4 with poor to moderate
yields (Scheme 2, method A). Alternatively, deprotection of the
hydrazine 10a−f followed by coupling with the acid chloride
derived from 7 and cyanation of the 2-chloropyrimidine hybrid
precursors 12 afforded the final products in moderate to good
yields (Scheme 2, method B).
To overcome selectivity issues often associated with
irreversible inhibitors, it is desirable to develop nonpeptidic
1
2,13
reversible inhibitors.
Nitriles inhibit cysteine proteases by
forming a reversible thioimidate intermediate resulting from the
14−18
nucleophilic attack of the catalytic cysteine residue.
Several
heterocyclic and peptidomimetic compounds containing a
nitrile warhead displayed excellent inhibitory activity against
Iron(II) Activation. Compound 13, lacking the tetraoxane
core, was prepared from the reductive cleavage of tetraoxane 4a
19
22
falcipain-2 and against cultured P. falciparum. In particular,
appropriately decorated pyrimidine and triazine scaffolds were
shown to position vectors for the S , S , and S binding pockets
using iron(II) bromide in dichloromethane (Scheme 3). This
reaction is intended to simulate the intraparasitic endoperoxide
iron(II) mediated bioactivation process in the digestive vacuole
1
2
3
11,22
and to direct the thioimidate adduct toward the oxyanion hole
of the parasite.
To confirm the proposed mechanism, an
1
8,19
of the enzyme.
An interesting feature of pyrimidine nitriles
aqueous solution of 4a was treated with high concentration of
is the possibility to accommodate a wide variety of substituents
iron(II) sulfate, and the resulting mixture was analyzed by LC-
MS. Decomposition of 4a into ketone 13 followed first-order
19
22
at P , without significantly compromising enzyme inhibition.
3
This observation prompted us to hypothesize that tetraoxane−
decay kinetics, with a half-life of 5.1 h (Figure 3). This value is
of the same order of magnitude as those observed with
tetraoxanes and 1,2,4-trioxolane analogues with basic side
chains, suggesting that the nature of the endoperoxide side
chain does not significantly affect the rate of iron(II)-triggered
pyrimidine nitrile constructs with the general structure 4
(
Figure 2), in which the peroxide moiety occupies the P3
position, could be endowed with both falcipain and P.
falciparum inhibitory activities, in contrast to their hybrid
11
22
vinyl sulfone counterparts 3. This approach could provide a
new class of hybrid compounds whose ability to inhibit
falcipains would not be dependent on the rate of peroxide
activation triggered by theiron(II) present in the digestive
vacuole of the malaria parasite. We now report the synthesis of
tetraoxane−pyrimidine nitrile hybrids 4, and the evaluation of
these compounds against the blood and liver stages of malaria
infection, against falcipain-2, against mammalian cells, and in
activation.
As depicted in Figure 3, ketone 13 accounts for 50% of the
initial concentration of 4a, indicating that another decom-
position pathway is operating under the conditions used.
Tetraoxane activation may involve coordination of iron(II)
either with O2, leading to the formation of 13, or with O1,
which leads to 2-adamantanone (Supporting Information
Scheme S1). Although we were not able to detect 2-
B
dx.doi.org/10.1021/jm5004528 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 2. Synthesis of Tetraoxanes−Pyrimidine Nitrile Hybrids 4a−g
a
Reagents and conditions: (a) (1) aldehyde, dry DCM, molecular sieves 3 Å (2) Et SiH, Pd/C, dry MeOH; (b) aldehyde or ketone, dry DCM,
3
glacial AcOH, NaBH(OAc) , rt, ov; (c) 2,4-dichloropyrimidines, DIPEA, iPrOH, reflux, ov; (d) KCN, DABCO, DMSO/H O (9:1), rt, 3 h; (e) (1)
3
2
pTsOH, ACN, rt, ov, (2) acid chloride of tetraoxane 7a−b, DIPEA, dry THF, rt, ov; (f) (1) pTsOH, ACN, rt, ov, (2) acid chloride of tetraoxane 7a,
DIPEA, dry THF, rt, ov; (g) KCN, DABCO, DMSO/H O (9:1), rt, 3 h.
2
a
Scheme 3. Synthesis of Pyrimidine Nitrile 13
adamantanone, these results are in agreement with similar
assays, where radicals derived from both activation pathways
were trapped with TEMPO and identified.
2
0,22
Falcipain-2 Inhibition. Compounds 4 were screened
against recombinant falcipain-2. They displayed a wide range
of inhibitory activities, with IC s ranging from 978 nM for 4g
50
to 3.4 nM for 4e (Table 1). These results suggest that
a
pyrimidine nitriles, in contrast to their vinyl sulfone hybrid
Reagents and conditions: (a) FeBr , ACN/DCM (1:1), rt, 24 h.
2
11
counterparts 3, can accommodate the bulky adamantane−
tetraoxane moiety at P without significantly affecting the
3
enzyme inhibitory activity. Structure−activity data revealed that
substituents at C-5 of the pyrimidine, which interact with the S
1
pocket, markedly affect the potency, with compounds bearing a
chlorine and bromine 270 and 55 times more potent than their
unsubstituted counterparts, respectively (4a and 4e versus 4f).
In contrast, a chlorine atom at C-6 had a deleterious effect on
the inhibitory capacity, compared to the unsubstituted
compound (4g versus 4f), suggesting that substituents at this
position do not interact adequately with the S binding site.
1
Extending the linker between the two pharmacophoric moieties
decreased the activity by 6-fold (4a versus 4b). Finally, varying
the P moiety suggested that the isobutyl and cyclohexyl groups
2
Figure 3. Time-dependence profile for the activation of hybrid 4a by
are well accommodated by the S binding pocket, a result in
2
1
8 equiv of FeSO in aqueous acetonitrile.
4
19
́
line with that reported by Coteron et al.
The predicted binding modes of hybrids 4 were further
supported by covalent molecular docking calculations. The
C
dx.doi.org/10.1021/jm5004528 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 1. Antiplasmodial Activity against Chloroquine-Sensitive (3D7), Chloroquine-Resistant (W2), and Atovaquone-Resistant
(
FCR3) Strains of P. falciparum, Falcipain-2 (FP-2) Inhibition, and Digestive Vacuole (DV) Swelling Induced by Hybrids 4a−g
and Ketone 13
IC50 (nM)
R1
R2
3D7
W2
FCR3
FP-2
DV swelling (123 nM)
a
compd
X
b
4
4
4
4
4
4
4
1
a
b
c
d
e
f
CO
−CH CH(CH )
5-Br
5-Br
5-Br
5-Br
5-Cl
5-H
6-Cl
58.1 ± 7.8
18.9 ± 4.2
70.7 ± 1.2
78.5 ± 8.4
80.9 ± 17.2
13.1 ± 0.6
28.4 ± 7.9
48.2 ± 11.1
36.2 ± 4.7
9.25 ± 3.5
9.8 ± 1.5
22.8 ± 2.8
29.5 ± 11.7
22.4 ± 12.3
33.8 ± 0
47.3 ± 1.7
48.9 ± 2.5
48.1 ± 3.2
14.1 ± 1.4
64.7 ± 5.5
ND
16.4 ± 4.2
95.1 ± 0.4
112 ± 9
25.3 ± 7.1
3.4 ± 0.6
909 ± 93
978 ± 205
58.4 ± 0.4
yes
yes
2
3
2
2
4
b
CH CO
−CH CH(CH )
ND
2
2
3
b
b
CO
CO
CO
CO
CO
−CH CH(CH )
ND
ND
2
2
−CH(CH2)5
81.2 ± 7.2
53.3 ± 7.6
30.3 ± 9.4
37.7 ± 6.4
yes
yes
no
−CH CH(CH )
2
3
2
2
2
−CH CH(CH )
2
3
g
3
−CH CH(CH )
no
2
3
b
ND
yes
ART
CQ
7.7 ± 8.7
36.2 ± 0.4
a
b
Observation of DV swelling at 123 nM concentration. ND: not determined.
Figure 4. (A) Superimposition of docked conformations of hybrids 4a (green) and 4e (light-gray) inside FP-2 binding pocket. (B) Docked
conformation of ketone 13 inside FP-2 binding pocket. (C) Closer view of the interactions between compound 4e and residues present in the FP-2
binding pocket.
crystal structure of FP-2 complexed with the epoxysuccinate
E64 at a resolution of 2.9 Å (PDB code: 3BPF) was employed
in the docking calculations. Inspection of the top ranking
solutions of 4a, 4b, 4e−g, and 13 inside the falcipain-2 binding
pocket (Figure 4A; see Supporting Information for a closer
view), selected according to their goldscore fitness function,
revealed that these compounds present the pyrimidine
thioimidate adduct resulting from the nucleophilic attack of
establishes a hydrogen bond with Gly83 (O···H ∼ 1.3 Å). But
the most prominent feature observed for derivatives 4a, 4b, and
4
e is the strong halogen bond established between 5-Br/5-Cl
and the Asn81 oxygen atom, ranging from 2.8 to 3.2 Å (Figure
C). These values are lower than the sum of the van der Waals
4
23
radii of 3.27 Å and contrasts with the value of 3.54 Å
observed for the less potent 6-Cl counterpart 4g.
Target engagement in the parasites was confirmed by the
observation that cultured parasites incubated with compounds
the active site cysteine sitting in the S binding pocket, while
1
the isobutyl group and tetraoxane moiety fit into the S and S3
2
4
at 123 nM for 24 h beginning at the ring stage developed
binding sites, respectively. Remarkably, the parent ketone 13
adopts the same pose as 4a and 4e (Figure 4B), indicating that
the adamantane group do not significantly disturb the
positioning of the pyrimidine inside the active site. The best
poses reveal that nitrogen atom of the thioimidate adduct is
stabilized by hydrogen bonding with Gln36 (N···H ∼ 2.4 Å)
and Cys42 (N···H ∼ 1.8 Å), while the carbonyl oxygen atom
swollen digestive vacuoles with the staining characteristics of
erythrocyte cytoplasm (Table 1). It has been previously shown
that this specific abnormality is indicative of a block in
24
hemoglobin hydrolysis caused by inhibition of falcipains.
Remarkably, compounds 4 with low nanomolar IC50 values
against falcipain-2 were able to block hemoglobin degradation
D
dx.doi.org/10.1021/jm5004528 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
at 123 nM to the same extent as the parent pyrimidine nitrile
compounds 4 significantly decreased the parasite load in
Huh-7 cells when compared to untreated controls and were
more active than primaquine at 10 μM (Figure 5) but less
active than atovaquone at 2 μM (see Figure S3 in Supporting
Information). A similar activity profile was observed for the
parent compound 13. The endoperoxide artemisinin was
inactive at both dose levels. To our knowledge, this is the
first report of endoperoxides and pyrimidine nitriles active
against liver stage malaria. So far, only hybrid compounds based
1
3.
In Vitro Antimalarial Activity. Hybrid compounds 4 were
screened against chloroquine-sensitive (3D7), chloroquine-
resistant (W2), and atovaquone-resistant (FCR-3) P. falciparum
strains to assess their potential as blood stage antimalarials
(Table 2). Compounds 4a−g inhibited the growth of the three
Table 2. In Vitro Cytotoxicity and Selectivity Index of
Hybrids 4a−g and Ketone 13
27−29
on known liver stage antimalarials
both blood and liver stage activity.
were noted to have
a
CC50 (μM)
Hek293T
selectivity index (SI)
In Vivo Efficacy. Hybrid 4e was selected to evaluate the in
vivo efficacy in a murine model of infection as it displayed
excellent FP-2 inhibitory activity, high potency against all P.
falciparum strains, low cytotoxicity, and very good activity in the
liver stage assay. Hybrid 4e was tested at 30 mg/kg/day,
administered intraperitoneally for five consecutive days
beginning when parasitemia reached 4% at day 4 postinfection.
Infected mice receiving compound 4e responded initially to the
treatment, but parasitemia increased after day 8, to reach a
mean valueof 51% at day 18 postinfection. However, all animals
treated with compound 4e survived to day 18 after initial
inoculation with parasites (Figure 6).
compd
NIH3T
Hek293T
NIH3T
4
4
4
4
4
4
4
1
a
b
c
d
e
f
>100
85.5 ± 6.7
>100
8.0 ± 0.7
2.3 ± 1.0
5.5 ± 0.5
1.4 ± 0.1
4.5 ± 1.3
0.9 ± 0.1
1.7 ± 0.8
>100
>10000
3750
816
101
186
63
>3390
>4464
>2959
>2114
>2045
>2079
>100
>100
133
19
>100
g
3
>100
35
>100
>2079
a
SI was calculated as the ratio of IC50 for in vitro cytotoxicity on
Hek293T and NIH3T to the IC50 of plasmodial inhibition against the
strain W2.
CONCLUSIONS
■
P. falciparum strains with IC50 values ranging from 9.8 to 81.2
nM, while displaying negligible cytotoxicity, with EC50 values
against HEK293T mammalian cells ranging from 85 to >100
μM.
In conclusion, we developed a series of tetraoxane−pyrimidine
nitrile hybrids 4 endowed with two distinct mechanisms of
action, a feature predicted to help prevent selection of drug
resistance. Remarkably, appending a large adamantane-based
Activity against liver-stage malaria parasites is highly
desirable, in particular for radical cure of Plasmodium vivax
and Plasmodium ovale infections and in drugs used for the
tetraoxane moiety to the P position of the pyrimidine scaffold
3
did not affect inhibitory potency against falcipain-2 or general
structure−activity relationship trends when compared to
previously reported pyrimidine nitriles. Although this enzyme
inhibitory profile translated into excellent in vitro potency
against the erythrocytic stage malaria parasites, there was only
moderate in vivo efficacy. Overall, this new class of hybrid
antimalarial compounds displayed good activity against the
blood and liver stages of malaria parasites and it is thus
2
5,26
prevention of malaria.
Thus, compounds 4 were also
evaluated for their activity against liver stages of the rodent
parasite Plasmodium berghei and for cytotoxicity to Huh-7
human hepatoma cells, employing previously reported
2
7
methods. Primaquine, the only approved drug to treat liver
stage/relapsing malaria, and atovaquone were also included as
positive controls for liver stage activity. Most hybrid
26
deserving of further exploration.
Figure 5. In vitro antimalarial activity against the liver stage of infection. In vitro inhibition of hepatic P. berghei infection by compounds 4a−g and
3. Compounds were added to Huh-7 hepatoma cells 1 h before infection with luciferase-expressing sporozoites. An amount of DMSO equivalent to
1
that in the highest compound concentration tested was used as a control. Then 48 h after the addition of P. berghei sporozoites, cell confluence (dots
on bar plots) was assessed by AlamarBlue fluorescence, and the infection rate (bars) was measured by quantifying the luciferase activity by
luminescence. The effects of two concentrations (2 and 10 μM) of compounds 4a−g and 13 are shown. Results are expressed as means ± standard
deviations.
E
dx.doi.org/10.1021/jm5004528 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 6. In vivo mouse efficacy studies for compound 4e in the P. berghei mouse model. Mice were infected with parasites on day 0, and compound
e dosing began on day 4 and lasted for a total of 5 consecutive days (indicated by the arrows). Dosing was by ip administration at 30 mg/kg bw. (A)
4
Parasitemia curve; (B) survival curve.
EXPERIMENTAL SECTION
ice water (30 mL) and extracted with EtOAc (2 × 30 mL). The
combined organic phases were washed with saturated solution of
NaHCO (30 mL), dried over Na SO , filtered, and concentrated.
■
Chemistry. All chemicals and solvents were of analytical reagent
grade and were purchased from Alfa Aesar or Sigma-Aldrich.
Tetrahydrofuran was dried before use. Thin layer chromatography
was performed using Merck silica gel 60F254 aluminum plates and
visualized by UV light, iodine, potassium permanganate dip, and/or p-
anisaldehyde dip. Flash column chromatography was performed using
Merck silica gel 60 (230−400 mesh ASTM) eluting with various
solvent mixtures and using an air aquarium pump to apply pressure.
NMR spectra were recorded on a Bruker 400 Ultra-Shield (400 MHz)
3
2
4
Purification by flash chromatography gave the pure compound.
Compound 4a. White solid (method A: 38% yield/method B: 60%
1
yield); mp 113−114 °C. H NMR (400 MHz, CDCl ) δ = 8.47 (s,
3
1
0
1
3
H), 7.79 (s, 1H), 3.67 (bs, 2H), 2.38 (bs, 1H), 2.11−1.46 (m, 23H),
.99 (d, J = 6.5 Hz, 6H) ppm. C NMR (101 MHz, CDCl ) δ =
71.9, 160.7, 160.7, 141.6, 115.4, 110.7, 106.7, 106.2, 59.0, 41.6, 36.9,
3.1, 27.0, 26.3, 20.2 ppm. HRMS-ESI: m/z [M + Na] calcd for
13
3
+
C H BrN O , 598.1641; found, 598.1618.
26
34
5
5
in CDCl ; chemical shifts, δ, are expressed in ppm, and coupling
3
Compound 4b. White solid (method A: 27% yield); mp 94−96 °C.
constants, J, are expressed in Hz. Mass analyses were determined using
a Micromass Quattro Micro API spectrometer, equipped with a
Waters 2695 HPLC module and Waters 2996 photodiode array
detector. High resolution mass spectra were performed in Unidade de
Espectrometria de Masas (HMRS-ESI-TOF), Santiago de Composte-
la, Spain. All compounds tested in the biological assays were
determined to be >95% pure by elemental analysis (for C, H, and
N). Melting points were determined using a Kofler Bock Monoscop M
and are uncorrected.
1
H NMR (400 MHz, CDCl ) δ = 8.46 (s, 1H), 7.63 (s, 1H), 3.67 (bs,
3
2
2
1
3
H), 2.19 (d, J = 6.7 Hz, 2H), 2.09−1.46 (m, 22H), 1.41−1.20 (m,
H), 0.96 (d, J = 6.7 Hz, 6H) ppm. 13C NMR (101 MHz, CDCl ) δ =
3
69.6, 160.7, 160.6, 141.6, 115.4, 110.6, 107.5, 106.0, 59.1, 39.5, 36.9+,
3.3, 33.1, 33.1, 27.0, 27.0, 26.3, 20.2 ppm. HRMS-ESI: m/z [M + H]
calcd for C H BrN O , 590.1978; found, 590.1961.
27 37
5
5
Compound 4c. White solid (method A: 10% yield/method B: 66%
1
yield); mp 190−191 °C. H NMR (400 MHz, CDCl ) δ = 8.44 (s,
3
1
1
H), 7.69 (s, 1H), 3.75 (bs, 2H), 2.42−2.31 (m, 1H), 2.25−2.15 (m,
Preparation of Compounds 4 (Method A). (1) To a solution of
compound 11a−f (0.2 mmol) in ACN (1.5 mL) was added p-
toluenesulfonic acid (0.8 mmol). The mixture was stirred overnight at
room temperature. Then the solvent was removed under reduced
pressure and the crude taken up in EtOAc (30 mL) and washed with 1
M solution of Na CO (30 mL). The aqueous phase was further
13
H), 2.07−1.47 (m, 26H), 1.27 (m, 4H) ppm. C NMR (101 MHz,
CDCl ) δ = 172.0, 160.7, 160.7, 141.8, 115.5, 110.9, 106.8, 106.2, 56.4,
3
4
+
1.7, 37.5, 37.0, 33.2, 30.7, 27.1, 27.1, 25.2 ppm. HRMS-ESI: m/z [M
+
H] calcd for C H BrN O , 602.1978; found, 602.1962.
28 37 5 5
Compound 4d. White solid (method A: 8% yield); mp 148−149
2
3
1
°
C. H NMR (400 MHz, CDCl ) δ = 8.41 (s, 1H), 7.47 (s, 1H), 4.54
extracted with EtOAc (2 × 20 mL). The combined organic phases
3
(
bs, 1H), 2.42 (bs, 1H), 2.13−1.20 (m, 31H), 1.20−1.01 (m, 2H)
were dried over Na SO , filtered, and concentrated to give the free
2
4
13
ppm. C NMR (101 MHz, CDCl ) δ = 172.5, 160.4, 159.6, 141.7,
amine. (2) A solution of tetraoxane 7a−b (0.24 mmol) in thionyl
chloride (2 mL) was refluxed for 2 h. The mixture was concentrated
under reduced pressure, and then the acid chloride was taken up in dry
THF (1 mL) in nitrogen atmosphere. A solution of the appropriate
free amine (0.2 mmol) in dry THF (1 mL) was added to the acid
chloride. After stirring for 30 min at room temperature under nitrogen,
DIPEA (0.6 mmol) was added, followed by overnight stirring in the
same conditions. The mixture was diluted with DCM (30 mL) and
water (30 mL), and the phases were separated. The aqueous phase was
further extracted with DCM (2 × 20 mL). The combined organic
extracts were dried over Na SO , filtered, and concentrated.
3
1
15.5, 110.7, 106.7, 106.3, 59.6, 36.9, 33.1, 27.0, 25.5 ppm. HRMS-
+
ESI: m/z [M + H] calcd for C H BrN O , 602.1978; found,
28 37
5
5
602.1971.
Compound 4e. White solid (method B: 57% yield); mp 134−137
1
°C. H NMR (400 MHz, CDCl
(bs, 2H), 2.40−2.27 (m, 1H), 2.08−1.46 (m, 23H), 0.97 (d, J = 6.7
Hz, 6H) ppm. 13C NMR (101 MHz, CDCl
) δ = 172.2, 159.8, 157.7,
141.1, 117.7, 115.3, 110.7, 106.7, 59.2, 41.6, 36.9, 33.1, 27.0, 27.0, 26.4,
) δ = 8.28 (s, 1H), 7.79 (s, 1H), 3.66
3
3
+
20.2 ppm. HRMS-ESI: m/z [M + H] calcd for C26H35ClN O ,
5 5
532.2327; found, 532.2312.
2
4
Purification by flash chromatography gave the pure compound.
Preparation of Compounds 4 (Method B). To a solution of
compound 12a−e (0.2 mmol) and DABCO (0.2 mmol) in DMSO/
water (9:1, 2 mL) was added KCN (0.22 mmol). The solution was
stirred for 5 h at room temperature. Then the mixture was poured into
Compound 4f. White solid (method B: 43% yield); mp 196−197
1
°C. H NMR (400 MHz, CDCl ) δ = 8.22 (d, J = 6.0 Hz, 1H), 8.06
3
(bs, 1H), 6.61 (d, J = 6.0 Hz, 1H), 3.97−3.25 (m, 2H), 2.47−2.33 (m,
1
3
1H), 2.07−1.47 (m, 23H), 0.93 (d, J = 6.5 Hz, 6H) ppm. C NMR
(101 MHz, CDCl ) δ = 158.5, 156.2, 144.1, 116.0, 110.7, 106.7, 105.6,
3
F
dx.doi.org/10.1021/jm5004528 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
4
1.8, 36.9, 33.1, 29.7, 27.0, 26.7, 20.1 ppm. HRMS-ESI: m/z [M −
steadily until day 6 postinfection, when all mice died with symptoms of
experimental cerebral malaria.
−
H] calcd for C H N O , 496.2560; found, 496.2568.
26
34
5
5
Compound 4g. White solid (method B: 37% yield); mp 106−108
1
°
C. H NMR (400 MHz, CDCl ) δ = 7.58 (s, 1H), 6.60 (s, 1H),
ASSOCIATED CONTENT
Supporting Information
Experimental details for the preparation of compounds 7a−b
and 11−13, the description of iron(II) activation of hybrid 4a,
and computational methodology. This material is available free
of charge via the Internet at http://pubs.acs.org.
3
■
3
6
1
.98−3.13 (m, 2H), 2.40 (bs, 1H), 2.14−1.46 (m, 23H), 0.95 (d, J =
*
S
13
.4 Hz, 6H) ppm. C NMR (101 MHz, CDCl ) δ = 160.7, 143.5,
3
15.0, 110.8, 106.6, 104.6, 41.9, 36.9, 33.1, 27.0, 26.7, 20.1 ppm.
−
HRMS-ESI: m/z [M − H] calcd for C H ClN O , 530.2170;
26
33
5
5
found, 530.2173.
In Vitro Activity Assay against Blood Stage of Infection.
Synchronized ring-stage P. falciparum strain W2 parasites were
cultured with multiple concentrations of test compounds (added
from 1000× stocks in DMSO) in RPMI 1640 medium with 10%
human serum. After 48 h of incubation, when control cultures
contained new rings, parasites were fixed with 1% formaldehyde in
phosphate-buffered saline (PBS), pH 7.4, for 48 h at room
temperature; then they were labeled with YOYO-1 (1 nM; Molecular
Probes) in 0.1% Triton X-100 in PBS. Parasitemia was determined
from dot plots (forward scatter versus fluorescence) acquired on a
FACSort flow cytometer using CellQuest software (Becton Dick-
inson). Fifty percent inhibitory concentrations (IC50 values) for
growth inhibition were determined with GraphPad Prism software
from plots of the percentage of parasitemia of the control relative to
the inhibitor concentration. In each case, the goodness of the curve fit
AUTHOR INFORMATION
Corresponding Authors
For P.M.O.: phone, 0151-794-3553; E-mail, p.m.oneill01@
liverpool.ac.uk.
For R.M.: fax, + 351 217946470; E-mail, rmoreira@ff.ul.pt.
■
*
*
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
2
was documented by R values of 0.95.
ACKNOWLEDGMENTS
This work was supported by Fundaca
Tecnologia, Portugal: grants PTDC/SAU-FAR/118459/2010,
PEst-OE/SAU/UI4013/2014, REDE/1501/REM/2005, and
PTDC/SAU-MIC/117060/2010; fellowship SFRH/BD/
In Vitro Activity against Liver Stage of Infection. Inhibition of
liver-stage infection by test compounds was determined by measuring
the luminescence intensity in Huh-7 cells infected with a firefly
luciferase expressing P. berghei line, PbGFP-Luccon, as previously
■
̧
o
̃
para a Cien
̂
cia e
30
described. Huh-7 cells, from a human hepatoma cell line, were
cultured in 1640 rpmI medium supplemented with 10% v/v fetal calf
serum, 1% v/v nonessential amino acids, 1% v/v penicillin/
streptomycin, 1% v/v glutamine, and 10 mM 4-(2-hydroxyethyl)-1-
6
3200/2009 to R.O.), and Fundaca
to R.M.).
̃
̧ oLuso−Americana (award
piperazineethanesulfonic acid (HEPES), pH 7, and maintained at 37
ABBREVIATIONS USED
ACT, artemisinin-based combination therapy; ART, artemisi-
nin; CQ, chloroquine; FP-2, falcipain-2
4
■
°
C with 5% CO . For infection assays, Huh-7 cells (1.2 × 10 per well)
2
were seeded in 96-well plates the day before drug treatment and
infection. The medium was replaced by medium containing the
appropriate concentration of each compound approximately 1 h prior
to infection with sporozoites freshly obtained through disruption of
salivary glands of infected female Anopheles stephensi mosquitoes.
Sporozoite addition was followed by centrifugation at 1700g for 5 min.
Then 24 h after infection, the medium was replaced by fresh medium
containing the appropriate concentration of each compound. Parasite
infection load was measured 48 h after infection. The effect of the
compounds on the viability of Huh-7 cells was assessed by the
AlamarBlue assay (Invitrogen, U.K.) using the manufacturer’s
protocol.
REFERENCES
■
(
1) World Malaria Report 2013; World Health Organization: Geneva,
013.
2
(
2) White, N. J.; Pukrittayakamee, S.; Hien, T. T.; Faiz, M. A.;
Mokuolu, O. A.; Dondorp, A. M. Malaria. Lancet 2014, 383, 723−735.
3) White, N. J. Artemisinin ResistanceThe Clock Is Ticking.
Lancet 2010, 376, 2051−2052.
4) Yeung, S.; Socheat, D.; Moorthy, V. S.; Mills, A. J. Artemisinin
Resistance on the Thai−Cambodian Border. Lancet 2009, 374, 1418−
1419.
5) Dondorp, A. M.; Nosten, F.; Yi, P.; Das, D.; Phyo, A. P.; Tarning,
J.; Lwin, K. M.; Ariey, F.; Hanpithakpong, W.; Lee, S. J.; Ringwald, P.;
Silamut, K.; Imwong, M.; Chotivanich, K.; Lim, P.; Herdman, T.; An,
S. S.; Yeung, S.; Singhasivanon, P.; Day, N. P. J.; Lindegardh, N.;
Socheat, D.; White, N. J. Artemisinin Resistance in Plasmodium
falciparum Malaria. N. Engl. J. Med. 2009, 361, 455−467.
(6) Guidelines for the Treatment of Malaria, 2nd ed.; World Health
Organization: Geneva, 2010.
(
(
Falcipain-2 Inhibition Assay. IC s against falcipain-2 were
50
24
determined as described earlier. Briefly, equal amounts (∼1 nM) of
recombinant falcipain-2 were incubated with different concentrations
of vinyl sulfones (added from 100× stocks in DMSO) in 100 mM
sodium acetate (pH 5.5)−10 mM dithiothreitol for 30 min at room
temperature before addition of the substrate benzoxycarbonyl-Leu-
Arg-7-amino-4-methyl-coumarin (final concentration, 25 μM). Fluo-
rescence was continuously monitored for 30 min at room temperature
in a Labsystems Fluoroskan II spectrofluorometer. IC s were
(
5
0
determined from plots of activity over enzyme concentration with
GraphPad Prism software.
(7) O’Neill, P. M.; Barton, V. E.; Ward, S. A. The Molecular
Mechanism of Action of ArtemisininThe Debate Continues.
Molecules 2010, 15, 1705−1721.
In Vivo Assay. C57Bl/6J mice were infected by intraperitoneal
6
inoculations of 2 × 10 GFP-expressing P. berghei ANKA-infected
(8) Meunier, B. Hybrid Molecules with a Dual Mode of Action:
Dream or Reality? Acc. Chem. Res. 2008, 41, 69−77.
erythrocytes. Parasitemias were monitored daily by microscopy and
flow cytometry. Drug treatment was initiated when blood parasitemias
were ca. 3.9%. At this point, 30 mg/kg bw of compound 4a were
administered once daily by intraperitoneal injection for 5 days. An
equivalent amount of drug vehicle was injected in control mice.
Parasitemias, disease symptoms, and survival were monitored daily
from the onset of treatment until the end of the experiment. The
experiment was terminated following 10 consecutive of growing blood
parasitemia in treated mice. Parasitemias of control mice increased
(9) Ariey, F.; Witkowski, B.; Amaratunga, C.; Beghain, J.; Langlois,
A.-C.; Khim, N.; Kim, S.; Duru, V.; Bouchier, C.; Ma, L.; Lim, P.;
Leang, R.; Duong, S.; Sreng, S.; Suon, S.; Chuor, C. M.; Bout, D. M.;
Men
Ringwald, P.; Le Bras, J.; Berry, A.; Barale, J.-C.; Fairhurst, R. M.;
Benoit-Vical, F.; Mercereau-Puijalon, O.; Menard, D. A Molecular
́
ard, S.; Rogers, W. O.; Genton, B.; Fandeur, T.; Miotto, O.;
́
Marker of Artemisinin-Resistant Plasmodium falciparum Malaria.
Nature 2014, 505, 50−55.
G
dx.doi.org/10.1021/jm5004528 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(
10) Martinelli, A.; Moreira, R.; Ravo, P. V. L Malaria Combination
Therapies: Advantages and Shortcomings. Mini-Rev. Med. Chem. 2008,
, 201−212.
11) Oliveira, R.; Newton, A. S.; Guedes, R. C.; Miranda, D.; Amewu,
(26) malERA Consultative Group on Drugs. A Research Agenda for
Malaria Eradication: Drugs. PLoS Med. 2011, 8, e1000402.
(27) Miranda, D.; Capela, R.; Albuquerque, I. S.; Meireles, P.; Paiva,
8
(
I.; Nogueira, F.; Amewu, R.; Gut, J.; Rosenthal, P. J.; Oliveira, R.;
R. K.; Srivastava, A.; Gut, J.; Rosenthal, P. J.; O’Neill, P. M.; Ward, S.
A.; Lopes, F.; Moreira, R. An Endoperoxide-Based Hybrid Approach
to Deliver Falcipain Inhibitors Inside Malaria Parasites. ChemMed-
Chem 2013, 8, 1528−1536.
̂
Mota, M. M.; Moreira, R.; Marti, F.; Prudencio, M.; O’Neill, P. M.;
Lopes, F. Novel Endoperoxide-Based Transmission-Blocking Anti-
malarials with Liver- and Blood-Schizontocidal Activities. ACS Med.
Chem. Lett. 2013, 5, 108−112.
(28) Capela, R.; Cabal, G. G.; Rosenthal, P. J.; Gut, J.; Mota, M. M.;
(
12) Rosenthal, P. J.; Sijwali, P. S.; Singh, A.; Shenai, B. R. Cysteine
Moreira, R.; Lopes, F.; Prudencio, M. Design and Evaluation of
̂
Proteases of Malaria Parasites: Targets for Chemotherapy. Curr.
Pharm. Des. 2002, 8, 1659−1672.
Primaquine−Artemisinin Hybrids as a Multistage Antimalarial
Strategy. Antimicrob. Agents Chemother. 2011, 55, 4698−4706.
(
13) Ettari, R.; Bova, F.; Zappala, M.; Grasso, S.; Micale, N.
(
̈
29) Lodige, M.; Lewis, M. D.; Paulsen, E. S.; Esch, H. L.; Pradel, G.;
Falcipain-2 Inhibitors. Med. Res. Rev. 2010, 30, 136−167.
Lehmann, L.; Brun, R.; Bringmann, G.; Mueller, A.-K. A Primaquine−
Chloroquine Hybrid with Dual Activity against Plasmodium Liver and
Blood Stages. Int. J. Med. Microbiol. 2013, 303, 539−547.
(
14) Greenspan, P. D.; Clark, K. L.; Cowen, S. D.; McQuire, L. W.;
Tommasi, R. A.; Farley, D. L.; Quadros, E.; Coppa, D. E.; Du, Z. M.;
Fang, Z.; Zhou, H. H.; Doughty, J.; Toscano, K. T.; Wigg, A. M.;
Zhou, S. Y. N-Arylaminonitriles as Bioavailable Peptidomimetic
Inhibitors of Cathepsin B. Bioorg. Med. Chem. Lett. 2003, 13, 4121−
̂
(30) Ploemen, I. H. J.; Prudencio, M.; Douradinha, B. G.; Ramesar,
J.; Fonager, J.; van Gemert, G. J.; Luty, A. J. F.; Hermsen, C. C.;
Sauerwein, R. W.; Baptista, F. G.; Mota, M. M.; Waters, A. P.; Que, I.;
Lowik, C.; Khan, S. M.; Janse, C. J.; Franke-Fayard, B. M. D.
Visualisation and Quantitative Analysis of the Rodent Malaria Liver
Stage by Real Time Imaging. PLoS One 2009, 4, e7881.
4
(
124.
15) Altmann, E.; Cowan-Jacob, S. W.; Missbach, M. Novel Purine
Nitrile Derived Inhibitors of the Cysteine Protease Cathepsin K. J.
Med. Chem. 2004, 47, 5833−5836.
(
16) Ehmke, V.; Heindl, C.; Rottmann, M.; Freymond, C.;
Schweizer, W. B.; Brun, R.; Stich, A.; Schirmeister, T.; Diederich, F.
Potent and Selective Inhibition of Cysteine Proteases from
Plasmodium falciparum and Trypanosoma brucei. ChemMedChem
2
(
011, 6, 273−278.
17) Ehmke, V.; Kilchmann, F.; Heindl, C.; Cui, K.; Huang, J.;
Schirmeister, T.; Diederich, F. Peptidomimetic Nitriles as Selective
Inhibitors for the Malarial Cysteine Protease Falcipain-2. MedChem-
Comm 2011, 2, 800−804.
(
18) Ehmke, V.; Quinsaat, J. E. Q.; Rivera-Fuentes, P.; Heindl, C.;
Freymond, C.; Rottmann, M.; Brun, R.; Schirmeister, T.; Diederich, F.
Tuning and Predicting Biological Affinity: Aryl Nitriles as Cysteine
Protease Inhibitors. Org. Biomol. Chem. 2012, 10, 5764−5768.
(
19) Coteron
B.; Fernandez, E.; Ferrer, S.; Gamo, F. J.; Gordo, M.; Gut, J.; de Las
Heras, L.; Legac, J.; Marco, M.; Miguel, J.; Munoz, V.; Porras, E.; de La
́
, J. M.; Catterick, D.; Castro, J.; Chaparro, M. J.; Díaz,
́
̃
Rosa, J. C.; Ruiz, J. R.; Sandoval, E.; Ventosa, P.; Rosenthal, P. J.;
Fiandor, J. M. Falcipain Inhibitors: Optimization Studies of the 2-
Pyrimidinecarbonitrile Lead Series. J. Med. Chem. 2010, 53, 6129−
6
(
152.
20) O’Neill, P. M.; Amewu, R. K.; Nixon, G. L.; Bousejra-El Garah,
F.; Mungthin, M.; Chadwick, J.; Shone, A. E.; Vivas, L.; Lander, H.;
Barton, V.; Muangnoicharoen, S.; Bray, P. G.; Davies, J.; Park, B. K.;
Wittlin, S.; Brun, R.; Preschel, M.; Zhang, K.; Ward, S. A. Identification
of a 1,2,4,5-Tetraoxane Antimalarial Drug-Development Candidate
(
RKA 182) with Superior Properties to the Semisynthetic
Artemisinins. Angew. Chem., Int. Ed. Engl. 2010, 49, 5693−5697.
21) Ghorai, P.; Dussault, P. H. Broadly Applicable Synthesis of
,2,4,5-Tetraoxanes. Org. Lett. 2009, 11, 213−216.
22) Bousejra-El Garah, F.; Wong, M. H.-L.; Amewu, R. K.;
(
1
(
Muangnoicharoen, S.; Maggs, J. L.; Stigliani, J.-L.; Park, B. K.;
Chadwick, J.; Ward, S. A.; O’Neill, P. M. Comparison of the Reactivity
of Antimalarial 1,2,4,5-Tetraoxanes with 1,2,4-Trioxolanes in the
Presence of Ferrous Iron Salts, Heme, and Ferrous Iron Salts/
phosphatidylcholine. J. Med. Chem. 2011, 54, 6443−6455.
(
23) Sirimulla, S.; Bailey, J. B.; Vegesna, R.; Narayan, M. Halogen
Interactions in Protein−Ligand Complexes: Implications of Halogen
Bonding for Rational Drug Design. J. Chem. Inf. Model. 2013, 53,
2
(
781−2791.
24) Rosenthal, P. J.; Olson, J. E.; Lee, G. K.; Palmer, J. T.; Klaus, J.
L.; Rasnick, D. Antimalarial Effects of Vinyl Sulfone Cysteine
Proteinase Inhibitors. Antimicrob. Agents Chemother. 1996, 40, 1600−
1
603.
(
25) Derbyshire, E. R.; Mota, M. M.; Clardy, J. The Next
Opportunity in Anti-Malaria Drug Discovery: The Liver Stage. PLoS
Pathog. 2011, 7, e1002178.
H
dx.doi.org/10.1021/jm5004528 | J. Med. Chem. XXXX, XXX, XXX−XXX