Structure-activity relationships and blood distribution of antiplasmodial aminopeptidase-1 inhibitors

Article abstract of DOI:10.1021/jm301506h

Malaria is a severe infectious disease that causes between 655 000 and 1.2 million deaths annually. To overcome the resistance to current drugs, new biological targets are needed for drug development. Aminopeptidase M1 (PfAM1), a zinc metalloprotease, has

Full text of DOI:10.1021/jm301506h

Article
pubs.acs.org/jmc
Structure−Activity Relationships and Blood Distribution of
Antiplasmodial Aminopeptidase‑1 Inhibitors
Rebecca Deprez-Poulain,*,†,‡,§ Marion Flipo,†,‡,§ Catherine Piveteau,†,‡,§ Florence Leroux,†,‡,§
Sandrine Dassonneville,†,‡,§ Isabelle Florent,∥ Louis Maes,⊥ Paul Cos,⊥ and Benoit Deprez*,†,‡,§
†INSERM U761, Biostructures and Drug Discovery and Faculte
Lille F-59000, France
́ ́
de Pharmacie, Universite Lille Nord de France, 3 rue du Pr Laguesse,
‡Institut Pasteur de Lille, IFR 142, Lille F-59021, France
§PRIM, Lille F-59000, France
∥CNRS/MNHN, UMR7245, Molec
Environnent, F-75231 Paris, France
́ ̀
ules de Communication et Adaptation des Micro-organismes, Adaptation des Protozoaires a leur
⊥Laboratory of Microbiology, Parasitology & Hygiene, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of
Antwerp, B-2020 Antwerp, Belgium
ABSTRACT: Malaria is a severe infectious disease that causes between 655 000 and 1.2 million deaths annually. To overcome
the resistance to current drugs, new biological targets are needed for drug development. Aminopeptidase M1 (PfAM1), a zinc
metalloprotease, has been proposed as a new drug target to fight malaria. Herein, we disclosed the structure−activity
relationships of a selective family of hydroxamate PfAM1 inhibitors based on the malonic template. In particular, we performed a
“fluoro-scanning” around hit 1 that enlightened the key positions of the halogen for activity. The docking of the best inhibitor 2 is
consistent with in vitro results. The stability of 2 was evaluated in microsomes, in plasma, and toward glutathione. The in vivo
distribution study performed with the nanomolar hydroxamate inhibitor 2 (BDM14471) revealed that it reaches its site of action.
However, it fails to kill the parasite at concentrations relevant to the enzymatic inhibitory potency, suggesting that killing the
parasite remains a challenge for potent and druglike catalytic-site binding PfAM1 inhibitors. In all, this study provides important
insights for the design of inhibitors of PfAM1 and the validity of this target.
Florent et al. characterized the aminopeptidase M1 (PfAM1
or PfM1AAP) that shows a strict aminopeptidase activity at pH
7.4 and a broad substrate spectrum.17,18 PfAM1 localizes
differently in trophozoites and schizonts, suggesting a particular
role in the intraerythrocytic life of the parasite. PfAM1 degrades
oligopeptides resulting from hemoglobin digestion either in the
vacuole19 or the cytosol.20 The inhibition of PfAM1 by bestatin
analogues induces swollen digestive vacuoles due to the
accumulation of oligopeptides.21 Also, a role in the reinvasion
of erythrocytes has been found, in agreement with the
observation of Kitjaroenthan et al.22 Studies on PfAM1
localization suggest that it is trafficked via the parasitophorous
INTRODUCTION
■
The WHO/TDR initiative promotes drug target validation for
neglected diseases.1 As such, innovative strategies are being
developed to discover new antiplasmodial targets and may
include a target-driven approach by cell-based screening,2 drug
repositioning, and chemogenomics.3,4 In this context, the
Plasmodium proteases are putative targets.5−8 Their study
benefits from the successful work done on proteases in other
pathologies, such as AIDS, hypertension, and type-II
diabetes.9,10 Among the metalloproteases of Plasmodium,
falcilysin is involved in the intravacuolar digestion of globin
and has no reported inhibitor.11 On the other hand,
aminopeptidases degrading oligopeptides resulting from
hemoglobin digestion are well-studied.12−14 Recently, inhibitors
of LAP (leucine-aminopeptidase), a hexameric aminopeptidase
of the M17-family, have been described.15,16
Received: July 12, 2012
Published: November 23, 2012
© 2012 American Chemical Society
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vacuole.23 PfAM1 is also present in the nucleus, but so far no
a
Scheme 1. Synthesis of Carboxylic Acid Precursors 5a−e
function can be related to its nuclear localization.19
Given these results, PfAM1 can be considered an attractive
candidate to initiate screening and lead optimization. Our
research group was the first to describe PfAM1 screening and
the first two series of hydroxamic nonpeptidic nonselective
PfAM1 inhibitors with a mixed mode of action.24 The screening
of a focused library of zinc-binding molecules25 resulted in a
malono-hydroxamic druggable hit, 1.26 Chemical modifications
explored the introduction of steric constrains at the malonic
position and led to the discovery of 2, a nanomolar selective
inhibitor of PfAM1. 2 shows good physicochemical, in vitro
plasma stability and promising antiplasmodial activity in vitro.26
Other inhibitors, such as phosphonopeptides, were subse-
quently published27,28 and were designed as mixed inhibitors of
the aminopeptidases of the M1 and M17 families. The binding
of one nonselective compound to PfAM1 was assessed by
cocrystallization with the recombinant enzyme whose X-ray
structure had been published.29 Recently, peptide-based
bestatin analogues were also disclosed.28
This paper reports the further optimization of our inhibitors
whereby initial medicinal chemistry experiments revealed that
the introduction of a fluorine on the scaffold had a significant
impact on the inhibitory activity.26 First, the effect on potency
of systematic moving of fluorine (“fluorine scanning”)30,31
around compounds 1 and 2 was evaluated. Also substitution at
the malonic carbon and the introduction of a pyridine to
introduce a positive charge, which is a hallmark of many
antiplasmodials, were explored. Analogues on PfAM1 covering
a 2.5 log range of IC50s were tested in vitro on the
intraerythrocytic cycle. We analyze these results in the light
of structure−activity relationships on the isolated enzyme and
distribution/stability data of the most active PfAM1 inhibitor.
a
(a) p-Fluorobenzaldehyde, piperidine, abs EtOH, reflux, 3.5 h; (b)
NaBH3CN, CH3CN, 24 h, rt; (c) KOH, abs EtOH, overnight, rt; (d) 1
equiv LiOH, THF, H2O, 4 h, rt; (e) (i) 1.2 equiv oxalyl chloride,
DCM, cat. DMF, 45 min, 0 °C, (ii) 3 equiv DIEA, 0.85 equiv O-
tritylhydroxylamine, DCM, 0 °C then rt, 3 h; (f) 4 equiv LiOH, THF,
H2O, overnight, rt; (g) (i) EtONa, EtOH, 1 h, 50 °C, (ii) benzyl
bromide, 2 h, 50 °C.
a
Scheme 2. Synthesis of PfAM1 inhibitors 6−14
RESULTS AND DISCUSSION
■
Synthesis of Inhibitors 1−14. Inhibitors were synthesized
as described in Schemes 1 and 2. Precursors 5a,b,d,e, 1−4, and
12 were synthesized as previously described.26,32 5c was
synthesized by a Knoevenagel reaction between 4-fluoroben-
zaldehyde and ethyl N-tert-butoxymalonamate in refluxed
ethanol in the presence of piperidine. The resulting ester was
saponified to give carboxylic acid 5c as a single E-isomer as
determined by ROESY NMR.
5a−d reacted with the corresponding amine after activation
of the carboxylic acid function using either ethylchloroformate
or EDCI/HOBt. The deprotection of the hydroxamate was
performed in acidic conditions to give compounds 6-11, 13,
and 14.
Compound 12 was obtained from 5e by coupling first the 4-
fluorobenzylamine. Then, the ester was saponified, and the
resulting carboxylic acid was activated and reacted with trityle-
protected hydroxylamine. The final deprotection was per-
formed in TFA/DCM.
PfAM1 Inhibition and Selectivity toward pAPN. In
these series, hit 1 (IC50 = 27 nM) and its analogue 2 (IC50 = 6
nM) bearing a p-fluorobenzene ring were more active than 4
(IC50 = 125 nM) deprived of a halogen (Table 1).26 Fluorine is
often used in medicinal chemistry33−35 as it is sterically small
and most often chemically inert. It is the most electronegative
halogen and forms stable bonds with carbon that are poorly
polarizable. Compounds 6−8, resulting from “fluorine scan-
ning”, display a 10-fold decrease in potency. This illustrates the
importance of the presence of fluorine in the para-position of
a
(a) (i) 1.1 equiv ethylchloroformate, 1.1 equiv TEA, DCM, 30 min, 0
°C, (ii) 1.1 equiv R1−CH2NH2, 1 h, rt; (b) R1−CH2NH2, EDCI/
HOBt, DMF/DIEA, 5 h, rt; (c) BTFA (0.75 M/TFA; 25 equiv) with
0.4% H2O, rt for t-Bu; (d) TFA 2%/DCM, TIS, 10 min, rt for trityl;
(e) KOH, abs EtOH, overnight, rt; (f) (i) 1.1 equiv ethyl
chloroformate, 1.1 equiv TEA, DCM, 30 min, 0 °C, (ii) 1.1 equiv
H2N-OTrt, HCl, 1 h, rt.
the benzylamido moiety rather than in ortho- or meta- position
or on the other phenyl ring. Interestingly, compound 9, which
has a fluorine atom at this critical position and an additional
fluorine, is less active than compound 1. This finding suggests
that fluorine on the benzyl malonyl moiety is exposed to a
fluorophobic region. The same trend is observed for
unsaturated compounds 10 and 11, analogues of 2 (IC50 = 6
nM), which are 7−10-fold less potent. Nevertheless, the
beneficial unsaturation compensates for bad fluorine position.
Substitution of a hydrogen atom by a methyl group on the
malonic carbon of 1 (compound 12) induced a 2-fold loss in
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substituent of 2 or larger substituents.26 The introduction of
a fluorine atom at this position, although tolerated, is not
critical for activity (compound 1 vs 8 and 9, Table 1).
Interestingly, a T-staking37,38 between Tyr575 and phenylmeth-
(Z)-ylidene moeity is observed (Figure 1), explaining why the
Z-isomer (2) is more active than the E-isomer (3).
Table 1. Inhibition of PfAM1, pAPN by Compounds 1−3
and 5−13
The amide function is engaged in two hydrogen bonds with
Gly460 and Ala461 backbones. The S′1 pocket is also delimited
with hydrophobic residues (Thr492, Val493, and Val523).29
Although a cation−π interaction is observed between the
benzyl group of 2 and Arg489, the introduction of the electron-
withdrawing fluorine has a positive impact on activity (2 vs 4,
Table 1). This may be the result of an increased desolvation
entropy upon binding in this pocket. Also, the presence of the
positively charged Arg489 explains why pyridine is less
tolerated on that position (compounds 13, 14, Table 1).
Parasite Growth Inhibition and Cytotoxicity. None of
the compounds tested was cytotoxic for MRC5 cells (IC50 > 32
μM, Table 2). Compounds 1, 2, 6−9, 11, and 12 inhibited
Table 2. Inhibition of Parasite Growth by Compounds 1, 2,
6−9, 11−13, and 15
a
b
PfAM1 inhibn
parasite growth
IC50 (μM)
cytotoxicity IC50
compound
IC50 (nM)
(μM)
f
1
2
6
7
8
9
27
17.4 (59 )
>64.0
32.0
f
6
11.0 (24 )
210
256
310
258
43
24.7
37.9
36.5
41.6
30.4
50.1
>64.0
>64.0
>64.0
>64.0
>64.0
32.2
11
12
56
>64.0
>64.0
13
1050
d
e
g
c
bestatine
478
8−21
_
e
g
c
hPheP[CH2]
79
24−62
_
d
Phe
d
e
g
c
15
43
6.4
_
a
b
a
b
IC50 of bestatine (nM) was 284. IC50 of bestatine (nM) was 2700.
Inative enzyme. Mean of at least three experiments; K1 strain, IC50
c
c
d
Mean of three experiments; standard error was below 10%.
of chloroquine (nM) was 154. Not determined. Reference 28 or 29.
e
f
Recombinant enzyme. FcB1 strain, IC50 of chloroquine (nM) was
g
activity, while the substitution of p-fluorophenyl by a pyridine
ring36 in compounds 1 and 2 enabled the exploration of the
properties of the enzyme pocket and generated a positive
charge. Compounds 13 and 14 were about 50 times less active
than their analogue.
All compounds were shown to be selective for the parasitic
enzyme versus the mammalian enzyme APN (neutral amino-
peptidase). The highest selectivity ratios were observed for
compounds 2 and 11.
To rationalize structure−activity relationships, compound 2
was docked in PfAM1 (using PDB ID 3T8V) (Figure 1). The
hydroxamate moiety binds the Zn2+ ion and is involved in a
hydrogen bond with Glu497. The S1 pocket of the enzyme is
hydrophobic and able to undergo major conformational
changes upon ligand binding.28 It is characterized by hydro-
phobic residues (Gln317, Val459, Met462, Glu572, Tyr575,
Met1034)29 that can accommodate the benzylmalonyl
110. 3D7 strain, IC50 of chloroquine (nM) was 10.
parasite growth at IC50 values ranging between 11 and 50 μM
(Table 2), which is comparable to other PfAM1-selective or
mixed PfAM1/LAP inhibitors12,16,27,29 or bestatine analogues.28
However, there is a lack of correlation between enzymatic and
parasite activities. Compounds very similar in structure, yet
displaying low nanomolar to micromolar potencies on the
enzymatic assay, show poor activity on the parasite (i.e., 6 vs 1;
10 vs 8).
To elucidate this large activity gap and the lack of correlation
between the in vitro inhibition of PfAM1 and the parasite
growth inhibition, in vitro ADME parameters were explored for
a set of analogues to select a compound for in vivo
pharmacokinetics.
in Vitro ADME Parameters for 2. Some key physico-
chemical parameters were measured for the most potent and
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Figure 1. Binding of 2 in PfAM1. (A) Docking of 2 in the enzyme (PDB ID: 3T8V). Zinc ion is colored magenta. The atoms oxygen and nitrogen
are colored red and blue, respectively, fluorine is colored green, and carbon is shown in light pink or light gray for, respectively, 2 or key residues of
PfAM1. Specific hydrogens are colored white. (B) Schematic representation of the interactions of 2 with PfAM1, featuring the three-letter codes of
amino acids lining the binding pocket. Polar residues are pink (blue circles for positively charged, red circles for negatively charged), and apolar
residues are green. Metal contacts are represented as black dotted lines. Hydrogen bonds in blue (acceptor) or green (donor) arrows, respectively.
Arene-H and arene-positive charge are represented as green dotted lines.
selective compounds 1, 2, and 11 (Table 3). Compounds 1 and
2 have comparable solubilities and log D, while 11 is slightly
time intervals after dosing (Figure 2). Plasma and red blood
cells (RBCs) were fractionated, and quantification of 2 was
performed using LC−MS/MS.
Table 3. Solubility, log D, and Plasma Stabilities for 1, 2, and
11
b
plasma stability (h)
a
a
compd
solubility (μM)
log D (pH 7.4)
rat
human
1
>200
198
1.2
1.1
1.7
0.8
22
>24
>24
>24
2
11
190
3.0
a
Solubility and log D are measured from a DMSO stock solution.
Half-life plasma stability at 37 °C using LC−MS/MS (MRM
b
detection mode).
more hydrophobic and less soluble. All three compounds are
stable in human plasma, but compound 2 is by far the most
stable compound in rodent plasma, which is critical for in vivo
pharmacokinetics. These stabilities are coherent with previous
data on hydroxamate stabilities.32
Figure 2. Concentration−time profile for compound 2. Concen-
Before the pharmacokinetic evaluation in mice, 2 was further
characterized in vitro. 2 displays a Michael acceptor function
that may be sensitive to soft nucleophiles like thiols. An in vitro
glutathione alkylation study39 revealed that compound 2 is
isomerized in vitro in its E-isomer 3 through the GSH-adducts
production (Z/E 54/46, after 24 h). The proportion of
combined GSH-adducts of the two unsaturated isomers reaches
50% at 24 h. 2 was also tested in vitro for mouse microsomal
stability and was found to be very stable in a classic 30-min
assay. Interestingly, this stability was observed for both the
oxidative and the nonoxidative metabolism, suggesting that no
hydrolysis of the hydroxamate moiety occurs through esterase
activity.
◇
◆
trations in plasma ( ) or red blood cells ( ) found in female CD1
mice receiving single ip administration at 50 mg/kg. Symbols represent
averages from two mice. Plasma and red blood cell data are presented
as μmol/L.
Following a single dose, 2 could be measured readily after
dosing and until the end of the experiment (6 h postdose)
(Figure 2), showing a plasma elimination half-life of 1 h (from
1 to 6 h; Table 4). As expected from the stability studies, only
traces of carboxylic acid were formed, indicating that the
hydroxamate was not a primary site of metabolism. Fortunately,
as opposed to in vitro results, no GSH-mediated isomerization
Pharmacokinetics of 2 in Mice. Female CD1 mice
received 2 at 50 mg/kg intraperitoneally to evaluate whether it
would be concentrated enough for in vivo activity against
Plasmodium berghei infection. As the parasite is located in
erythrocytes, both the plasma concentration and the distribu-
tion in erythrocytes were investigated. 2 was formulated in 50/
50 DMSO/PBS at 10 mg/mL. Blood was collected at regular
Table 4. Pharmacokinetic Parameters of 2 in Female Mice
dose (mg/
kg ip)
Tlast
(h)
AUC0→last
(μmol h/L)
AUC0→∞
(μmol h/L)
T1/2
(h)
matrix
plasma
RBCs
1 × 50
6
66
90
68
98
1.0
1.5
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occurred in mice as E-isomer 3 was not detected in the in vivo
experiment either in plasma or in red blood cells. 2 penetrates
red blood cells with a significant AUC0→∞ of 98 μmol h/L
measured in red cells (Figure 2, Table 4).
functions of PfAM1 may have to be targeted to produce
antimalarial drug candidates.
MATERIALS AND METHODS
■
The global AUC for 2 in red blood cells is even higher than
that in plasma, suggesting that 2 enters and accumulates in
RBCs. Thus, the gap between in vitro and in vivo activity could
not be explained by a lack of host cell penetration. However, it
could possibly be explained by the trapping of the compound
by a component of red blood cells before it is able to reach the
parasite. It is known that Zn2+ ion ligands, such as dorzolamide,
can be trapped in RBC by carbonic anhydrase.40 Carbonic
anhydrase is a zinc metallohydrolase that is located in red blood
cells in high concentrations (150 μM total isoforms).
Nevertheless, 2 does not inhibit hCAII (0% inhibition at 10
and 50 μM, tested in duplicate), indicating that it does not bind
carbonic anhydrase and likely is free to enter the parasitic cell
from the cytoplasm of red blood cells. In all, this shows that the
gap between enzymatic inhibitory potency and antiplasmodial
activity cannot be explained by any of the molecular properties
of 2 tested by us.
1
General Chemistry. H and 13C NMR were recorded at room
temperature on a Bruker DPX 300 at 300 and 75 MHz, respectively.
Chemical shifts are in parts per million (ppm). The assignments were
made using one-dimensional (1D) 1H and 13C spectra or two-
dimensional (2D) HSQC and COSY spectra. Mass spectra were
recorded on a LC−MS system (Waters). HPLC analysis were
performed using a C18 TSK-GEL Super ODS 2 μm particle size
column, dimensions 50 × 4.6 mm. A LC−MS gradient starting from
100% H2O/0.1% formic acid and reaching 20% H2O/80% MeOH/
0.08% formic acid within 5 min at a flow rate of 1 mL/min was used.
purity (%) was determined by reversed-phase chromatography HPLC
using UV detection (215 nm), and all compounds showed purities
greater than 95%. All commercial reagents and solvents were used
without further purification.
Coupling Reactions for Amide Bond formation. (a)
Carboxylic acid (0.66 mmol, 1 equiv) was dissolved in DCM (7
mL), and TEA (102 μL, 0.73 mmol, 1.1 equiv) was added. The
mixture was cooled at 0 °C (ice bath) and ethylchloroformate (70 μL,
0.73 mmol, 1.1 equiv) was added dropwise. The reaction mixture was
stirred 30 min at 0 °C and then the desired amine (0.73 mmol, 1.1
equiv) was added. The organic layer was stirred for 1 h at room
temperature and then evaporated. The crude product was dissolved in
ethyl acetate; washed twice with aq NaHCO3 5%. once with water, and
once with aq NaCl; dried over MgSO4; filtered; and evaporated. The
product was purified by thick-layer chromatography (DCM 100%)
and/or precipitated in petroleum ether (40−60) to give the expected
amide. Alternatively, (b) carboxylic acid (0.1M/DMF, 1 equiv), DIEA
(2.4 equiv), EDCI (1.1 equiv), and HOBt (1.1 equiv) were stirred for
5 min at room temperature. Then the appropriate amine (0.1 M/
DMF, 1 equiv) and DIEA (2 equiv) were added. The mixture was
stirred for 5 h at room temperature, and then solvents were removed
under reduced pressure. The residue was dissolved in AcOEt and the
organic phase was washed with aqueous NaHCO3 5% (six times) and
NaCl (once). The organic layer was dried over MgSO4 and evaporated
under reduced pressure. The residue was precipitated in petroleum
ether.
tert-Butyl Deprotection. tert-Butylated intermediate was dis-
solved in a suspension of BTFA (0.75 M/TFA, 25 equiv) with 0.4%
H2O. The reaction mixture was stirred for 4−5.5 h at room
temperature. Solvents were removed under reduced pressure and the
residue was dissolved in water. The aqueous phase was basified with 1
M NaOH to pH 7 and extracted three times with AcOEt. The organics
layers were dried over MgSO4, filtered, and evaporated. The residue
was precipitated in ether−pentane. Yields given are those of the
deprotection step.
Trityl Deprotection. Tritylated intermediate was dissolved in TFA
2%/DCM, and triisopropylsilane was added drop-by-drop until the
yellow color disappeared. The reaction mixture was stirred for 5 min at
room temperature, solvents were removed under reduced pressure,
and the residue was washed with ether−pentane. Yields given are those
of the deprotection step.
(E)-2-tert-Butoxycarbamoyl-3-(4-fluorophenyl)acrylic Acid
(5c). 4-Fluorobenzaldehyde (32.0 mmol, 1 equiv) was added to a
solution of ethyl N-tert-butoxymalonate (7.163 g, 35.2 mmol, 1.1
equiv) and piperidine (1 mL) in abs ethanol (150 mL). The solution
was refluxed for 3.5 h and solvent was removed under reduced
pressure. The crude product was purified by flash chromatography
(DCM/MeOH 98/2) to give ethyl (Z/E)-2-tert-butoxycarbamoyl-3-
(4-fluorophenyl)acrylate in 71% yield (85% purity). 1H NMR
(CDCl3) δ ppm: 1.21−1.29 (m, 12H), 4.24 (q, J = 7.1 Hz, 2H),
7.01 (t, J = 8.6 Hz, 2H), 7.51−7.55 (m, 2H), 7.69 (s, 1H), 7.86 (s,
NH). MS: (MH)+ m/z 310. MS: (MNa)+ m/z 332. The later ethyl
ester (3.478 g, 11.25 mmol) is dissolved in abs ethanol (115 mL), and
KOH (2.57 g, 45 mmol, 4 equiv) is added. The mixture is stirred
overnight at room temperature and then evaporated. The crude
CONCLUSION
■
On the basis of literature data, PfAM1 has been regarded as
essential to the malaria parasite. However, none of its described
inhibitors, including ours, was able to kill the parasite at
concentrations below 1 μM, raising the question of the
pharmacological relevance of inhibiting PfAM1 to treat malaria.
To formulate a conclusion, we must take into account that the
pharmacological activity of an inhibitor is not only determined
by its binding to the target and its inhibitory potency, but also
by its ability to reach the target at a sufficient concentration for
a sufficient period of time. The lack of pharmacokinetic data on
inhibitors reported earlier made it difficult to interpret their
poor antiplasmodial potency. In this paper, we report the first
structurally homogeneous family of inhibitors demonstrating a
100-fold range of enzyme potency, together with antiplasmodial
activities and key pharmacokinetic parameters.
Structure−activity relationships established by “fluorine
scanning” showed that the best position for fluorine is para
on the benzylamido group. Substitution at the malonic carbon
is tolerated, whereas introduction of a pyridine is deleterious for
activity. The range of compounds tested here spanned a 2.5 log
scale of inhibitory potency on the isolated enzyme. However, in
this population, we were not able to observe any correlation
between enzymatic potency and antimalarial activity. To rule
out potential compound-related issues in the course of the
validation of PfAM1 as a drug target, we performed stability and
distribution studies with 2 (BDM14471), our most promising
inhibitor, taking into account potency, physicochemical
parameters, and metabolic stability. Pharmacokinetic experi-
ments in mice evidenced a significant distribution of 2 in
plasma and red blood cells until 6 h after dosing. In this
experiment, 2 reached micromolar concentrations within the
red blood cells that harbor the parasites in culture. Altogether,
these results show that the compound reaches the site of action
but fails to kill the parasite at concentrations relevant to the
enzymatic inhibitory potency, suggesting that killing the
parasite remains a challenge for potent and druglike catalytic
site binding PfAM1 inhibitors. A working hypothesis to explain
these results could be that the catalytic activity of the PfAM1 is
not critical for the survival of the parasite at the intraerythrocyte
stage. In this situation, other nonproteolytic regulatory
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product is dissolved in water. The aqueous phase is acidified with 1 N
aq HCl until pH 1 and then the carboxylic acid is extracted with ethyl
acetate (4 × 20 mL). The organic layers are pooled, dried over MgSO4
and evaporated to give 5c as a yellow powder in 92% yield (95%
purity). 1H NMR (DMSO-d6) δ ppm: 1.17 (s, 9H), 7.28 (t, J = 8.9 Hz,
2H), 7.61 (s, 1H), 7.64−7.69 (m, 2H), 10.83 (s, NH), 12.85 (s, OH).
trLC−MS: 4.19 min. MS: (MH)+ m/z 282. Mp: 188−190 °C.
2-Benzyl-N-(4-fluorobenzyl)-N′-hydroxymalonamide (1).
White powder, yield 88% (100% purity). 1H NMR (DMSO-d6) δ
ppm: 2.96−3.10 (m, 2H), 3.35−3.40 (m, 1H), 4.16 (dd, J = 15, J = 5.7
Hz, 1H), 4.27 (dd, J = 15, J = 6 Hz, 1H), 7.04−7.27 (m, 9H), 8.40 (t, J
= 5.6 Hz, NHCO), 8.93 (s, OH), 10.58 (s, CONHO). 13C NMR
(DMSO-d6) δ ppm: 35.0, 42.1, 52.5, 115.5 (d, JCF = 21.1 Hz), 126.8,
128.8, 129.4, 129.5 (d, JCF = 8.4 Hz), 136.0 (d, JCF = 2.3 Hz), 139.6,
161.7 (d, JCF = 240.4 Hz), 166.3, 168.9. trLC−MS: 4.31 min. MS: (MH)+
m/z 317. Mp: 193−194 °C.
N-(4-Fluorobenzyl)-N′-hydroxy-2-[1-phenylmeth-(Z)-
ylidene]malonamide (2). White powder, yield 37% (overall) (99%
purity). 1H NMR (DMSO-d6) δ ppm: 4.37 (d, J = 6.0 Hz, 2H), 7.12−
7.18 (m, 2H), 7.32−7.40 (m, 5H), 7.43 (s, 1H), 7.51−7.54 (m, 2H),
8.26 (t, J = 6.0 Hz, NHCO), 9.13 (s, OH), 11.01 (s, CONHO). 13C
NMR (DMSO-d6) δ ppm: 42.6, 115.6 (d, JCF = 21 Hz), 129.3, 129.5,
129.9, 130.1, 130.6, 134.2, 136.2, 137.3, 161.8 (d, JCF = 246 Hz), 163.5,
164.6. trLC−MS: 4.23 min. MS: (MH)+ m/z 315.
N-(4-Fluorobenzyl)-N′-hydroxy-2-[1-phenylmeth-(E)-
ylidene]malonamide (3). White powder, yield 50% (overall) (99%
purity). 1H NMR (DMSO-d6) δ ppm: 4.30 (d, J = 6.0 Hz, 2H), 7.08−
7.37 (m, 10H), 8.86 (t, J = 6.0 Hz, NHCO), 9.08 (br s, OH), 10.70 (br
s, CONHO). 13C NMR (DMSO-d6) δ ppm: 42.3, 115.3 (d, JCF = 20
Hz), 128.9, 129.5, 129.6, 129.8, 130.3 (d, JCF = 12 Hz), 132.0, 134.3,
135.2, 161.4 (d, JCF = 225 Hz), 163.3, 166.2. trLC−MS: 4.41 min. MS:
(MH)+ m/z 315.
2,N-Dibenzyl-N′-hydroxymalonamide (4). White powder, yield
84% (100% purity). 1H NMR (DMSO-d6) δ 3.00−3.08 (m, 2H), 3.30
(t, J = 6.75 Hz, 1H), 4.20 (dd, J = 5.1 Hz, J = 15 Hz, 1H), 4.30 (dd, J =
5.4 Hz, J = 15 Hz, 1H), 7.10−7.23 (m, 10H), 8.20−8.23 (m, NHCO),
10.44 (s, CONHO). 13C NMR (DMSO-d6) δ 35.1, 42.7, 52.7, 126.6,
N-(4-Fluorobenzyl)-2-[1-(4-fluorophenyl)meth-(Z)-ylidene]-
N′-hydroxymalonamide (11). Beige powder, yield 15% (95%
purity). 1H NMR (DMSO-d6) δ ppm: 4.36 (d, J = 5.7 Hz, 2H),
7.12−7.36 (m, 6H), 7.43 (s, 1H), 7.56−7.60 (m, 2H), 8.27 (t, J = 5.7
Hz, NHCO), 9.14 (s, OH), 11.03 (s, CONHO). trLC−MS: 4.36 min.
MS: (MH)+ m/z 333.
2-Benzyl-N-(4-fluorobenzyl)-N′-hydroxy-2-methylmalona-
mide (12). White powder, yield 60% (99% purity). 1H NMR
(CD2Cl2) δ ppm: 1.24 (s, 3H), 2.99 (s, 2H), 4.34 (s, 2H), 7.00−7.23
(m, 9H). trLC−MS: 4.66 min. MS: (MH)+ m/z 331.
2-Benzyl-N-hydroxy-N′-pyridin-4-ylmethylmalonamide (13).
White powder, yield 91% (99% purity). 1H NMR (DMSO-d6) δ ppm:
3.06−3.10 (m, 2H), 3.41 (t, J = 7.7 Hz, 1H), 4.47 (d, J = 5.7 Hz, 2H),
7.20−7.31 (m, 5H), 7.62 (d, J = 6.0 Hz, 2H), 8.63 (t, J = 5.7 Hz,
CONH), 8.76 (d, J = 6.0 Hz, 2H), 10.64 (s, CONHO). trLC−MS: 2.08
min. MS: (MH)+ m/z 300.
2-Benzylidene-N-hydroxy-N′-pyridin-4-ylmethylmalona-
mide (14). White powder, yield 90% (98% purity). 1H NMR
(DMSO-d6) δ ppm: 4.58 (d, J = 5.7 Hz, 2H), 7.10−7.56 (m, 7H), 7.76
(s, 1H), 8.45−8.78 (m, 2H + NHCO), 11.09 (s, CONHO). trLC−MS
:
2.14 min. MS: (MH)+ m/z 298.
PfAM1 Inhibition. Native PfAM1 was purified according to the
procedure described by Allary et al.17 and diluted 10 times in Tris−
HCl buffer (25 mM, pH 7.4) before use. The assays were set up in 96-
well plates. The compounds were tested at the concentration of 10
μM, and 33 μL of purified PfAM1 was preincubated for 10 min at
room temperature with 33 μL of the inhibitor (30 μM in Tris−HCl
buffer, 0.3% DMSO). Then 33 μL of the substrate Leu-pNA (Km
=
0.099 mM) (0.3 mM in Tris−HCl buffer) was added. The reaction
kinetics performed at room temperature was followed on a UV-
microplate reader MultiskanRC (Labsystems) at 405 nm. The control
activity was determined by incubating the enzyme under the same
conditions without inhibitor. Bestatin was used as the reference
inhibitor (IC50 = 284 nM). The statistical Z′ factor for the test was
0.82, allowing activities to be determined with a single point with a
95% confidence. Initial velocities are expressed in μmol min−1. Data
were normalized to the controls that represent Vmax. For the
determination of IC50s, initial velocities were plotted as a function of
inhibitor concentration, using XLfit software from IDBS.
127.1, 127.5, 128.6, 128.7, 129.3, 139.4, 139.6, 166.2, 168.6. trLC−MS
:
4.25 min. MS: (MH)+ m/z 299. Mp: 167−169 °C.
pAPN Inhibition. Microsomal neutral aminopeptidase (APN)
from porcine kidney was purchased from Sigma Inc. as an ammonium
sulfate suspension [3.5 M (NH4)2SO4 solution containing 10 mM
MgCl2, 10−40 units/mg protein]. The enzyme suspension was diluted
600-fold in Tris−HCl buffer (25 mM, pH 7.4) before use. The assays
were performed in 96-well plates. The compounds were tested at the
concentration of 10 μM, and 33 μL of purified APN was preincubated
for 10 min at room temperature with 33 μL of the inhibitor (30 μM in
Tris−HCl buffer, 0.3% DMSO). Then 33 μL of the substrate Leu-pNA
(Km = 0.099 mM) (0.3 mM in Tris−HCl buffer) was added. The
reaction kinetics performed at room temperature was followed on a
UV-microplate reader MultiskanRC (Labsystems, Finland) at 405 nm.
The control activity was determined by incubating the enzyme in the
same conditions without inhibitor. Bestatin was used as the reference
inhibitor (IC50 = 2.7 μM). The statistical Z′ factor for the test was
0.75, allowing activities to be determined with a single point with a
95% confidence.39 Initial velocities are expressed in μmol min−1. Data
were normalized to the controls that represent Vmax. For the
determination of IC50s, initial velocities were plotted as a function of
inhibitor concentration, using XLfit software from IDBS.
2-Benzyl-N-(3-fluorobenzyl)-N′-hydroxymalonamide (6).
White powder, yield 90% (100% purity). 1H NMR (DMSO-d6) δ
ppm: 2.98−3.12 (m, 2H), 3.29−3.35 (m, 1H), 4.21 (dd, J = 15.6 Hz, J
= 6.0 Hz, 1H), 4.30 (dd, J = 15.6 Hz, J = 6.3 Hz, 1H), 6.93−7.06 (m,
3H), 7.18−7.33 (m, 6H), 8.31 (t, J = 6.0 Hz, NH), 8.95 (s, OH), 10.49
(s, CONHO). trLC−MS: 4.51 min. MS: (MH)+ m/z 317. Mp: 167.5−
168.8 °C.
2-Benzyl-N-(2-fluorobenzyl)-N′-hydroxymalonamide (7).
White powder, yield 70% (100% purity). 1H NMR (DMSO-d6) δ
ppm: 3.03−3.07 (m, 2H), 3.30−3.35 (m, 1H), 4.23 (dd, J = 15.6 Hz, J
= 5.6 Hz, 1H), 4.32 (dd, J = 15.6 Hz, J = 6.0 Hz, 1H), 7.02−7.32 (m,
9H), 8.25 (t, J = 5.7 Hz, NH), 8.95 (s, OH), 10.47 (s, CONHO).
trLC−MS: 4.20 min. MS: (MH)+ m/z 317. Mp: 182.3−184.7 °C.
N-Benzyl-2-(4-fluorobenzyl)-N′-hydroxymalonamide (8).
White powder, yield 23% (95% purity). 1H NMR (DMSO-d6) δ
ppm: 3.01−3.05 (m, 2H), 3.28 (t, J = 7.8 Hz, 1H), 4.18 (dd, J = 5.4
Hz, J = 15.3 Hz, 1H), 4.29 (dd, J = 6.0 Hz, J = 15.3 Hz, 1H), 7.04−
7.11 (m, 4H), 7.19−7.29 (m, 5H), 8.24 (t, J = 6.0 Hz, NHCO), 8.95
(s, 1H, OH), 10.46 (s, CONHO). trLC−MS: 4.36 min. MS: (MH)+ m/z
317.
In Vitro Plasmodium falciparum Culture and Drug Assay.
Chloroquine-resistant P. falciparum 2/K 1-strain was cultured in
human erythrocytes O+ at 37 °C under a low oxygen atmosphere (3%
O2, 4% CO2, and 93% N2) in RPMI-1640, supplemented with 10%
human serum. Infected human red blood cells (200 μL, 1%
parasitaemia, 2% hematocrit) were added to each well and incubated
for 72 h. After incubation, test plates were frozen at −20 °C. Parasite
multiplication was measured by the Malstat method. An amount of
100 μL of Malstat reagent was transferred to a new plate and mixed
with 20 μL of the hemolysed parasite suspension for 15 min at room
temperature. After addition of 20 μL of NBT/PES solution and 2 h of
2,N-Bis(4-fluorobenzyl)-N′-hydroxymalonamide (9). Beige
powder, yield 21% (96% purity). 1H NMR (DMSO-d6) δ ppm:
3.00−3.04 (m, 2H), 3.26 (t, J = 7.8 Hz, 1H), 4.16 (dd, J = 5.7 Hz, J =
15.0 Hz, 1H), 4.26 (dd, J = 6.0 Hz, J = 15.0 Hz, 1H), 7.04−7.23 (m,
8H), 8.27 (t, J = 6.0 Hz, NHCO), 8.94 (s, 1H, OH), 10.47 (s,
CONHO). trLC−MS: 4.50 min. MS: (MH)+ m/z 335.
N-Benzyl-2-[1-(4-fluorophenyl)meth-(Z)-ylidene]-N′-hydrox-
ymalonamide (10). Beige powder, yield 15% (99% purity). 1H NMR
(DMSO-d6) δ ppm: 4.39 (d, J = 5.4 Hz, 2H), 7.22−7.36 (m, 7H), 7.44
(s, 1H), 7.56−7.60 (m, 2H), 8.23 (t, J = 5.4 Hz, NHCO), 9.15 (s,
OH), 11.03 (s, CONHO). tr LC−MS: 4.17 min. MS: (MH)+ m/z 315.
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Journal of Medicinal Chemistry
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incubation in the dark, the absorbance was spectrophotometrically
read at 655 nm (Biorad 3550 UV microplate reader). Percentage
growth inhibition was calculated compared to the negative blanks. IC50
values are calculated from the duplicate determinations with relative
difference below 25%.
in methanol at a concentration of 0.1 mM. Pooled male mouse (CD-
1) liver microsomes were purchased from BD Gentest (Le Pont de
Claix, France). All incubations were performed in duplicate in a
shaking water bath at 37 °C. The incubation mixture was prepared in
polypropylene tubes and contained 1 μM test compound (1%
methanol), mouse liver microsomes (0.6 mg of microsomal protein/
mL), 5 mM MgCl2, 1 mM NADP, 5 mM glucose-6-phosphate, 0.4 U/
mL glucose-6-phosphate dehydrogenase, and 50 mM potassium
phosphate buffer (pH 7.4) in a final volume of 1.5 mL. Sampling
points were taken at 5, 10, 15, 20, 25, 30, 45, and 60 min, and reactions
were terminated by adding ice-cold acetonitrile containing 1 μM
internal standard (1 vol). The samples were centrifuged for 10 min at
4000 g, 4 °C to pellet precipitated microsomal protein, and the
supernatant was subjected to LC−MS/MS analysis. Control
incubations were performed with denaturated microsomes with
acetonitrile containing 1 μM internal standard, and sampling points
were taken at 0 and 60 min (to evaluate the compound chemical
stability under the experimental conditions). Quantitation of each
compound was achieved by conversion of the corresponding analyte/
internal standard peak area ratios in LC−MS/MS (MRM mode) to
the percentage drug remaining, using the T0 ratio values as 100%. In
vitro intrinsic clearance (CLint expressed as μL/min/mg) was
calculated according to CLint = (dose/AUC∞), where dose is the
initial amount of drug in the incubation mixture (1 μM) and AUC∞ is
the area under the concentration versus time curve extrapolated to
infinity. The slope of the linear regression from log percentage
remaining versus incubation time relationships (−k) was used in the
conversion to in vitro T1/2 values by in vitro T1/2 = −0.693/k.
GSH Sensitivity. In a 1 mL polypropylene tube are introduced 10
μL of a 10 μM solution of compound in acetonitrile with 145 μL of
phosphate buffer (0.067 M at pH 7.4) and 145 μL of glutathione (5
mM/H2O). The final compound concentration is 330 μM. After 1 h of
room-temperature incubation, samples are analyzed by LC−MS to
look for the glutathione conjugate.
Cytotoxicity Test. MRC-5 SV2 cells, human fetal lung fibroblast,
were cultivated in MEM supplemented with L-glutamine (20 mM),
16.5 mM sodium hydrogen carbonate, and 5% FCS at 37 °C and 5%
CO2. For the assay, 104 MRC-5 cells/well were seeded onto the test
plates containing the prediluted compounds and incubated at 37 °C
and 5% CO2 for 72 h. After 72 h of incubation, parasite growth was
assessed fluorimetrically by adding resazurin for 24 h at 37 °C.
Fluorescence was measured using a GENios Tecan fluorimeter
(excitation 530 nm, emission 590 nm). IC50 values are calculated
from duplicate determinations with relative difference below 25%.
LC−MS/MS analysis. The LC−MS/MS system consisted of a
Varian 1200L (Varian, Les Ulis, France) a Prostar 430 autosampler, a
triple quadrupole mass spectrometer (Varian, Les Ulis, France)
equipped with an electrospray ionization source, and a Prostar 325
detector. Analytes in incubation mixtures were separated by HPLC
using a gelTSK C18 Super-ODS, 5 μm, 50 × 4.6 mm column
(Interchim, Montluco̧ n France). The mobile phase solvents used were
(A) 0.01% formic acid in water and (B) 0.01% formic acid in
acetonitrile. The following mobile phase gradient was applied: 0−
100% B in 7.30 min, hold at 100% B for 1 min, 0−100% A in 0.30 min,
and 100% A hold for 10 min. The injection volume was 10 μL and the
flow rate of 1 mL/min. Approximately 30% of the eluent was
introduced into the triple quadrupole mass spectrometer source. The
source temperature of the mass spectrometer was maintained at 300
°C, the declustering potential was 50 V, and the curtain gas pressure
was 1.5 mTorr. Collision energy and observed transitions were
respectively individually optimized for each compound (MRM mode).
Docking. The docking of BDM14471 in PfAM1 (using PDB
structure 3T8V) was performed using MOE 2009.10 software from
Chem Computing Group, Inc. Briefly, the protein was prepared within
the MOE package using Gasteiger partial charges, and the generalized
Born/volume integral formalism for protonation at pH 7. The protein
atoms were kept as rigid and the ligands as flexible. No restraints were
used during docking (conformational search using MM Iteration Limit
1000; all other standard parameters were set on default), but the
binding modes were chosen on the basis of both the most favored
predicted interaction energy with PfAM1 and a proper chelating of the
zinc ion by the hydroxamic function.
hCA Inhibition.41 human erythrocyte carbonic anhydrase (hCA)
activity was measured by following the reaction kinetics of hydrolysis
of 4-nitrophenyl acetate (450 μM) on a UV-microplate reader at 400
nm, at CEREP, SA. The control activity was determined by incubating
the enzyme under the same conditions without inhibitor. Acetazola-
mide (IC50 = 29 nM) was used as the reference inhibitor. The results
are expressed as a percent inhibition of control specific activity (100 −
(measured specific activity/control specific activity) × 100). This
analysis was performed using a software developed at CEREP, SA.
Pharmacokinetic Study.42 BDM14471 was formulated in 50/50
DMSO/PBS at 10 mg/mL and solubilized by short ultrasonic
treatment (bath) if necessary. Twelve female CD-1 mice (approx-
imately body weight 25−30 g) were given a single 50 mg/kg
administration by intraperitonal injection and two mice served as
negative control [no treatment (blank plasma)]. Food and drinking
water remained available ad libitum throughout the trial. Blood
samples (two mice per data point) were collected at 15 and 30 min
and 1, 2, 4, and 6 h. The blood was collected intracardially at the stated
time point using a heparinized syringe (1 droplet, 2% heparin) and
transferred into an Eppendorf tube. The tubes were put on ice and
centrifuged at 2600 rpm for 10 min to separate the plasma. The
fractions of red cells or plasma were stored at 4 °C before analysis.
Oasis-HLB 1 cm3 (30 mg) flangeless cartridges from Waters Inc. were
activated with 1 mL of methanol and then equilibrated with 1 mL of
water Milli-Q. Samples were treated as follows. To 100 μL of plasma
were added 2 μL of internal standard and 400 μL of water. To 100 μL
of red blood cells was added 400 μL of water. The resulting suspension
was sonicated and centrifuged, 400 μL of the supernatant was
collected, and 2 μL of internal standard was added. The resulting
mixtures (from plasma or red blood cells) were eluted on an HLB-
column. The column was then washed with 0.25 mL of water/MeOH
(95/5 v/v). The compounds were eluted with a solution of
acetonitrile/water (95/5 v/v) containing 0.1% of formic acid. The
eluate was evaporated and redissolved in 200 μL of methanol/mobile
phase 60/40 (v/v) for LC−MS/MS analysis.
Solubility. A 40 μL portion of the 10 mM solution in DMSO of
the sample was added to 1.960 mL of MeOH or PBS at pH 7.4. The
samples were gently shaken for 24 h at room temperature, centrifuged
for 5 min, and filtered over 0.45 μm filters. A 20 μL aliquot of each
solution was added to 180 μL of MeOH and analyzed by LC−MS/
MS. The solubility was determined by the ratio of mass signal areas
PBS/MeOH.
log D. A 40 μL portion of the 10 mM solution in DMSO of the
sample was added to 1.960 mL of a 1/1 octanol/PBS at pH 7.4
solution. The mixture was gently shaken 2 h at room temperature, and
then the two phases were separated. A 20 μL aliquot of each solution
was added to 180 μL of MeOH and analyzed by LC−MS/MS. log D
was determined as the logarithm of the ratio of concentrations of
product in octanol and PBS, determined by mass signals.
Plasma Stability. A 40 μL portion of the 5 mM solution in DMSO
of the sample was added to 1.960 mL of rat plasma (male rat from
Charles River Laboratories) to obtain a 100 μM final solution. The
mixture was gently stirred for 96 h at 37 °C. Aliquots of 200 μL were
taken at various times (from 0 to 96 h) and diluted with 200 μL of
phosphoric acid (0.14 M), and 10 μL of the 2 mM solution in
methanol of the internal standard was added. Compounds were
extracted three times with 2 mL of AcOEt. The organic layer was
evaporated, diluted with 200 μL of methanol, and quantified by LC−
MS/MS, using a calibration curve.
Microsome Stability. All chemicals were obtained from Sigma-
Aldrich (Steinheim, Germany). Solvents were from common sources
and of HPLC grade. Stock solutions of all compounds were prepared
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Journal of Medicinal Chemistry
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(12) Skinner-Adams, T. S.; Stack, C. M.; Trenholme, K. R.; Brown,
C. L.; Grembecka, J.; Lowther, J.; Mucha, A.; Drag, M.; Kafarski, P.;
McGowan, S.; Whisstock, J. C.; Gardiner, D. L.; Dalton, J. P.
Plasmodium falciparum neutral aminopeptidases: New targets for anti-
malarials. Trends Biochem. Sci. 2009, 35, 53−61.
(13) Gardiner, D. L.; Skinner-Adams, T. S.; Brown, C. L.; Andrews,
K. T.; Stack, C. M.; McCarthy, J. S.; Dalton, J. P.; Trenholme, K. R.
Plasmodium falciparum: New molecular targets with potential for
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1087−1098.
(14) Gavigan, C. S.; Dalton, J. P.; Bell, A. The role of
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AUTHOR INFORMATION
Corresponding Author
*Phone: (+33) 320 964 947. Fax: (+33) 320 964 709. E-mail:
rebecca.deprez@univ-lille2.fr (R.D-P.); benoit.deprez@univ-
lille2.fr (B.D.).
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank Dr. N. Willand and B. Villemagne for their
help with docking and visualization software. We are grateful to
■
Inserm, Universite
work was specifically funded by ≪Reg
Calais≫ and EC.
́
de Lille2, and Institut Pasteur de Lille. This
ion Nord-Pas-de-
́
(16) Skinner-Adams, T. S.; Lowther, J.; Teuscher, F.; Stack, C. M.;
Grembecka, J.; Mucha, A.; Kafarski, P.; Trenholme, K. R.; Dalton, J. P.;
Gardiner, D. L. Identification of phosphinate dipeptide analog
inhibitors directed against the Plasmodium falciparum M17 leucine
aminopeptidase as lead antimalarial compounds. J. Med. Chem. 2007,
50, 6024−6031.
ABBREVIATIONS USED
■
AcOEt, ethyl acetate; AUC, area-under-curve; BTFA, boron
tris(trifluoroacetate); APN, neutral aminopeptidase; CA,
carbonic anhydrase; CH3CN, acetonitrile; DCM, dichloro-
methane; DIEA, diisopropylethylamine; DMF, dimethylforma-
mide; DMSO, dimethyl sulfoxide; EDCI, N-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; EtOH, ethanol; GSH,
glutathione; HOBt, N-hydroxybenzotriazole; MeOH, meth-
anol; PBS, phosphate-buffered saline; RBC, red blood cells; rt,
room temperature; SAR, structure−activity relationship; TEA,
triethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran;
TIS, triisopropylsilane; Trt, trityl.
(17) Allary, M.; Schrevel, J.; Florent, I. Properties, stage-dependent
expression and localization of Plasmodium falciparum M1 family zinc-
aminopeptidase. Parasitology 2002, 125, 1−10.
(18) Florent, I.; Derhy, Z.; Allary, M.; Monsigny, M.; Mayer, R.;
Schrevel, J. A Plasmodium falciparum aminopeptidase gene belonging
to the M1 family of zinc-metallopeptidases is expressed in erythrocytic
stages. Mol. Biochem. Parasitol. 1998, 97, 149−160.
(19) Ragheb, D.; Dalal, S.; Bompiani, K. M.; Ray, W. K.; Klemba, M.
Distribution and biochemical properties of an M1-family amino-
peptidase in Plasmodium falciparum indicate a role in vacuolar
hemoglobin catabolism. J. Biol. Chem. 2011, 286, 27255−27265.
(20) Klemba, M.; Gluzman, I.; Goldberg, D. E. A Plasmodium
falciparum dipeptidyl aminopeptidase I participates in vacuolar
hemoglobin degradation. J. Biol. Chem. 2004, 279, 43000−43007.
(21) Harbut, M. B.; Velmourougane, G.; Dalal, S.; Reiss, G.;
Whisstock, J. C.; Onder, O.; Brisson, D.; McGowan, S.; Klemba, M.;
Greenbaum, D. C. Bestatin-based chemical biology strategy reveals
distinct roles for malaria M1- and M17-family aminopeptidases. Proc.
Natl. Acad. Sci. U. S. A. 2011, 108, E526−E534.
(22) Kitjaroentham, A.; Suthiphongchai, T.; Wilairat, P. Effect of
metalloprotease inhibitors on invasion of red blood cell by Plasmodium
falciparum. Acta Trop. 2006, 97, 5−9.
(23) Azimzadeh, O.; Sow, C.; Geze, M.; Nyalwidhe, J.; Florent, I.
Plasmodium falciparum PfA-M1 aminopeptidase is trafficked via the
parasitophorous vacuole and marginally delivered to the food vacuole.
Malaria J. 2010, 9, 189.
(24) Flipo, M.; Florent, I.; Grellier, P.; Sergheraert, C.; Deprez-
Poulain, R. Design, synthesis and antimalarial activity of novel,
quinoline-based, zinc metallo-aminopeptidase inhibitors. Bioorg. Med.
Chem. Lett. 2003, 13, 2659−2662.
(25) Flipo, M.; Beghyn, T.; Charton, J.; Leroux, V. A.; Deprez, B. P.;
Deprez-Poulain, R. F. A library of novel hydroxamic acids targeting the
metallo-protease family: Design, parallel synthesis and screening.
Bioorg. Med. Chem. 2007, 15, 63−76.
(26) Flipo, M.; Beghyn, T.; Leroux, V.; Florent, I.; Deprez, B. P.;
Deprez-Poulain, R. F. Novel selective inhibitors of the zinc plasmodial
aminopeptidase PfA-M1 as potential antimalarial agents. J. Med. Chem.
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(27) Cunningham, E.; Drag, M.; Kafarski, P.; Bell, A. Chemical target
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