Novel selective inhibitors of the zinc plasmodial aminopeptidase PfA-M1 as potential antimalarial agents

Article abstract of DOI:10.1021/jm061169b

Proteases that are expressed during the erythocytic stage of Plasmodium falciparum are newly explored drug targets for the treatment of malaria. We report here the discovery of potent inhibitors of PfA-M1, a metallo- aminopeptidase of the parasite. These

Full text of DOI:10.1021/jm061169b

1322
J. Med. Chem. 2007, 50, 1322-1334
Novel Selective Inhibitors of the Zinc Plasmodial Aminopeptidase PfA-M1 as Potential
Antimalarial Agents
Marion Flipo,† Terence Beghyn,† Virginie Leroux,† Isabelle Florent,‡ Benoit P. Deprez,*,† and Rebecca F. Deprez-Poulain†
Inserm, U761, Biostructures and Drug DiscoVery, Lille F-59006 France, Faculte´ de Pharmacie, UniVersite´ de Lille2, Lille F-59006 France,
Institut Pasteur de Lille, Lille F-59019 France, and USM504/EA3335 Muse´um National d’Histoire Naturelle, Regulations, DeVelopment,
Molecular diVersity, Paris, F-75005 France
ReceiVed October 9, 2006
Proteases that are expressed during the erythocytic stage of Plasmodium falciparum are newly explored
drug targets for the treatment of malaria. We report here the discovery of potent inhibitors of PfA-M1, a
metallo-aminopeptidase of the parasite. These compounds are based on a malonic hydroxamic template and
present a very good selectivity toward neutral aminopeptidase (APN-CD13), a related protease in mammals.
Structure-activity relationships in these series are described. Further optimization of the best inhibitor yielded
a nanomolar, selective inhibitor of PfA-M1. This inhibitor displays good physicochemical and pharmacokinetic
properties and a promising antimalarial activity.
Introduction
Malaria is a major disease in developing countries, causing
every year 1-2 million deaths, a majority of cases being young
children in Africa.1 This burdensome disease is caused by
protozoa parasites Plasmodium. The species P. falciparum is
by itself responsible for the majority of deaths. The parasite is
Figure 1. Markush formula of the library members.
transmitted by the female Anopheles mosquito and transits
through the liver and the blood of the mammalian host. The
and metallo-proteases, all of them being expressed at the
erythocytic stage.15-17
blood stages of the parasites are those that cause the symptoms.
At present, the protease inhibitors described are inhibitors of
Apart from quinoline-based antimalarials, antifolates, and
the two classes of acidic endopeptidases (plasmepsins and
antibiotics, the endoperoxide artemisinin and its synthetic
falcipains) that initiate the degradation of globin in the digestive
vacuole. Hallberg et al. have developed potent and selective
inhibitors of the aspartyl-proteases plasmepsin I, II, and IV,
derived from an hydroxyethylene scaffold or a related transition
state mimick.18-20 Inhibitors of the entire family of plasmepsins,
including the related histoaspartyl-protease (HAP), have also
been described.21 In parallel, HIV protease inhibitors have been
shown to inhibit plasmepsin II and IV in the submicromolar
range.22 The teams of Rosenthal and Avery have designed, or
identified by virtual screening, irreversible or reversible inhibi-
tors of falcipain-2 and -3.23-26 Greenbaum et al. designed
thiosemicarbazones inhibitors of several parasitic falcipains
including falcipain-2, and recently, Bogyo et al. designed
electophilic azapeptides as cysteine protease probes.27,28
Among the metalloproteases of Plasmodium, both endo- and
exo-peptidases have been described along with falcilysin,
methionine aminopeptidase 1b, leucylaminopeptidase (LAP),
and PfA-M1.29-32 Falcilysin is an endopeptidase with dual
specificity. It is involved in the intravacuolar digestion of globin,
in peptide degradation in apicoplast and has no reported
inhibitor.29,33 Methionine aminopeptidase 1b has recently been
screened for inhibitors.32 LAP is a tripeptide aminopeptidase
of the M17-family.30 PfA-M1 belongs to the M1 family and
displays a strict aminopeptidase activity at pH 7.4 and a broad
substrate spectrum.34,35 It is involved both in haemoglobin
breakdown and erythrocyte reinvasion. Indeed, PfA-M1 localizes
differently in trophozoites and schizonts, suggesting two dif-
ferent roles in critical steps of the intraerythrocytic life of the
parasite.35 (1) In the trophozoite, it accumulates at the cyto-
plasmic side of the digestive vacuole and could be involved in
the cytoplasmic degradation of oligopeptides that are the end-
derivatives are widely used to combat the disease.2 Unfortu-
nately, there has been a spread of resistant strains of the parasite
to the current therapeutics in numerous countries.3-4 This
antimalarial drug resistance, which is also an economic burden
to these countries, has become one of the greatest challenges
in malaria control.5 There is, thus, a need for new therapies,
which development can be accelerated by the recent publication
of Plasmodium genome.6-8
Targeting the erythrocytic stage of the parasite is widely
investigated for the design of antimalarials.9-10 In this context,
host hemoglobin degradation pathways are of high interest. They
produce both heme, detoxified by the parasite as hemozoin (the
malarial pigment), and globin, the source of amino acids for
the parasite. This offers two possible strategies to combat the
disease: (1) inhibition of heme detoxification, so that heme
accumulates as its lethal form for the parasite and (2) inhibition
of proteases responsible for globin digestion. Interestingly, some
proteases are not only involved in globin hydrolysis, but also
in parasite release and reinvasion of red blood cells.11-13 As a
consequence of these important functions and the successful
work done on proteases in other pathologies (Aids, hyperten-
sion), proteases expressed in the erythrocyte cycle of Plasmo-
dium are now considered as promising molecular targets for
drug discovery.12,14 So far, 92 proteases have been found in the
genome of the parasite, including serine-, aspartyl-, cysteine-,
* To whom correspondence should be addressed. INSERM U761, Faculte´
de Pharmacie, 3 rue du Pr. Laguesse, F-59006 Lille Cedex, France. Tel.:
+33(0)-320-964-024. Fax: +33(0)-320-964-709. E-mail: benoit.deprez@
univ-lille2.fr.
† INSERM U761; Universite´ Lille 2; Institut Pasteur de Lille.
‡ USM504/EA3335 Muse´um National d’Histoire Naturelle.
10.1021/jm061169b CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/28/2007

Inhibitors of PfA-M1 as Potential Antimalarial Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1323
Figure 2. Amine series used for library synthesis.
Table 1. Percentage of Library Members in the Corresponding Range
Table 2. Percentage of Library Members in the Corresponding Range
of Inhibition of PfA-M1, Sorted by R1 Group
of Inhibition of PfA-M1, Sorted by Series of Amine Building Blocks
range of % inhibition at 10 µM
range of % inhibition at 10 µM
R1
<50%
[50-80%]
>80%
series of amines
<50%
[50-80%]
>80%
H
i-butyl
benzyl
m-phenoxy-benzyl
NH2
96
60
49
43
94
2
24
25
24
6
2
16
26
33
0
A
B
C
D
E
F
54
77
44
60
73
81
65
13
13
28
20
27
11
24
33
10
28
20
0
8
11
G
products of globin degradation in the food vacuole. Hemoglobin
digestion is an important process for the generation and
regulation of free amino acids that are used for protein anabolism
and for maintaining osmotic stability within the infected
erythrocyte. Because there is no detectable aminopeptidase
activity in the food vacuole, it is probable that globin degradation
does not go to completion in the food vacuole and that small
oligo and dipeptides need to be hydrolyzed by extravacuolar
aminopeptidases, such as PfA-M1.16 (2) At the schizont stage,
an apparent vesicular readdressing leads to the formation of

1324 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Scheme 1. Synthesis of Compounds 32 to 63a
Flipo et al.
a Reagents and conditions: (a) acid 0.5 M/DMF (1 equiv), DIEA (1.1 equiv), CDI (1.1 equiv) 0.25 M/THF, 2 h, rt, then amine 0.1 M/DMF (1 equiv),
DIEA (2 equiv), rt, 2 h; or acid 0.5 M/DMF (1 equiv), DIEA (1.2 equiv), and PyBrop 0.2M/DCM (1.2 equiv) 30 s, rt, then amine 0.1 M/DMF (1 equiv),
DIEA (2 equiv), rt, overnight; or acid 0.1 M/DMF (1 equiv), DIEA (2.4 equiv), EDCI (1.1 equiv), HOBt (1.1 equiv), rt, 5 min then amine 0.1 M/DMF (1
equiv), DIEA (2 equiv), rt, 5 h; (b) BTFA 0.75M/TFA 0.4% H2O (25 equiv), 0 °C to rt, 4-6 h or TFA 2%/DCM (0.03 M) and triisopropylsilane, rt, 5 min.
Table 3. Inhibition of PfA-M1 by iso-Butyl Compounds 32-41
Table 4. Inhibition of PfA-M1 by m-Phenoxy-benzyl Compounds
42-48
a Mean of two experiments. SD was <10% in most cases.
nanomolar, selective inhibitor of PfA-M1, with a good physi-
cochemical and in vitro stability profile that displays a promising
antimalarial activity in vitro.
Library
a Mean of two experiments. SD was <10% in most cases.
We previously reported the design and synthesis of a diverse
library of zinc chelating hydroxamates targeting metalloproteases
(Markush formula in Figure 1).39 Amine series (Figure 2) were
selected to generate diverse pharmacophoric patterns and
geometries. We selected various chain lengths between an
aromatic ring and the amino function. Cyclic amines were also
incorporated into the library, as well as bulkier amines displaying
two phenyl rings. The malonic building blocks (R1 in Figure 1)
were synthesized through various procedures to display a variety
of R1 substituents on the malonic carbon: hydrogen, a polar
amino group, and various hydrophobic groups (an isobutyl group
and two different aromatic groups) to target diverse metallo-
proteases. In total, 155 compounds were prepared and screened.
strong fluorescence spots in the merozoites, suggesting a role
in the reinvasion of erythrocytes, in agreement with the
observation of Kitjaroenthan et al.36 Among metallo-aminopep-
tidases identified in P. falciparum and expressed during the
erythrocytic stage, the zinc aminopeptidase PfA-M1 is the first
enzyme characterized at the protein level.31,35 It is, therefore,
an attractive candidate to initiate a program of screening and
lead optimization.37
Our group has already described three nonpeptidic, nonselec-
tive inhibitors of PfA-M1, with a mixed mode of action.38 Our
goal is to identify a selective and potent series of inhibitors of
PfA-M1 by screening a library targeting the metalloprotease
family.39 We report here the identification of several potent and
selective PfA-M1 inhibitors and describe structure-activity
relationships in these series. Further optimization produced a
Screening
PfA-M1 was extracted from FcB1 strain as previously
described.35 The library of compounds was screened at 10 µM

Inhibitors of PfA-M1 as Potential Antimalarial Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1325
Table 5. Inhibition of PfA-M1 by Benzyl Compounds 49-63
Table 7. Inhibition of PfA-M1 and Specificity Toward Mammalian
APN of Compounds 40-42, 47, 52, 57, and 59
a Mean of two experiments. SD was <10% in most cases.
Table 6. Influence of the Malonic Substituent on the Inhibition of
PfA-M1
a Mean of two experiments. SD was <10% in most cases.
Statistical Results
Table 1 displays the screening results sorted according to the
substituent of the malonic carbon (R1 in Figure 1). Compounds
bearing a benzyl or a m-phenoxy-benzyl moiety gave the highest
number of hits above 80% of inhibition. The low hit rates
obtained for H or NH2 substituents evidence the need for an
isobutyl or better, an aromatic substituent on the malonic chain,
to inhibit PfA-M1. Among the aromatic substituents, the
m-phenoxyphenyl group gives the best activity. Binding of the
hydroxamate to the Zn2+ ion is expected to be the primary
anchoring point of the potential inhibitor. Thus, the enzyme
should discriminate more dramatically among substituents close
to the hydroxamate than among those remotely bound by a
flexible chain. Table 2 displays the screening results sorted
according to the amine building-blocks series (R2 and R′2 in
Figure 1). Several amine series gave lower hit rates than the
others: cyclic and constrained amines (series F) and amines
with two aromatic groups (series G). Results for other series
suggest that the distance between the nitrogen atom and the
aromatic ring is critical for inhibition of PfA-M1. Benzylamines
(series A), phenylpropylamines (series C), and phenylbutyl-
amines (series D) give higher numbers of hits above 80% of
inhibition than phenethylamines (series B). The lowest hit-rates
are obtained for amines bearing a tertiary nitrogen atom on the
lateral chain (series E). The same library was also screened
against mammalian APNa (neutral aminopeptidase microsomal).
a Mean of two experiments. SD was <10% in most cases. ND: not
determined.
against the native enzyme. The enzyme activity was monitored
at 405 nm by the release of the chromogenic p-nitro-aniline,
resulting from the cleavage of the Leu-p-nitro-anilide substrate.
A Z′ factor of 0.82 was obtained using these conditions, thus
screening was performed in single point determination.40 Tables
of inhibition percentages are given in the Supporting Informa-
tion. As expected from an array of hydroxamates, our library
yielded a significant numbers of hits on PfA-M1.
a Abbreviations: AcOEt, ethylacetate; AcOH, acetic acid; APN, neutral
aminopeptidase; BTFA, boron tris(trifluoroacetate); CDI, N,N′carbonyldi-
imidazole; CH3CN, acetonitrile; DCM, dichloromethane; DIEA, diisopro-
pylethylamine; DMF, dimethylformamide; DMSO, dimethylsulfoxide;
EDCI, N-ethyl-3-(3-dimethyaminopropyl)carbodiimide; EtOH, ethanol;
HOBT, N-hydroxybenzotriazole; Leu-pNA, leucine-para-nitroanilide; MeOH,
methanol; PyBrop, bromo-trispyrrolidinophosphonium hexafluorophosphate;
TEA, triethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofurane.

1326 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Flipo et al.
Table 8. Activities and Preliminary ADME Parameters of Compounds 52, 57, 66-68, 71, and 72
PfA-M1
inhibition IC50
(nM)
mammalian APN
inhibition IC50
(nM)
solubilitya
(µM)
LogDa
(pH ) 7.4)
plasma stabilityb
cpd
(hrs)
57
66
68
71
27
6
330
>10 000
3616
1372
>10 000
>10 000
>200
198
1.2
0.75
22
1.1
NDc
NDc
NDc
NDc
NDc
>48
52
67
72
46
13
>10 000
>10 000
3238
>10 000
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
a Solubility and LogD are measured from a DMSO stock solution. b Half-life rat plasma stability at 37 °C using LC-MS-MS (MRM detection mode).
c ND ) not determined.
Scheme 2. Synthesis of Compounds 66-68a
Structure-Activity Relationships
Library members displaying a percentage of inhibition above
80% were selected for resynthesis and further biological
characterization (dose response curve and IC50 determination).
Some structurally close but less active library members were
prepared as well to support structure-activity relationships. Only
results with these purified and characterized compounds will
be used in the following discussion. Two protocols were used
for the synthesis of the compounds depending on the protecting
group of the malonic precursor (Scheme 1).39
A rapid analysis shows that SARs depend primarily on the
nature of the substituent of the malonic carbon. IC50 values given
in Tables 3-5 summarize results for compounds incorporating,
respectively, an isobutyl, a benzyl, and a m-phenoxy-benzyl
substituent on the malonic carbon. Table 6 allows a comparison
between the malonic substituents.
Compounds Bearing an Isobutyl on the Malonic Carbon
(Table 3). The length and conformation of the carbon chain
between the nitrogen of the amine and the aromatic ring appears
to be critical for bioactivity (compounds 32-35, and 38-41).
Globally, the best activities are obtained for arylmethyl chains.
Compounds 36 and 37 are cyclic analogues of 35. Both are
more active than 35. Compound 37 is 4 times more active than
36. This may be due to the hydrogen remaining on the amide
function or to its more constrained structure.
In the N-benzyl series, substitution at the para position on
the benzyl ring of 32 seems to be critical. A fluorine or chlorine
atom, in compounds 38 and 39, leads to a decrease of activity.
This decrease is more pronounced for fluorine, suggesting that
steric hindrance is not the only factor affecting the binding of
the aromatic ring. Compound 37 is an analogue of reduced
flexibility of both 32 and 35. Interestingly, it represents the
intersection between the conformational spaces of 32 and 35,
and it is more active than these two compounds (IC50 ) 109
nM).
a Reagents and conditions: (a) LiOH (1 equiv), THF, H2O, 4 h, rt; (b)
(i) 1.2 equiv oxalylchloride, DCM, cat. DMF, 45 min, 0 °C; (ii) 3 equiv
DIEA, 0.85 equiv O-tritylhydroxylamine, DCM, 0 °C then rt, 3 h; (c) 4
equiv LiOH, THF, H2O, overnight, rt, isomerization; (d) (i) 1.1 equiv
ethylchloroformate, 1.1 equiv TEA, DCM, 30 min, 0 °C; (ii) 1.1 equiv
4-fluorobenzylamine or phenylbutylamine, 1 h, rt; (e) TFA 2%/DCM,
triisopropylsilane, 5 min, rt; (f) (i) 1.1 equiv ethylchloroformate, 1.1 equiv
TEA, DCM, 30 min, 0 °C; (ii) 1 equiv 4-fluorobenzylamine, DCM, 1 h, rt,
isomerization; (g) 4 equiv LiOH, THF, H2O, overnight, rt; (h) (i) 1.1 equiv
ethylchloroformate, 1.1 equiv TEA, DCM, 30 min, 0 °C; (ii) 0.85 equiv
O-tritylhydroxylamine, 1 h, rt.
In this case, results were less clear-cut, suggesting that selectivity
against APN, which belongs to the host and should be left intact,
can be tractable.39

Inhibitors of PfA-M1 as Potential Antimalarial Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1327
Table 9. Antimalarial Activities of 57 and 66
Scheme 3. Synthesis of Compounds 71 and 72a
PfA-M1
inhibition
IC50 (nM)
FcB1a
IC50
(µM)
F32c
IC50
(µM)
b
b
selectivity ratio
(IC50 APN/ IC50 PfA-M1)
cmpd
57
66
27
6
134
229
59
24
57
13
a FcB1: chloroquine-resistant strain. b Inhibition of parasite growth; mean
of at least three experiments. c F32: chloroquine-sensitive strain.
54 by piperazine (compound 55) leads to a 10-fold decrease in
activity. Introduction of larger groups (compounds 61-63) leads
to a sharp decrease of activity.
Introducing a constraint via cyclization of benzylamine (49)
into 1,2,3,4-tetrahydroisoquinoline (56) leads to a small loss of
activity. Interestingly, substitution on the benzyl ring of 49 is
allowed: p-chlorobenzylamine (compound 58) and 1-naphta-
lenemethylamine (compound 60) have the same activity as
benzylamine (compound 49), whereas introduction of a fluorine
atom in the para-position (compound 57) or a methyl group in
the ortho-position (compound 59) strongly increases activity.
The impact of chlorine on activity is in sharp contrast with the
results obtained for compounds in other series. Compound 57
with a p-fluoro-benzylamine group is the most active compound
(IC50 ) 27 nM) in our array.
Table 6 summarizes the influence of the malonic substituent
on the activity of PfA-M1. Isobutyl derivatives display a lower
activity than their aromatic counterparts. This suggests that an
aromatic substituent on the malonic chain is necessary to
improve the activity of the inhibitors. In all cases, compounds
bearing a benzyl group on the malonic carbon display better or
same activities than compounds bearing m-phenoxybenzyl
group. The best hit, 57, derives from benzylmalonic acid and
displays an IC50 of 27 nM on the target. It is 150 times more
active than its analogue 38 bearing an isobutyl side chain.
a Reagents and conditions: (a) 1.2 equiv diethylmalonic ester, 0.4 equiv
acetic acid, 0.12 equiv piperidine, benzene, 72 h, reflux; (b) PtO2, H2, AcOH,
24 h, rt; (c) 4-fluorobenzylamine or 4-phenylbutylamine, 4 h, 60 °C.
In contrast with the results described above, larger groups
are tolerated in the meta position (compounds 40 and 41). In
this series, compound 41 bearing a 3-phenoxybenzylamine group
has the best activity (IC50 ) 45 nM).
Specificity. We also evaluated the specificity (IC50) of some
compounds against the mammalian APN to identify potential
interactions with host biology (Table 7). As expected from
previous results on screening on APN, isobutyl derivatives tend
to be less specific for PfA-M1 than benzyl derivatives.39 The
most important result is that compounds 52 and 57, which are
the most active inhibitors of PfA-M1, are also highly selective.
Compounds Bearing a 3-Phenoxy-benzyl on the Malonic
Carbon (Table 4). In the N-(phenylalkyl) series, chain length
with one or four methylene groups is preferred. Compound 43
is 2.5 times less active than benzylamine and phenylbutylamine
analogues (42 and 44, respectively). Interestingly, cyclic
compound 45 displays an activity similar to its noncyclic
analogue 44, whereas cyclization of benzylamine (42) into
1,2,3,4-tetrahydroisoquinoline (46) strongly affects activity,
compound 46 being 6 times less active than 42. This suggests
that both flexibility and distance between the nitrogen atom and
the aryl moiety are important criteria for the activity in this
series. Substitution of the benzyl ring by a chlorine atom or a
trifluoromethyl group in the para-position (compounds 47 and
48) appears to impact only slightly on the activity, in contrast
with the previous series. In this series, compounds 42 and 47
display the best activities (164 and 130 nM, respectively).
Compounds Bearing a Benzyl on the Malonic Carbon
(Table 5). In the N-(phenylalkyl) series (compounds 49-52),
the longest chain (compound 52) gives the best activity.
Shortening the chain to two methylene groups (compound 50)
leads to a marked decrease of activity. This observation is
confirmed with compound 53, a cyclic analogue of 50. This
suggests that distance between the nitrogen atom and the aryl
moiety is an important criterion for the activity.
Optimization of 52 and 57 and Antimalarial Activity. To
increase the activity on the target and to remove the chiral carbon
(malonic), we synthesized unsaturated analogues of 52 and 57:
compounds 66-68 and 71 and 72 (Schemes 2 and 3, respec-
tively). Carboxylic acid 65 was obtained from the corresponding
ethyl ester using LiOH in a mixture of THF and H2O (Scheme
2). The configuration of this acid was determined by a
nondecoupled 13C NMR experiment. Further reaction of 65 with
O-trityl-hydroxylamine, hydrolysis of the residual ester function,
and reaction with the desired amines gave compounds 66 and
67. ROESY experiments allowed us to attribute a Z configu-
ration to 66 and 67. E-Isomer 68 was obtained from the same
intermediate 65 by reversing the sequence of introduction of
the amine and the protected hydroxylamine (Scheme 2). Cyclic
compounds 71 and 72 were obtained in three steps (Scheme
3). First, o-nitro-benzaldehyde and diethylmalonate react in a
classical Knoevenagel reaction. Then 70 is obtained via a one-
pot reduction and cyclization.41 Finally, aminolysis of the
remaining ester function, with the corresponding amines, yielded
71 and 72 (Scheme 3). The determination of the activities of
these compounds allowed expanding our structure-activity
relationships.
Constrained compound 54 is four times less active than its
flexible analogue (52), derived of phenylbutylamine. Interest-
ingly, in this series the replacement of piperidine of compound

1328 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Flipo et al.
DMSO). An amount equal to 33 µL of the substrate Leu-pNA
(Km ) 0.099 mM), 0.3 mM in Tris-HCl buffer, was then 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 ) 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.40 Initial velocities are expressed
in µmol‚min-1. Data were normalized to the controls that represent
While cyclization of 52 and 57 (respectively, compounds 72
and 71) is detrimental for the inhibition of the target (Table 8),
introduction of an insaturation with a Z configuration at the
malonic carbon increases the activity on PfA-M1 (compounds
66 and 67; Table 8). Selectivity is either maintained (67 vs 52)
or enhanced (66 vs 57). In the 4-fluorobenzyl series, E-isomer
68 is 12 times less active than the saturated 57 and 55 times
less active than Z-isomer 66. Nevertheless, 66 and 68 are both
selective of PfA-M1 versus APN. Compound 66 displays the
best activity on the target (IC50 ) 6 nM).
V
max. For the determination of IC50 values, initial velocities were
plotted as a function of inhibitor concentration using XLfit software
from IDBS.
Various parameters were calculated for compounds 52, 57,
66-68, 71, and 72 to evaluate drug-likeness: molecular weight,
312-340; HBD, 2 or 3; HBA, 3; PSA, 70-86; and bioavail-
ability score ABS, 0.55.42 Solubility and LogD (pH 7.4) were
measured for 57 and 66 and found acceptable (Table 8). Plasma
stability at 37 °C was measured for compounds in the 4-fluo-
robenzyl series 57, 66, and 71 using LC-MS-MS. Electronic
and steric environment of the hydroxamic acid function, here
insaturation and cyclization, strongly influences the stability as
previously observed.43,44 In plasma, compound 57 is hydrolyzed
to carboxylic acid, with a half-life of 45 min. Cyclization in
1-hydroxy-2-oxo-1,2-dihydroquinoline prevents hydrolysis to
carboxylic acid and stabilizes compound 71 (t1/2 > 48 h).
Interestingly, introduction of the double bond (compound 66)
further increases stability (t1/2 ) 22 h). Generally, these
preliminary ADME studies confirmed that this series, and
compound 66 in particular, displays reasonable drug-like
properties and deserves further biological characterization.
The best inhibitor 57 directly issued from screening and its
optimized analogue 66 were tested on Plasmodium falciparum
FcB1 and F32 strains (Table 9). Both compounds demonstrated
an inhibition of parasite growth in the order of magnitude of
somepreviouslyreportedinhibitorsofplasmepsinsorfalcipains.19-23
The differences of activity between the inhibition of PfA-M1
and the in vitro antimalarial activity may be due to low cell
permeation or trapping of these compounds in red blood cells
by carbonic anhydrase, a zinc metallo-enzyme found at a high
cellular concentration in red blood cells.45 Other analogues are
currently being synthesized to optimize cell permeation. Com-
pound 66, more active on PfA-M1 and more stable than 57, is
also twice as active as its analogue. Both compounds display
good (>100 fold) selectivity toward the related mammalian
enzyme APN.
Inhibition of APN. Microsomal 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. Purified APN (33 µL) was preincubated
10 min at room temperature with 33 µL of the inhibitor (30 µM in
Tris-HCl buffer, 0.3% DMSO). The substrate Leu-pNA (33 µL;
Km ) 0.099 mM), 0.3 mM in Tris-HCl buffer, was then 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.40 Initial velocities are expressed
in µmol‚min-1. Data were normalized to the controls that represent
V
max. For the determination of IC50 values, initial velocities were
plotted as a function of inhibitor concentration, using XLfit software
from IDBS.
Parasite Growth Inhibition. P. falciparum strains were main-
tained continuously in culture on human erythrocytes, as described
by Trager and Jensen.46 In vitro antiplasmodial activity was
determined using a modification of the semiautomated microdilution
technique of Desjardins et al.47 P. falciparum chloroquine (CQ)-
resistant FcB1 (Colombia) strain (CQ: IC50 ) 115 ( 5 nM) or
chloroquine (CQ)-sensitive F32 (Tanzania) strain (CQ: IC50 ) 21
( 5 nM) was used. Stock solutions of test compounds were prepared
in sterile distilled water and DMSO, respectively. Drug solutions
were serially diluted with culture medium and introduced to
asynchronous parasite cultures (0.5% parasitemia and 1% final
hematocrite) on 96-well plates for 24 h at 37 °C prior to the addition
of 0.5 mCi of [3H]hypoxanthine (1-5 Ci/mmol; Amersham, France)
per well. The growth inhibition of each drug concentration was
determined by comparison of the radioactivity incorporated into
the treated culture with that in the control culture (without drug)
maintained on the same plate. The IC50 value, as obtained from
the drug concentration-response curve, was expressed as the mean
of three independent experiments, SD were <10% in most cases.
The DMSO concentration never exceeded 0.1% and did not inhibit
the parasite growth.
Conclusion
We have screened a library of hydroxamates with a malonic
scaffold for its inhibition of PfA-M1, a zinc amino-peptidase
of the M1 family of Plasmodium falciparum. Several inhibitors
displaying promising in vitro activity on the enzyme have been
identified. Furthermore, most of them showed the desired
specificity against APN, the host M1 family prototype. Espe-
cially, we have identified compound 57 as a promising hit,
displaying activity on Plasmodium falciparum FcB1 and F32
strains. Further optimization produced 66, the best inhibitor of
PfA-M1 so far described, which demonstrated antiplasmodial
activity.
Solubility. Log D. Plasma Stability. These experiments used a
LC-MS-MS triple-quadrupole system (Varian 1200ws) under MRM
detection using the following mass parameters: mode of ionization,
electrospray; declustering potential, 50 V; collision-activated dis-
sociation, 1.5 mTorr; collision energy, 20 eV. Transitions observed
were, respectively: 57 (317 > 109); 66 (315 > 109); 71 (313 >
109).
Solubility. The 10 mM solution (40 µL) 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, then centrifuged
for 5 min, and filtered over 0.45 µm filters. An amount equal to 20
µL 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.
Experimental Section
Biology. Inhibition of PfA-M1. Native PfA-M1 (FcB1 strain)
was purified according to the procedure described by Allary et al.35
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. An amount equal to 33 µL of
purified PfA-M1 was preincubated 10 min at room temperature
with 33 µL of the inhibitor (30 µM in Tris-HCl buffer, 0.3%
Log D. A 40 µL aliquot 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

Inhibitors of PfA-M1 as Potential Antimalarial Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1329
solution. The mixture was gently shaken for 2 h at room temper-
ature, 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.
were added. The mixture was stirred 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% (6 times) and NaCl (once). The organic layer
was dried over MgSO4 and evaporated under reduced pressure. The
residue was precipitated in petroleum ether.
Plasma Stability. A 40 µL aliquot of the 5 mM solution in
DMSO of the sample was added to 1.960 mL of rat plasma (male
Zucker rat FA/FA plasma from Charles River Laboratories) to
obtain a 100 µM final solution. The mixture was gently stirred 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). A
10 µL aliquot 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.
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 4 h to 5 h 30 min at room
temperature. Solvents were removed under reduced pressure and
the residue was dissolved in water. The aqueous phase was basified
with NaOH 1 M to pH ) 7 and extracted three times with AcOEt.
The organic 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 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.
Chemistry. General Information. NMR spectra were recorded
on a Bruker DRX-300 spectrometer. Chemical shifts are in parts
per million (ppm). The assignments were made using one-
1
dimensional (1D) H and 13C spectra and two-dimensional (2D)
HSQC and COSY spectra. Mass spectra were recorded on a
MALDI-TOF Voyager-DE-STR spectrometer or with a LC-MS-
MS triple-quadrupole system (Varian 1200ws). HPLC analysis was
performed using a C18 TSK-GEL Super ODS 2 µm particle size
column, dimensions 50 × 4.6 mm. A gradient starting from 100%
H2O/0.05% TFA and reaching 20% H2O/80% CH3CN/0.05% TFA
within 10 min at a flow rate of 1 mL/min was used. LCMS gradient
starting from 100% H2O/0.1% formic acid and reaching 20% H2O/
80% CH3CN/0.08% formic acid within 10 min at a flow rate of 1
mL/min was used. Melting points were determined on a Bu¨chi
B-540 apparatus and are uncorrected. All commercial reagents and
solvents were used without further purification. Organic layers
obtained after extraction of aqueous solutions were dried over
MgSO4 and filtered before evaporation in vacuo. Purification yields
were not optimized. Bond-Elut SAX-columns were purchased from
Varian, Inc. All the 5 µmol scale reactions were performed in 96-
well (round-bottomed) polypropylene plates (1200 µL).
N-Benzyl-N′-hydroxy-2-isobutyl-malonamide (32; Method B):
White powder; yield 75%; purity 100%; H NMR (DMSO-d6) δ
1
0.85 (d, J ) 6.0 Hz, 6H), 1.39-1.52 (m, 1H), 1.53-1.70 (m, 2H),
3.04 (t, J ) 7.5 Hz, 1H), 4.26 (d, J ) 5.7 Hz, 2H), 7.21-7.34 (m,
5H), 8.15 (t, J ) 5.7 Hz, NHCO), 8.97 (s, 1H, OH), 10.53 (s, 1H,
CONHO); 13C NMR (DMSO-d6) δ 22.6, 23.0, 26.0, 38.9, 42.7,
49.4, 127.2, 127.5, 128.7, 139.8, 167.2, 169.5; tR(LCMS) 3.90 min;
MS (MH)+ m/z 265 and (MNa)+ m/z 287.
N-Hydroxy-2-isobutyl-N′-phenethyl-malonamide (33; Method
B): White powder; yield 74%; purity 100%; 1H NMR (DMSO-d6)
δ 0.82 (d, J ) 6.6 Hz, 6H), 1.30-1.43 (m, 1H), 1.45-1.58 (m,
2H), 2.69 (t, J ) 7.2 Hz, 2H), 2.94 (t, J ) 7.5 Hz, 1H), 3.27 (q,
J ) 6.9 Hz, 2H), 7.18-7.31 (m, 5H), 7.68 (t, J ) 5.7 Hz, NHCO),
8.94 (s, 1H, OH), 10.47 (s, 1H, CONHO); tR(LCMS) 4.11 min; 13C
NMR (DMSO-d6) δ 22.6, 22.9, 25.9, 35.5, 39.2, 41.0, 49.4, 126.5,
128.7, 129.1, 139.8, 167.3, 169.3; MS (MH)+ m/z 279 and (MNa)+
m/z 301.
Synthesis from N-tert-Butoxy-malonamic Acids Using PyBrop
as Coupling Agent (Method A): Synthesis of N-tert-butoxy-
malonamic acid precursors is described in reference 36. Carboxylic
acid (0.5 M/DMF; 1 equiv), DIEA (1.2 equiv), and PyBrop (0.2
M/DCM; 1.2 equiv) were stirred 30 s at room temperature. Then
the appropriate amine (0.1 M/DMF; 1 equiv) and DIEA (2 equiv)
were added, the mixture was stirred overnight at room temperature,
and then solvents were removed under reduced pressure. The residue
was dissolved in DCM, and the organic phase was washed with
aqueous HCl 1 N (3 times), aqueous NaHCO3 5% (3 times), and
water (once). The organic layer was dried over MgSO4 and
evaporated under reduced pressure. The residue was purified by
thick layer chromatography on silica gel (DCM/MeOH 95/5).
Synthesis from N-Trityloxy-malonamic Acids Using CDI as
Coupling Agent (Method B): Synthesis of N-trityloxy-malonamic
acid precursors is described in reference 36. Carboxylic acid (0.5
M/DMF; 1 equiv), DIEA (1.1 equiv), and N,N′-carbonyldiimidazole
(0.25 M/THF; 1.1 equiv) were stirred 2 h at room temperature.
Then the appropriate amine (0.1 M/DMF; 1 equiv) and DIEA (2
equiv) were added, the mixture was stirred 2 h at room temperature,
and then solvents were removed under reduced pressure. The residue
was dissolved in the minimum AcOEt, and the organic phase was
washed with aqueous KHSO4 (pH ) 3) and water (3 times). The
organic layer was dried over MgSO4 and evaporated under reduced
pressure. The residue was purified on Bond-Elut SAX column using
DCM to remove residual carboxylic acid. The powder obtained
was triturated in ether-pentane.
N-Hydroxy-2-isobutyl-N′-(3-phenyl-propyl)-malonamide (34;
Method B): White powder; yield 75%; purity 100%; H NMR
1
(DMSO-d6) δ 0.85 (d, J ) 6.0 Hz, 6H), 1.38-1.51 (m, 1H), 1.55-
1.73 (m, 4H), 2.48-2.52 (m, 2H), 2.98 (t, J ) 7.5 Hz, 1H), 3.00-
3.07 (m, 2H), 7.18-7.31 (m, 5H), 7.69 (br s, NHCO), 8.93 (s, 1H,
OH), 10.47 (s, 1H, CONHO);13C NMR (DMSO-d6) δ 22.6, 22.9,
26.1, 31.3, 32.9, 38.7, 38.9, 49.5, 126.2, 128.7, 128.8, 142.1, 167.3,
169.4; tR(LCMS) 4.63 min; MS (MH)+ m/z 293.
N-Hydroxy-2-isobutyl-N′-(4-phenyl-butyl)-malonamide (35;
Method A): The compound was deprotected using TFA 80%/DCM,
room temperature, 72 h. It was purified by reverse-phase preparative
1
HPLC and lyophilized; white powder; yield 7%; purity 100%; H
NMR (DMSO-d6) δ 0.75 (dd, J ) 1.2 Hz, J ) 6.5 Hz, 6H), 1.28-
1.35 (m, 3H), 1.40-1.50 (m, 4H), 2.48 (t, J ) 7.3 Hz, 2H), 2.87
(t, J ) 7.5 Hz, 1H), 2.95-3.00 (m, 2H), 7.07-7.12 (m, 3H), 7.16-
7.20 (m, 2H), 7.54 (t, J ) 5.7 Hz, NHCO), 8.82 (br s, OH), 9.30
(br s, CONHO); tR(LCMS) 5.10 min; MS (MH)+ m/z 307.
2-(4-Benzyl-piperidine-1-carbonyl)-4-methyl-pentanoic Acid
Hydroxyamide (36; Method C): White powder; yield 30%; purity
1
100%; H NMR (CD2Cl2) δ 0.91-0.98 (m, 6H), 1.11-1.22 (m,
2H), 1.55-1.59 (m, 1H), 1.61-1.82 (m, 5H), 2.52-2.59 (m, 3H),
3.03 (t, J ) 12.9 Hz, 1H), 3.73 (t, J ) 7.0 Hz, 1H), 3.98 (d, J )
13.2 Hz, 1H), 4.58 (d, J ) 12.9 Hz, 1H), 7.16-7.24 (m, 3H), 7.29-
7.34 (m, 2H); tR(LCMS) 5.30 min; MS (MH)+ m/z 333.
N-Hydroxy-2-isobutyl-N′-(1,2,3,4-tetrahydro-naphthalen-1-
yl)-malonamide (37; Method B): White powder; yield 24%; purity
100%; mixture of the cis and trans form of the amide function; 1H
NMR (DMSO-d6) δ 0.86 (d, J ) 6.3 Hz, 6H), 1.42-1.86 (m, 7H),
2.64-2.82 (m, 2H), 3.01-3.08 (m, 1H), 4.91-4.94 (m, 1H), 7.09-
7.17 (m, 4H), 7.95 (d, J ) 8.4 Hz, 0.6H, NHCO), 7.99 (d, J ) 8.4
Hz, 0.4H, NHCO), 8.96 (s, 1H, OH), 10.42 (s, 1H, CONHO);
tR(LCMS) 4.71 min; MS (MH)+ m/z 305.
Synthesis from N-Trityloxy-malonamic Acids Using HOBt/
EDCI as Coupling Agents (Method C): Synthesis of N-trityloxy-
malonamic acid precursors is described in reference 36. Carboxylic
acid (0.1 M/DMF; 1 equiv), DIEA (2.4 equiv), EDCI (1.1 equiv),
and HOBt (1.1 equiv) were stirred 5 min at room temperature. Then
the appropriate amine (0.1 M/DMF; 1 equiv) and DIEA (2 equiv)

1330 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Flipo et al.
N-(4-Fluoro-benzyl)-N′-hydroxy-2-isobutyl-malonamide (38;
Method A): Beige powder; yield 15%; purity 95%; 1H NMR
(DMSO-d6) δ 0.85 (d, J ) 5.4 Hz, 6H), 1.39-1.67 (m, 3H), 3.03
(t, J ) 7.5 Hz, 0.7H), 3.19 (t, J ) 6.9 Hz, 0.3H), 4.24 (d, J ) 5.7
Hz, 2H), 7.12-7.17 (m, 2H), 7.25-7.29 (m, 2H), 8.17 (t, J ) 5.7
Hz, 0.7H, NHCO), 8.40 (s, 0.3H, NHCO), 8.96 (s, 1H, OH), 10.54
(s, 1H, CONHO); tR(LCMS) 4.13 min; MS (MH)+ m/z 283.
N-(4-Chloro-benzyl)-N′-hydroxy-2-isobutyl-malonamide (39;
Method A): White powder; yield 30%; purity 95%; 1H NMR
(DMSO-d6) δ 0.85 (d, J ) 6.3 Hz, 6H), 1.42-1.64 (m, 3H), 3.03
(t, J ) 7.5 Hz, 0.5H), 3.20 (t, J ) 6.3 Hz, 0.5H), 4.24 (d, J ) 6
Hz, 2H), 7.23-7.39 (m, 4H), 8.20 (t, J ) 6.0 Hz, 0.5H, NHCO),
8.43 (m, 0.5H, NHCO), 8.96 (s, 1H, OH), 10.55 (s, 1H, CONHO);
tR(LCMS) 4.65 min; MS (MH)+ m/z 299.
N-(4-Chloro-benzyl)-N′-hydroxy-2-(3-phenoxy-benzyl)-mal-
onamide (47; Method B): Beige powder; yield 73%; purity 99%;
1H NMR (DMSO-d6) δ 3.00-3.06 (m, 2H), 3.30-3.35 (m, 1H),
4.13-4.28 (m, 2H), 6.82-7.36 (m, 13H), 8.27-8.30 (m, NHCO),
8.96 (s, OH), 10.45 (s, CONHO); 13C NMR (DMSO-d6) δ 34.9,
42.0, 52.6, 117.0, 118.8, 119.7, 123.7, 124.6, 128.6, 129.3, 130.2,
130.4, 131.6, 138.8, 141.6, 156.8, 157.1, 166.0, 168.6; tR(LCMS) 5.82
min; MS (MH)+ m/z 425; mp 169-171 °C.
N-Hydroxy-2-(3-phenoxy-benzyl)-N′-(4-trifluoromethyl-ben-
zyl)-malonamide (48; Method B): White powder; yield 78%;
1
purity 99%; H NMR (DMSO-d6) δ 3.02-3.07 (m, 2H), 3.32-
3.37 (m, 1H), 4.26 (dd, J ) 15.0 Hz, J ) 4.5 Hz, 1H), 4.36 (dd,
J ) 15.6 Hz, J ) 5.7 Hz, 1H), 6.82-7.00 (m, 5H), 7.12 (t, J ) 7.3
Hz, 1H), 7.25-7.39 (m, 5H), 7.60 (d, J ) 7.8 Hz, 2H), 8.37-8.40
(m, NHCO), 8.97 (s, OH), 10.49 (s, CONHO); 13C NMR (DMSO-
d6) δ 34.9, 42.3, 52.6, 116.9, 118.8, 119.7, 123.7, 124.6, 124.8 (d,
JCF ) 270 Hz), 125.4 (d, JCF ) 4 Hz), 127.8 (d, JCF ) 35 Hz),
128.0, 130.2, 130.4, 141.6, 144.7, 156.8, 157.1, 165.9, 168.7;
tR(LCMS) 6.05 min; MS (MH)+ m/z 459; mp 189-191 °C.
N-Hydroxy-2-isobutyl-N′-naphthalen-1-ylmethyl-malona-
mide (40; Method A): Beige powder; yield 59%; purity 95%; 1H
NMR (DMSO-d6) δ 0.85 (d, J ) 6.3 Hz, 6H), 1.41-1.50 (m, 1H),
1.64 (t, J ) 6.9 Hz, 2H), 3.08 (t, J ) 7.5 Hz, 1H), 4.68 (dd, J )
5.4, J ) 15.3 Hz, 1H), 4.79 (dd, J ) 5.4, J ) 15.3 Hz, 1H), 7.43-
7.57 (m, 4H), 7.86 (d, J ) 7.5 Hz, 1H), 7.94-7.97 (m, 1H), 8.04-
8.07 (m, 1H), 8.19 (t, J ) 5.4 Hz, 1H, NHCO), 8.97 (s, 1H, OH),
10.52 (s, 1H, CONHO); tR(HPLC) 5.03 min; MS (MH)+ m/z 315.
N-Hydroxy-2-isobutyl-N′-(3-phenoxy-benzyl)-malonamide (41;
2N-Dibenzyl-N′-hydroxy-malonamide (49; Method C): White
1
powder; yield 84%; purity 100%; H 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, 127.1, 127.5, 128.6, 128.7,
129.3, 139.4, 139.6, 166.2, 168.6; tR(LCMS) 4.25 min; MS (MH)+
m/z 299; mp 167-169 °C.
1
Method B): White powder; yield 52%; purity 100%; H NMR
(DMSO-d6) δ 0.82 (d, J ) 6.3 Hz, 6H), 1.35-1.44 (m, 1H), 1.51-
1.63 (m, 2H), 3.02 (t, J ) 7.5 Hz, 1H), 4.24-4.27 (m, 2H), 6.85-
6.90 (m, 2H), 6.98-7.01 (m, 3H), 7.14 (t, J ) 7.2 Hz, 1H), 7.32
(t, J ) 7.8 Hz, 1H), 7.39 (t, J ) 8.1 Hz, 2H), 8.19 (t, J ) 5.4 Hz,
NHCO), 8.95 (s, 1H, OH), 10.52 (s, 1H, CONHO); tR(LCMS) 5.32
min; MS (MH)+ m/z 357.
N-Benzyl-N′-hydroxy-2-(3-phenoxy-benzyl)-malonamide (42;
Method B): Beige powder; yield 89%; purity 99%; 1H NMR
(DMSO-d6) δ 2.96-3.10 (m, 2H), 3.29-3.32 (m, 1H), 4.17 (dd,
J ) 15.1 Hz, J ) 5.2 Hz, 1H), 4.29 (dd, J ) 15.1 Hz, J ) 6.1 Hz,
1H), 6.81-7.39 (m, 14H), 8.26 (t, J ) 5.7 Hz, NHCO), 8.95 (s,
OH), 10.47 (s, CONHO); tR(LCMS) 5.47 min; MS (MH)+ m/z 391;
mp 164-167 °C.
N-Hydroxy-2-(3-phenoxy-benzyl)-N′-(3-phenyl-propyl)-mal-
onamide (43; Method B): White powder; yield 68%; purity 99%;
1H NMR (DMSO-d6) δ 1.60-1.64 (m, 2H), 2.45-2.50 (m, 2H),
2.95-3.05 (m, 4H), 3.22 (t, J ) 7.0 Hz, 1H), 6.78-6.95 (m, 5H),
6.99-7.37 (m, 9H), 7.73-7.76 (m, NHCO), 8.93 (s, OH), 10.41
(s, CONHO); 13C NMR (DMSO-d6) δ 31.2, 32.8, 34.9, 38.8, 52.6,
116.9, 118.8, 119.7, 123.7, 124.6, 126.2, 128.7, 128.8, 130.1, 130.4,
141.8, 142.1, 156.7, 157.1, 166.2, 168.4; tR(LCMS) 5.90 min; MS
(MH)+ m/z 419; mp 126-128 °C.
N-Hydroxy-2-(3-phenoxy-benzyl)-N′-(4-phenyl-butyl)-mal-
onamide (44; Method B): White powder; yield 88%; purity 99%;
1H NMR (DMSO-d6) δ 1.31-1.38 (m, 2H), 1.43-1.50 (m, 2H),
2.52-2.55 (m, 2H), 2.96-3.04 (m, 4H), 3.19 (t, J ) 7.6 Hz, 1H),
6.78-6.97 (m, 5H), 7.10-7.39 (m, 9H), 7.69 (t, J ) 5.4 Hz,
NHCO), 8.92 (s, OH), 10.39 (s, CONHO); 13C NMR (DMSO-d6)
δ 28.6, 29.0, 34.9, 35.2, 39.2, 52.6, 117.0, 118.8, 119.7, 123.7,
124.6, 126.1, 128.7, 128.8, 130.1, 130.4, 141.7, 142.5, 156.7, 157.2,
166.2, 168.3; tR(LCMS) 6.26 min; MS (MH)+ m/z 433; mp 131-
133 °C.
2-Benzyl-N-hydroxy-N′-phenethyl-malonamide (50; Method
A): Beige powder; yield 17%; purity 100%; 1H NMR (DMSO-d6)
δ 2.63 (t,J ) 7.1 Hz, 2H), 2.96 (d, J ) 7.2 Hz, 2H), 3.17-3.22
(m, 3H), 7.12-7.29 (m, 10H), 8.07 (s, NHCO); tR(LCMS) 4.31 min;
MS (MH)+ m/z 313; mp 121-125 °C.
2-Benzyl-N-hydroxy-N′-(3-phenyl-propyl)-malonamide (51;
Method C): White powder; yield 53%; purity 98%; 1H NMR
(DMSO-d6) δ 1.58-1.68 (m, 2H), 2.45-2.50 (m, 2H), 2.99-3.05
(m, 4H), 3.22 (t, J ) 7.5 Hz, 1H), 7.14-7.29 (m, 10H), 7.74 (t,
J ) 5.4 Hz, NHCO), 8.91 (s, OH), 10.41 (s, CONHO); 13C NMR
(DMSO-d6) δ 31.2, 32.9, 35.1, 38.8, 52.8, 126.2, 126.6, 128.6,
128.7, 128.8, 129.2, 139.5, 142.1, 166.3, 168.5; tR(LCMS) 4.82 min;
MS (MH)+ m/z 327; mp 148-150 °C.
2-Benzyl-N-hydroxy-N′-(4-phenyl-butyl)-malonamide (52;
Method B): White powder; yield 18%; purity 99%; 1H NMR
(DMSO-d6) δ 1.29-1.39 (m, 2H), 1.43-1.53 (m, 2H), 2.53 (t,
J ) 7.5 Hz, 2H), 2.98-3.06 (m, 4H), 3.19 (t, J ) 7.8 Hz, 1H),
7.16-7.25 (m, 10H), 7.69 (t, J ) 5.4 Hz, 1H, NHCO), 8.91 (s,
1H, OH), 10.4 (br s, 1H, CONHO); 13C NMR (DMSO-d6) δ 28.6,
29.1, 35.1, 35.2, 39.2, 52.8, 126.1, 126.6, 128.6, 128.7, 128.8, 129.2,
139.5, 142.5, 166.3, 168.5; tR(LCMS) 5.20 min; MS (MH)+ m/z 341.
2-Benzyl-N-hydroxy-N′-indan-2-yl-malonamide (53; Method
1
B): White powder; yield 76%; purity 99%; H NMR (DMSO-d6)
δ 2.63 (dd, J ) 5.4 Hz, J ) 16 Hz, 1H), 2.75 (dd, J ) 5.4 Hz, J
) 16 Hz, 1H), 3.00 (d, J ) 7.2 Hz, 2H), 3.09 (t, J ) 7.2 Hz, 1H),
3.16 (dd, J ) 7.8 Hz, J ) 15.8 Hz, 2H), 4.35-4.46 (m, 1H), 7.12-
7.27 (m, 9H), 7.99 (d, J ) 7.2 Hz, 1H, NHCO), 8.62 (s, 1H, OH),
10.35 (br s, 1H, CONHO); tR(LCMS) 4.92 min; MS (MH)+ m/z 325.
2-Benzyl-3-(4-benzyl-piperidin-1-yl)-N-hydroxy-3-oxo-propi-
onamide (54; Method C): Yellow oil; yield 21% after purification
by thick layer chromatography; purity 95%; 1H NMR (DMSO-d6)
δ 0.73-1.07 (m, 2H), 1.32-1.55 (m, 2H), 1.60-1.75 (m, 1H),
2.27-2.50 (m, 3H), 2.73-2.96 (m, 2H), 3.01-3.11 (m, 1H), 3.63-
3.72 (m, 1H), 3.83-3.94 (m, 1H), 4.31 (d, J ) 12.7 Hz, 1H), 7.09-
7.27 (m, 10H), 8.87 (s, 0.55 OH), 8.92 (s, 0.45 OH), 10.43 (0.55
CONHO), 10.54 (s, 0.45 CONHO); tR(LCMS) 5.46 min; MS (MH)+
m/z 367.
2-Benzyl-3-(4-benzyl-piperazin-1-yl)-N-hydroxy-3-oxo-propi-
onamide (55; Method C): Orange powder; yield 72%; purity 96%;
1H NMR (DMSO-d6) δ 2.56-2.66 (m, 2H), 2.99 (d, J ) 6.3 Hz,
2H), 3.05-3.23 (m, 6H), 3.57-3.69 (m, 2H), 3.73-3.79 (m, 1H),
7.19-7.43 (m, 10H), 8.95 (s, OH), 10.56 (s, CONHO); tR(LCMS)
3.16 min; MS (MH)+ m/z 368.
3-(4-Benzyl-piperidin-1-yl)-N-hydroxy-3-oxo-2-(3-phenoxy-
benzyl)-propionamide (45; Method B): Beige powder; yield 74%;
purity 95%; H NMR (DMSO-d6) δ 0.75-1.04 (m, 2H), 1.38-
1
1.50 (m, 2H), 1.68-1.72 (m, 1H), 2.37-2.50 (m, 4H), 2.78-2.94
(m, 2H), 3.67-3.71 (m, 1H), 3.85-3.92 (m, 1H), 4.27-4.31 (m,
1H), 6.78-7.41 (m, 14H), 10.45 (s, 0.6 CONHO), 10.55 (s, 0.4
CONHO); tR(LCMS) 6.50 min; MS (MH)+ m/z 459; mp 73-77 °C.
3-(3,4-Dihydro-1H-isoquinolin-2-yl)-N-hydroxy-3-oxo-2-(3-
phenoxy-benzyl)-propionamide (46; Method B): Yellow oil; yield
85%; purity 95%; mixture of the cis and trans form of the amide
function (0.6/0.4); 1H NMR (CD2Cl2) δ 2.28-2.37 (m, 0.6H),
2.66-2.77 (m, 1.4H), 3.12-3.32 (m, 2.6H), 3.47-3.64 (m, 1H),
3.79-3.86 (m, 0.4H), 3.96-4.15 (m, 2H), 4.47-4.60 (m, 1H),
6.70-7.34 (m, 13H), 10.23 (s, CONHO); tR(LCMS) 5.77 min; MS
(MH)+ m/z 417.

Inhibitors of PfA-M1 as Potential Antimalarial Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1331
2-Benzyl-3-(3,4-dihydro-1H-isoquinolin-2-yl)-N-hydroxy-3-
oxo-propionamide (56; Method A): Beige powder; yield 46%;
purity 100%; H NMR (DMSO-d6) δ 2.67-2.79 (m, 2H), 3.00-
3.08 (m, 2.5H), 3.56-3.79 (m, 2.5H), 3.28-3.35 (m, 0.5H), 4.44-
4.50 (m, 0.5H), 4.61-4.69 (m, 1H), 7.10-7.23 (m, 9H), 8.95 (s,
OH), 10.62 (s, CONHO); tR(LCMS) 4.53 min; MS (MH)+ m/z 325;
mp 137-142 °C.
equiv). The reaction mixture was stirred 4 h at room temperature.
THF was evaporated under reduced pressure. A solution of NaHCO3
5% was added, and the remaining diethyl ester was extracted with
DCM. The aqueous phase was then acidified to pH ) 1 with HCl,
and the expected product was extracted three times with DCM.
The organic phases were joined, dried over MgSO4, and evaporated
under reduced pressure to give 2-[1-phenyl-meth-(Z)-ylidene]-
malonic acid monoethyl ester (65) as a white powder. The
configuration of this acid was determined by nondecoupled 13C
NMR experiment (see Supporting Information). Yield 85%; purity
1
2-Benzyl-N-(4-fluoro-benzyl)-N′-hydroxy-malonamide (57;
1
Method C): White powder; yield 88%; purity 100%; H NMR
(DMSO-d6) δ 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 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;
tR(LCMS) 4.31 min; MS (MH)+ m/z 317; mp 193-194 °C.
1
98%; H NMR (DMSO-d6) δ 1.20 (t, J ) 7.2 Hz, 3H), 4.26 (q,
J ) 7.2 Hz, 2H), 7.43-7.51 (m, 5H), 7.66 (s, 1H), 13.35 (s, 1H,
OH); MS (MH)+ m/z 221.
N-(4-Fluoro-benzyl)-N′-hydroxy-2-[1-phenyl-meth-(Z)-ylidene]-
malonamide (66). Acid (65; 2.225 g; 10.1 mmol; 1 equiv) was
dissolved in DCM (25 mL) with catalytic DMF (75 µL). The
mixture was cooled at 0 °C (ice bath), and oxalyl chloride (1.041
mL; 12.1 mmol; 1.2 equiv) was added dropwise. The reaction
mixture was stirred 45 min at 0 °C and then evaporated under
reduced pressure. The residue was dissolved in DCM (25 mL) and
cooled at 0 °C. DIEA (5 mL; 30 mmol; 3 equiv) was added and
then O-trityl-hydroxylamine (2.365 g; 8.6 mmol; 0.85 equiv) was
added. The reaction mixture was stirred 3 h at room temperature.
The organic phase was washed with NaHCO3 5%, dried over
MgSO4, and evaporated under reduced pressure. A minimum of
DCM was added and trituration with petroleum ether allowed (Z)-
3-phenyl-2-trityloxycarbamoyl-acrylic acid ethyl ester as a white
powder. The configuration of this compound was determined by
ROESY NMR experiment. Yield 45%; purity 95%; 1H NMR
(DMSO-d6) δ 1.19 (t, J ) 7.2 Hz, 3H), 4.13 (q, J ) 7.2 Hz, 2H),
7.21-7.35 (m, 20H), 7.51 (s, 1H), 10.37 (s, 1H, NH); MS (MNa)+
m/z 500. A total of 900 mg (1.9 mmol; 1 equiv) of ester was
dissolved in 20 mL of a mixture of THF and water (1/1). A total
of 320 mg of LiOH (7.6 mmol; 4 equiv) was added, and the mixture
was stirred overnight at room temperature. THF was evaporated
under reduced pressure. The aqueous phase was then acidified to
pH ) 6 with HCl 1 N, and the expected product was extracted
four times with DCM. The organic phases were joined, dried over
MgSO4, and evaporated under reduced pressure. As this stage, the
double bond is isomerized to give (E)-3-phenyl-2-trityloxycarbam-
oyl-acrylic acid as a white powder. The configuration of this acid
was determined by nondecoupled 13C NMR experiment. Yield 85%;
2-Benzyl-N-(4-chloro-benzyl)-N′-hydroxy-malonamide (58;
1
Method C): White powder; yield 90%; purity 97%; H NMR
(DMSO-d6) δ 3.00-3.08 (m, 2H), 3.30 (t, J ) 7.35 Hz, 1H), 4.19
(dd, J ) 4.8 Hz, J ) 14.7 Hz, 1H), 4.26 (dd, J ) 6.3 Hz, J ) 14.4
Hz, 1H), 7.10-7.32 (m, 9H), 8.26-8.28 (m, NHCO), 10.46 (s,
CONHO); 13C NMR (DMSO-d6) δ 35.1, 42.0, 52.8, 126.7, 128.5,
128.6, 129.2, 129.3, 131.6, 138.8, 139.3, 166.2, 168.7; tR(LCMS) 4.80
min; MS (MH)+ m/z 333; mp 198-200 °C.
2-Benzyl-N-hydroxy-N′-(2-methyl-benzyl)-malonamide (59;
1
Method C): White powder; yield 85%; purity 97%; H NMR
(DMSO-d6) δ 2.18 (s, 3H), 3.02-3.06 (m, 2H), 3.32 (t, J ) 7.5
Hz, 1H), 4.14-4.23 (m, 2H), 6.99-7.25 (m, 9H), 8.05 (t, J ) 5.4
Hz, NHCO), 8.95 (s, OH), 10.42 (s, CONHO); 13C NMR (DMSO-
d6) δ 19.0, 35.1, 41.0, 52.7, 126.1, 126.6, 127.3, 127.9, 128.6, 129.3,
130.3, 136.1, 137.0, 139.4, 166.3, 168.4; tR(LCMS) 4.53 min; MS
(MH)+ m/z 313; mp 191-193 °C.
2-Benzyl-N-hydroxy-N′-naphthalen-1-ylmethyl-malona-
mide (60; Method A): Beige powder; yield 25%; purity 99%; 1H
NMR (DMSO-d6) δ 3.03 (dd, J ) 7.9 Hz, J ) 13.6 Hz, 1H), 3.11
(dd, J ) 7.6 Hz, J ) 13.6 Hz, 1H), 3.32-3.35 (m, 1H), 4.62 (dd,
J ) 15.3, J ) 5.1 Hz, 1H), 4.77 (dd, J ) 15.3, J ) 6.0 Hz, 1H),
7.19-7.25 (m, 6H), 7.39 (t, J ) 7.6 Hz, 1H), 7.51-7.54 (m, 2H),
7.83 (d, J ) 8.1 Hz, 1H), 7.92-7.99 (m, 2H), 8.27 (t, J ) 5.4 Hz,
NHCO), 8.95 (s, OH), 10.44 (s, CONHO); 13C NMR (DMSO-d6)
δ 35.1, 40.1, 52.7, 123.8, 125.5, 125.8, 126.2, 126.6, 126.7, 127.9,
128.6, 128.9, 129.3, 131.2, 133.7, 134.6, 139.4, 166.2, 168.6;
tR(LCMS) 4.89 min; MS (MH)+ m/z 349; mp 156-159 °C.
1
purity 95%; H NMR (CD2Cl2) δ 7.16-7.39 (m, 20H), 7.90 (s,
1H); MS (MH)- m/z 448. This acid (297 mg; 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 4-fluorobenzylamine (84 µL; 0.73 mmol; 1.1 equiv) was added.
The organic layer was stirred 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%)
then precipitated in petroleum ether to give a white powder. Yield
20%; purity 95%; MS (MH)- m/z 555. The protected hydroxamic
acid (64 mg; 0.11 mmol) was dissolved in TFA 2%/DCM (0.03
M), and triisopropylsilane was added drop by drop until the yellow
color disappeared. The reaction mixture was stirred 5 min at room
temperature, solvents were removed under reduced pressure, and
the residue was washed with petroleum ether to give (66) as a white
powder. The configuration of this compound was determined by
ROESY NMR experiment (see Supporting Information). Yield 96%;
2-Benzyl-N-hydroxy-N′-(3-phenoxy-benzyl)-malonamide (61;
Method B): White powder; yield 64%; purity 99%; 1H NMR
(DMSO-d6) δ 3.01 (d, J ) 7.5 Hz, 2H), 3.29 (dd, J ) 4.2 Hz, J )
7.8 Hz, 1H), 4.18 (dd, J ) 5.7 Hz, J ) 15.3 Hz, 1H), 4.29 (dd,
J ) 5.7 Hz, J ) 15.3 Hz, 1H), 6.83-6.90 (m, 3H), 6.99 (d, J )
7.8 Hz, 2H), 7.10-7.30 (m, 7H), 7.39 (t, J ) 7.8 Hz, 2H), 8.26 (t,
J ) 6 Hz, NHCO), 8.94 (s, 1H, OH), 10.46 (s, 1H, CONHO); 13
C
NMR (DMSO-d6) δ 35.1, 42.4, 52.8, 117.3, 117.8, 119.0, 122.6,
123.8, 126.7, 128.6, 129.2, 130.3, 130.5, 139.4, 142.1, 157.0, 157.1,
166.2, 168.7; tR(LCMS) 5.45 min; MS (MH)+ m/z 391.
2-Benzyl-N-biphenyl-3-ylmethyl-N′-hydroxy-malonamide (62;
Method B): White powder; yield 71%; purity 99%; 1H NMR
(DMSO-d6) δ 3.03-3.09 (m, 2H), 3.28-3.32 (m, 1H), 4.28 (dd,
J ) 5.8 Hz, J ) 15.3 Hz, 1H), 4.37 (dd, J ) 6.0 Hz, J ) 15.0 Hz,
1H), 7.10-7.22 (m, 6H), 7.34-7.39 (m, 2H), 7.45-7.52 (m, 4H),
7.62-7.65 (m, 2H), 8.29 (t, J ) 5.4 Hz, NHCO), 8.94 (s, 1H, OH),
10.48 (s, 1H, CONHO); tR(LCMS) 5.43 min; MS (MH)+ m/z 375.
2-Benzyl-N-(2,2-diphenyl-ethyl)-N′-hydroxy-malonamide (63;
Method B): White powder; yield 50%; purity 98%; 1H NMR
(DMSO-d6) δ 2.84 (d, J ) 7.5 Hz, 2H), 3.11 (t, J ) 7.2 Hz, 1H),
3.59-3.78 (m, 2H), 4.11 (t, J ) 7.8 Hz, 1H), 7.06-7.29 (m, 15H),
7.69 (t, J ) 5.4 Hz, 1H, NHCO), 8.91 (s, 1H, OH), 9.85 (s, 1H,
CONHO); tR(LCMS) 5.49 min; MS (MH)+ m/z 389.
2-[1-Phenyl-meth-(Z)-ylidene]-malonic Acid Monoethyl Ester
(65). In a round-bottom flask containing a solution of LiOH (1.05
g; 25 mmol; 1 equiv) dissolved in THF (50 mL) and water (50
mL) was added diethyl benzylidenmalonate (5.60 mL; 25 mmol; 1
1
purity 99%; NMR H NMR (DMSO-d6) δ 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 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; tR(LCMS) 4.23 min; MS (MH)+ m/z
315.

1332 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Flipo et al.
N-Hydroxy-N′-(4-phenyl-butyl)-2-[1-phenyl-meth-(Z)-ylidene]-
malonamide (67). (E)3-Phenyl-2-trityloxycarbamoyl-acrylic acid
(see synthesis of 66; 180 mg; 0.4 mmol; 1 equiv) was dissolved in
DCM (4 mL) and TEA (62 µL; 0.44 mmol; 1.1 equiv) was added.
The mixture was cooled at 0 °C (ice bath) and ethylchloroformate
(42 µL; 0.44 mmol; 1.1 equiv) was added dropwise. The reaction
mixture was stirred 30 min at 0 °C, and then phenylbutylamine
(70 µL; 0.44 mmol; 1.1 equiv) was added. The organic layer was
stirred 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/MeOH 98/2) and then precipitated
in petroleum ether to give a white powder. Yield 20%; purity 99%;
MS (MNa)+ m/z 603. The protected hydroxamic acid (46 mg; 0.08
mmol) was dissolved in TFA 2%/DCM (0.03 M), and triisopro-
pylsilane was added drop by drop until the yellow color disappeared.
The reaction mixture was stirred 5 min at room temperature,
solvents were removed under reduced pressure, and the residue was
washed with petroleum ether to give (67) as a white powder. The
configuration of this compound was assessed by ROESY NMR
experiment. Yield 89%; purity 99%; 1H NMR (DMSO-d6) δ 1.47-
1.61 (m, 4H), 2.59 (t, J ) 7.2 Hz, 2H), 3.17-3.23 (m, 2H), 7.14-
7.30 (m, 5H), 7.36 (s, 1H), 7.37-7.40 (m, 3H), 7.48-7.52 (m,
2H), 7.71 (t, J ) 5.7 Hz, NHCO), 9.13 (br s, OH), 10.95 (s,
CONHO); 13C NMR (DMSO-d6) δ 28.8, 29.2, 35.3, 39.3, 126.1,
128.7, 128.8, 129.1, 129.8, 129.9, 130.7, 134.2, 136.6, 142.6, 163.6,
164.3; tR(LCMS) 5.07 min; MS (MH)- m/z 337.
by drop until the yellow color disappeared. The reaction mixture
was stirred 5 min at room temperature, solvents were removed under
reduced pressure, and the residue was washed with petroleum ether
to give (68) as a white powder. (See Supporting Information for
stereochemical assignment). Yield 70%; purity 99%; 1H NMR
(DMSO-d6) δ 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 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; tR(LCMS) 4.41 min; MS (MH)+ m/z
315.
2-(2-Nitro-benzylidene)-malonic Acid Diethyl Ester (69). To
a solution of diethylmalonic ester (3.843 mL; 27 mmol; 1.2 equiv),
acetic acid (519 µL; 9 mmol; 0.4 equiv), and piperidine (265 µL;
2.7 mmol; 0.12 equiv) in benzene (60 mL) was added 2-nitroben-
zaldehyde (3.40 g; 22.5 mmol; 1 equiv). The reaction mixture was
refluxed for 72 h and then evaporated. The crude product was
dissolved in ethyl acetate, washed four times with aq. NaHCO3
5%, and once with aq. NaCl, dried over MgSO4, filtered, and
evaporated. The product was purified over silica gel (BP SUP 110
g Silice Flashsmart) with cyclohexane/AcOEt (100/0 to 95/5) to
give 69 as a colorless oil. Yield 12%; purity 99%; 1H NMR
(DMSO-d6) δ 0.95 (t, J ) 7.2 Hz, 3H), 1.26 (t, J ) 7.2 Hz, 3H),
4.04 (q, J ) 7.2 Hz, 2H), 4.28 (q, J ) 7.2 Hz, 2H), 7.43 (dt, J )
7.5 Hz, J ) 0.9 Hz, 1H), 7.69-7.74 (m, 1H), 7.83 (td, J ) 7.5 Hz,
J ) 1.5 Hz, 1H), 8.17 (s, 1H), 8.24 (dd, J ) 1.2 Hz, J ) 8.1 Hz,
1H); MS (MH)+ m/z 294, (MNa)+ m/z 316.
1-Hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylic Acid Eth-
yl Ester (70). Compound 69 (307 mg, 1.05 mmol) was dissolved
in acetic acid (5 mL), and PtO2 (5 mg) was added. The reaction
mixture was stirred 24 h at room temperature under hydrogen, then
filtered over celite, and washed with methanol. The solvents were
evaporated under reduce pressure. The product was purified by thick
layer chromatography (DCM/TEA 9/1) to give 70 as a beige
powder. Yield 55%; purity 99%; 1H NMR (CD2Cl2) δ 1.43 (t, J )
7.2 Hz, 3H), 4.43 (q, J ) 7.2 Hz, 2H), 7.37-7.42 (m, 1H), 7.79-
7.83 (m, 3H), 8.59 (s, 1H); MS (MH)+ m/z 234.
N-(4-Fluoro-benzyl)-N′-hydroxy-2-[1-phenyl-meth-(E)-ylidene]-
malonamide (68). Acid (65; 110 mg; 0.5 mmol; 1 equiv) was
dissolved in DCM (5 mL), and TEA (77 µL; 0.55 mmol; 1.1 equiv)
was added. The mixture was cooled at 0 °C (ice bath), and
ethylchloroformate (53 µL; 0.55 mmol; 1.1 equiv) was added
dropwise. The reaction mixture was stirred 30 min at 0 °C, and
then 4-fluorobenzylamine (57 µL; 0.5 mmol; 1 equiv) was added.
The organic layer was stirred 1 h at room temperature and then
evaporated. The crude product was dissolved in ethyl acetate,
washed twice with water and once with aq. NaCl, dried over
MgSO4, filtered, and evaporated. At this stage, the double bond is
isomerized to give (E)-2-(4-fluoro-benzylcarbamoyl)-3-phenyl-
acrylic acid ethyl ester. Yield 95%; purity 95%; 1H NMR (DMSO-
d6) δ 1.24 (t, J ) 7.2 Hz, 3H), 4.22 (q, J ) 7.2 Hz, 2H), 4.37 (d,
J ) 6 Hz, 2H), 7.12-7.18 (m, 2H), 7.31-7.44 (m, 5H), 7.52-
7.55 (m, 2H), 7.58 (s, 1H), 8.99 (t, J ) 6 Hz, NH); MS (MH)+ m/z
328. A total of 147 mg (0.45 mmol; 1 equiv) of ester was dissolved
in 5 mL of a mixture of THF and water (1/1). A total of 76 mg of
LiOH (1.8 mmol; 4 equiv) was added, and the mixture was stirred
overnight at room temperature. THF was evaporated under reduced
pressure. The aqueous phase was washed three times with DCM
and then acidified to pH ) 1 with HCl. The expected product was
extracted three times with DCM. The organic phases were joined,
dried over MgSO4, evaporated under reduced pressure, and then
precipitated in petroleum ether to give (E)-2-(4-fluoro-benzylcar-
bamoyl)-3-phenyl-acrylic acid as a white powder. Yield 75%; purity
1-Hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylic Acid
4-Fluoro-benzylamide (71). Compound 70 (37 mg; 0.16 mmol)
was dissolved in 4-fluorobenzylamine (2 mL), and the reaction
mixture was heated at 60 °C for 4 h, and then cooled at room
temperature. Ethyl acetate was added, and the organic layer was
washed four times with HCl 1 N, once with water, once with brine,
dried over MgSO4, filtered, and evaporated. The residue was
precipitated in DCM and filtered to give 71 as a beige powder.
1
Yield 70%; purity 99%; H NMR (DMSO-d6) δ 4.58 (d, J ) 6.0
Hz, 2H), 7.14-7.20 (m, 2H), 7.38-7.41 (m, 3H), 7.76-7.81 (m,
2H), 8.05 (d, J ) 7.5 Hz, 1H), 8.85 (s, 1H), 10.06 (t, J ) 6.0 Hz,
NH), 11.82 (s, 1H, OH); 13C NMR (DMSO-d6) δ 42.5, 113.2, 115.8
(d, JCF ) 21 Hz), 118.4, 121.9, 123.9, 130.0 (d, JCF ) 8 Hz), 130.8,
134.0, 135.9, 139.7, 141.4, 158.1, 161.9 (d, JCF ) 240 Hz), 163.1;
tR(LCMS) 5.10 min; MS (MH)+ m/z 313.
1-Hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylic Acid (4-
Phenyl-butyl)-amide (72). Compound 70 (47 mg; 0.2 mmol) was
dissolved in phenylbutylamine (2 mL) and the reaction mixture was
heated at 60 °C for 4h, then cooled at room temperature. Ethyl
acetate was added, and the organic layer was washed four times
with HCl 1 N, once with water, once with brine, dried over MgSO4,
filtered, and evaporated. The residue was precipitated in DCM and
1
95%; H NMR (DMSO-d6) δ 4.34 (d, J ) 6 Hz, 2H), 7.10-7.15
(m, 2H), 7.26-7.40 (m, 5H), 7.46-7.49 (m, 2H), 7.50 (s, 1H),
8.90 (t, J ) 6 Hz, NH), 12.43 (s, OH); MS (MH)- m/z 298. The
previous synthesized acid (90 mg; 0.3 mmol; 1 equiv) was dissolved
in DCM (3 mL), and TEA (46 µL; 0.33 mmol; 1.1 equiv) was
added. The mixture was cooled at 0 °C (ice bath), and ethylchlo-
roformate (31 µL; 0.33 mmol; 1.1 equiv) was added dropwise. The
reaction mixture was stirred for 30 min at 0 °C, and then
O-tritylhydroxylamine (70 mg; 0.25 mmol; 0.85 equiv) was added.
The organic layer was stirred 1 h at room temperature and then
evaporated. The crude product was dissolved in DCM, washed three
times 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/MeOH 99/1) to give
a white powder. Yield 35%; purity 95%; MS (MH)- m/z 555. The
protected hydroxamic acid (20 mg; 0.036 mmol) was dissolved in
TFA 2%/DCM (0.03 M), and triisopropylsilane was added drop
1
filtered to give 72 as a beige powder. Yield 87%; purity 99%; H
NMR (DMSO-d6) δ 1.57-1.63 (m, 4H), 2.57-2.64 (m, 2H), 3.38-
3.43 (m, 2H), 7.14-7.29 (m, 5H), 7.36-7.41 (m, 1H), 7.76-7.81
(m, 2H), 8.04 (d, J ) 7.5 Hz, 1H), 8.82 (s, 1H), 9.70 (t, J ) 5.4
Hz, NH), 11.81 (s, 1H, OH); 13C NMR (DMSO-d6) δ 29.0, 29.2,
32.3, 39.1, 113.2, 118.3, 121.8, 123.9, 126.3, 128.8, 128.9, 130.7,
134.0, 139.7, 141.1, 142.6, 158.0, 162.7; tR(LCMS) 6.01 min; MS
(MH)+ m/z 337.
Acknowledgment. The authors would like to thank Profes-
sors Andre´ Tartar and He´le`ne Gras for fruitful discussions. We
are grateful to the institutions that support our laboratory

Inhibitors of PfA-M1 as Potential Antimalarial Agents
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1333
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(Inserm, Universite´ de Lille2 and Institut Pasteur de Lille).
Marion Flipo received a grant from CNRS and Re´gion Nord-
Pas-de-Calais. This work was specifically funded by Re´gion
Nord-Pas-de-Calais and EC. NMR spectra were recorded in the
Laboratoire d’Application RMN (LARMN) at Universite´ de
Lille2, Faculte´ des Sciences Pharmaceutiques de Lille. Data
management was performed using Pipeline Pilot from Scitegic.
Supporting Information Available: Screening results for the
whole library, spectral NMR, and other analytical data that support
configuration assignments of 65, 66, and 68. This material is
available free of charge via the Internet at http://pubs.acs.org.
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