High antiplasmodial activity of novel plasmepsins I and II inhibitors

Article abstract of DOI:10.1021/jm061033d

The aim of this study was to develop new antiplasmodial compounds acting through distinct mechanisms during both the liver and the blood stages of the parasite life cycle. Compounds were designed on the basis of the "double-drug" approach: primaquine, whi

Full text of DOI:10.1021/jm061033d

7440
J. Med. Chem. 2006, 49, 7440-7449
High Antiplasmodial Activity of Novel Plasmepsins I and II Inhibitors
Mario Dell’Agli,† Silvia Parapini,§ Germana Galli,† Nadia Vaiana,# Donatella Taramelli,§ Anna Sparatore,# Peng Liu,
Ben M. Dunn, Enrica Bosisio,† and Sergio Romeo*,#
Department of Pharmacological Sciences, Department of Public Health-Microbiology-Virology, and Institute of Medicinal Chemistry, School of
Pharmacy, UniVersity of Milan, Milan 20100, Italy, and Department of Biochemistry and Molecular Biology, College of Medicine, UniVersity
of Florida, GainesVille, Florida 32610
ReceiVed August 29, 2006
The aim of this study was to develop new antiplasmodial compounds acting through distinct mechanisms
during both the liver and the blood stages of the parasite life cycle. Compounds were designed on the basis
of the “double-drug” approach: primaquine, which has been linked to statine-based inhibitors of plasmepsins
(PLMs), the plasmodial aspartic proteases involved in degradation of hemeoglobin. The compounds were
tested in vitro for anti-PLM I/PLM II activities and against chloroquine-sensitive (D10) and chloroquine-
resistant (W2) strains of P. falciparum. An antiplasmodial activity (IC50) as low as 0.1 µM was obtained,
an excellent improvement in comparison with inhibitors previously reported (IC50 ) 2-20 µM). The killing
activity was equally directed against both P. falciparum strains and was correlated to lipophilicity (calculated
as ALogP), for all compounds but one (9). All compounds inhibited PLM I and PLM II in the nanomolar
range (Ki ) 1-700 nM). The most promising compounds (2, 6, 10) were not cytotoxic against human
fibroblasts at 100 µM and were highly selective for PLMs vs human cathepsin D.
Introduction
Malaria, with AIDS and tuberculosis, is one of the three major
communicable diseases linked to poverty. The unavailability
of a vaccine and the spread and intensification of drug resistance
over the past 15-20 years have led to a dramatic decline in the
efficacy of the most affordable antimalarial drugs.1-3 In order
to circumvent that situation, medications based on the associa-
tion of two substances acting against the parasite have been
developed.
The basis of this study was to develop new compounds acting
through distinct mechanisms during both the liver stage and
the blood stage of the life cycle of the parasite. Compounds
were designed on the basis of the “double-drug” approach:
primaquine (PQa), active against the gametocytes and hypno-
zoites of the relapsing malaria parasites P. ViVax and P. oVale
and which has been linked to statine-based inhibitors of
plasmepsins (PLMs), the aspartic proteases involved in degrada-
tion of hemoglobin during the blood stage of the parasite.4
Figure 1. P. falciparum growth inhibition by three plasmepsin
inhibitors encompassing a norstatine (A), a 1,2-dihydroxyethylene (B),
and a statine (PS777621) motif. Compound C represents the most potent
inhibitor in our previous series.
The therapy with PQ has been reconsidered after the
observation that PQ is a safe and effective agent for the
prophylaxis of malaria due to both P. falciparum and P. ViVax.5
Furthermore, the use of the prodrug Val-Leu-Lys-PQ reduced
toxicity and increased the activity of the drug.6
Several inhibitors of PLM II have already been designed by
replacement of the scissile peptide bond with a transition state
analogue,7,8 including PS777621 characterized by a statine-based
core (Figure 1).9 A drawback for the development of substrate-
based PLM II inhibitors was that while enzyme inhibition
occurred at the nanomolar level, effectiveness of the inhibitors
in killing the parasite was limited (IC50 ) 2-20 µM).9-11
Differences of this kind were attributed to low bioavailability
probably because most of these inhibitors are derived from
peptides. However, in view of recent publications, the low
potency against cultured parasites may also be explained by
the plasmepsins’ redundancy,12,13 which implies that blockade
of this enzyme family may not be lethal.14
* To whom correspondence should be addressed. Phone: +39 02
50317568. Fax: +39 02 50317565. E-mail: sergio.romeo@unimi.it.
† Department of Pharmacological Sciences, University of Milan.
§ Department of Public Health-Microbiology-Virology, University of
Milan.
The “double-drug” strategy might help to improve cell
permeability and pharmacological activity while reducing dose
and side effects. Following this strategy, we published the
antiplasmodial activity of a series of compounds that have a
statine moiety bound to Leu-Lys-PQ residue by means of
different linkers.15 The best IC50 value for the inhibition of P.
falciparum was 0.4 µM, thus achieving a remarkable improve-
ment with respect to other inhibitors of PLM II.8-11 We also
# Institute of Medicinal Chemistry, University of Milan.
University of Florida, College of Medicine.
a Abbreviations: PLM, plasmepsin; PQ, primaquine; IPTG, isopropyl
â-D-1-thiogalactopyranoside; LB, Luria Bertani medium; DMEM, Dulbec-
co’s modified Eagle’s medium; SDS, sodium dodecyl sulfate; CQ, chlo-
roquine; RPMI, Roswell Park Memorial Institute.
10.1021/jm061033d CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/16/2006

Plasmepsins I and II Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7441
demonstrated that the removal of the Lys-PQ moiety led to a
drastic reduction of the antiparasitic activity.
zyl ester of 4,4′-oxybis(benzoic acid) 14 to give dipeptide
intermediate 27, which was coupled to statine salt 13 to give
compound 10. Compound 9 was prepared by Nꢀ Boc depro-
tection of 10.
In this paper we report the synthesis and the biological
evaluation of nine new statine-based PLMs inhibitors (Table
1). Our efforts were mainly directed in improving the killing
on the parasite during the intraerythrocytic stage. All synthesized
compounds presented the statine moiety unchanged, while
modifications were made to the Leu-Lys-PQ unit (Table 1). In
particular, the first variations were directed toward the dipeptide
Leu-Lys by removing Lys (2) or both amino acids (3). In
compounds 4-8 the Leu-Lys dipeptide has been modified by
introducing Ala in place of the Lys and/or Leu residue or by
protecting the Nꢀ of Lys with a Boc group. In compounds 9
and 10 the PQ residue has been substituted with a propyl ester
and the Nꢀ of Lys was unsubstituted or protected with a Boc
group.
The compounds have been tested in vitro against recombinant
PLMs I and II, and against D10 (chloroquine sensitive) and
W2 (chloroquine resistant) strains of P. falciparum, using
compound 1 (representing the previously described PS777621)
as the reference molecule. The selectivity over human cathepsin
D, which shares a high homology with PLMs, and cell toxicity
on mammalian cells were also investigated for the most
promising compounds.
Effect of Compounds on PLM I and PLM II Activities.
All compounds inhibited both PLM I and PLM II in a
concentration-dependent fashion (Table 1). PLM I inhibition
was improved in comparison to 1 for all the compounds with
the exception of compound 2. In particular, compound 2,
deprived of Ala, and compound 7, where the Nꢀ of Lys is Boc-
protected, possessed the lowest affinity for PLM I (Ki of 765
and 230 nM, respectively). For compounds 2-6, Ki values for
PLM II inhibition ranged between 4 and 26 nM, which
represents an improvement with respect to 1, while for 7 and 8
the affinity decreased. Compounds 9 and 10 showed similar
values for inhibition of PLMs I and II, (Ki ) 10-17 nM).
Compound 5 deserves particular attention in view of the highest
inhibition against both PLMs (1.0 and 3.9 nM for PLM I and
PLM II, respectively), demonstrating a remarkable improvement
toward PLM I (>700-fold vs 1).
As assessed by SDS-PAGE (Figure 2), compounds 2, 6, and
10, the best inhibitors of the parasite growth (Table 1), were
able to prevent the human hemoglobin cleavage induced by
PLM II, a result that further supports the possibility that these
molecules are able to inhibit the enzyme when hemoglobin is
the substrate for P. falciparum PLMs.
Results
Effect of Compounds on P. falciparum Growth. All PQ-
statine compounds inhibited parasite growth in a dose-dependent
manner, and IC50 values against both strains (0.2-4 µM) are
significantly below that of the reference compound 1 (15-23
µM), confirming that the “double-drug” approach represents a
good strategy for improving the effectiveness of PLM inhibitors
to starve and kill the parasite. By comparison of compounds
4-8 characterized by a dipeptide linking PQ to the statine
portion of the molecule, compound 6 with the Leu-Ala dipeptide
was found to be the most active compound against both D10
chloroquine-sensitive and W2 chloroquine-resistant strains of
P. falciparum (0.2 and 0.3 µM, respectively). Modifications at
the Leu-Ala dipeptide decreased the antiplasmodial activity (4,
5, 7, 8). While compound 2 showed a lower activity against
the D10 strain than compound 6 and while the effect on W2
was comparable, compound 3 showed a sharp decrease in
activity against both strains. In our previous study15 we
demonstrated that the removal of aminoquinoline led to a
dramatic loss of inhibitory activity, while, as shown here,
compound 10, where PQ is replaced by a propyl ester group
and the amino group of Lys is protected, still maintains a very
high inhibitory activity (IC50 ) 0.1 µM). For compound 9, where
the amino group of Lys is not Boc-protected, the killing effect
worsened (IC50 ) 3.8-4.5 µM).
Molecular Modeling. The 3D structural models of compound
6, one of the most promising compounds, were constructed from
pepstatin A using the X-ray crystal coordinates of pepstatin A
in complex with plasmepsin II (PDB entry 1XDH) as a starting
point. Figure 3 shows a superimposition of the conformers of 6
with pepstatin A, while the conformers of 6, without pepstatin
A, in complex with PLM II are shown in Figure 4. Molecular
modeling shows that the side chains of Ile and Sta and the
N-butylammido group are easily superimposed with pepstatin
A (Figure 3) and are nicely accommodated within the active
site (Figure 4). The conformational freedom of the 4,4′-
oxybisbenzoic ring system allows the LeuAla-PQ part of the
molecule to assume several conformations in which PQ is
partially or totally exposed to the solvent (Figure 4). PQ does
Chemistry. In view of the high costs of commercially
available statine, a gram-scale synthesis of N-Boc-statine has
been developed in order to obtain compounds 1-10 (Schemes
1 and 2). Stereoselective addition of the zinc enolate of
ethylacetate16 to N-Boc-leucinal17 resulted in N-Boc-statine ethyl
ester 11, which after hydrolysis and coupling with butylamine
followed by Boc removal and coupling with BocIleOH gave
intermediate 12, easily purified by crystallization. This synthesis
required only one chromatographic purification step (after
enolate addition), and the overall yields from Boc-leucine were
15%. After deprotection of 12 to give amine 13 and coupling
with the monobenzyl ester of 4,4′-oxybisbenzoic acid 14,
obtained by selective monobenzylation of 4,4′-oxybisbenzoic
acid, compound 15, the statine-containing portion of inhibitors
3-8, was obtained (Scheme 1). The PQ-containing portions,
18-22, of inhibitors 4-8 were obtained by reaction of PQ
diphosphate with Boc-leucine or Boc-alanine using standard
coupling conditions to give compounds 16 and 17, which were
coupled to Cbz-leucine or Cbz-alanine to give PQ-containing
fragments 18-20. Lysine-containing fragments 21 and 22 were
obtained by coupling with 4′-N-(Nꢀ-Boc-lysyl)PQ6 (Scheme 1).
The PQ-containing fragments 18-20 and 22 and the statine-
containing fragment 15 were simultaneously deprotected and
coupled to afford final compounds 5-8. Compound 3 was
obtained by coupling of 15 with PQ and compound 4 by
coupling of 15 with 21 followed by deprotection of the lysine
Nꢀ Boc group and formation of the diphosphate salt (Scheme
1). For the synthesis of 2 a slightly different synthetic approach
was undertaken (Scheme 2). The monobenzyl ester of 4,4′-
oxybis(benzoic acid) 14 was first coupled to 4′-N-(Boc-leucyl)-
PQ 17 and subsequently coupled with the statine-containing
fragment 13.
The synthesis of compounds 1, 9, and 10, not containing PQ,
was carried out as described in Scheme 2. Compound 1 was
prepared by coupling 2-naphthoxyacetic acid to 13. Fully
protected lysine derivative 25 was prepared by Nꢀ Boc protection
of Cbz-lysine allyl ester,18 which was coupled with Cbz-leucine
to give compound 26, followed by coupling with the monoben-

7442 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Dell’Agli et al.
Table 1. Enzyme Inhibition, Antiplasmodial Activity, and Mammalian Cell Toxicity of Inhibitorsa
a Results are the mean ( SD. N.D., not detectable.

Plasmepsins I and II Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7443
Scheme 1. Synthesis of Inhibitors 3-8a
Figure 2. Hemoglobin degradation on SDS-PAGE, showing the effect
of compounds 2, 6, and 10 on the cleavage of human hemeoglobin by
PLM II: (lanes 1-4) hemoglobin incubated with the vehicle and PLM
II for 0, 10, 30, and 60 min; (lanes 5-8) hemoglobin incubated with
the inhibitor and PLM II for 0, 10, 30, and 60 min.
Figure 3. Superimposition of pepstatin A (pink) with some plausible
conformations of compound 6 (various colors), showing the good
superimposition of the statine moiety of the molecules and the
conformational freedom of primaquine. The image was compiled using
the PyMol molecular visualization system.30
a Reagents: (i) LDA, EtOAc, ZnBr2; (ii) NaOH; (iii) C4H9NH2, HBTU,
HOBt; (iv) HCl; (v) BocIleOH, HBTU, HOBt; (vi) BnBr, DMF; (vii)
HBTU; (viii) BocLeuOH or BocAlaOH, HBTU, HOBt; (ix) CbzAlaOH or
CbzLeuOH, HBTU, HOBt; (x) H2, Pd-C; (xi) TFA; (xii) H3PO4.
Scheme 2. Synthesis of Inhibitors 1, 2, 9, 10a
Figure 4. Surface plot of PLM II (green) in complex with some
plausible conformations of compound 6 (various colors), showing the
conformational freedom of primaquine. The image was compiled using
the PyMol molecular visualization system.30
a Reagents: (i) 2-(naphthalen-2-yloxy)acetic acid, HBTU; (ii) HCl; (iii)
14, HBTU; (iv) H2, Pd-C; (v) HBTU; (vi) Boc2O; (vii) CbzLeuOH, HBTU,
HOBt.
log P values were calculated using the Accelrys DS ViewerPro
5.0 property calculator (trial version) and are indicated as
ALogP. The ratio of the IC50 values was chosen as an indicator
of cell penetration of the synthesized inhibitors; the number will
increase along with the impairment to cross cell membranes. A
linear correlation (r ) 0.86) between lipophilicity and activity
ratio was obtained for all compounds but one (9).
Selectivity toward Human Cathepsin D and Cytotoxicity.
For compounds 2, 6, and 10, cathepsin D inhibition was
negligible at the concentration required to inhibit PLM II by
50% (IC50) (data not shown). At concentrations 100-fold higher,
cathepsin D was inhibited by 30%, 50%, and 37%, respectively
have a great deal of conformational freedom in the active site,
and since it is exposed to the solvent, it is quite difficult to
predict an active conformation. The model shows that the
enzyme is easily able to accommodate large substituents after
P3 and the 4,4′-oxybisbenzoic ring system is fitted nicely within
the active site.
Correlation between Lipophilicity (log P) and Biological
Activities. To verify whether lipophilicity and therefore cell
penetration was correlated to the antiparasite activity, the degree
of lipophilicity expressed as the log P value of PLM inhibitors
was plotted against the ratio of the IC50 against the P. falciparum
W2 strain in RBCs to the IC50 against PLM II (Figure 5). The

7444 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Dell’Agli et al.
seen because the score function mainly evaluates the interactions
in the assigned boxes, i.e., in the active site cleft. Thus, structural
modifications of this part of the molecule should not greatly
affect the PLM II Ki values of the compounds. Regarding
compounds 7 and 8, which have the lowest affinity for PLM
II, their interaction with the enzyme may be impaired by their
high lipophilicity as deduced from the ALogP values (Figure
5).
The inhibition of P. falciparum growth in the erythrocytes
was drastically improved by all compounds in comparison to
1. IC50 values were below 1 µΜ for compounds 2, 4, 6, and 10
with a noteworthy value of 100 nM for compound 10, which
represents the most active transition state analogue inhibitor to
the best of our knowledge. Furthermore the PQ-statine com-
pounds are equally active against chloroquine-sensitive and
chloroquine-resistant strains of P. falciparum, thus suggesting
that the mechanism of action (i.e., PLM inhibition) and the
mechanism for chloroquine resistance are independent.
The degree of PLM inhibition is not predictive of the ability
to kill the parasite, since compounds with Ki values of 1-700
nM possess a range of potency against the parasite, falling within
a much narrower interval (IC50 values of 0.1-4 µM). If parasite
growth is not always correlated to PLM inhibition, then several
factors could contribute to the antiplasmodial activity of these
compounds and help explain the lack of correlation with
inhibition of PLMs. They include (i) the ability to penetrate
the cell and the food vacuole, where hemoglobin is degraded
by PLMs I and II and (ii) the blockage of other proteolytic
enzymes working in combination with PLM impairment.
In general, the crossing of cell membranes by a drug is
facilitated by the hydrophobicity of the molecule. Figure 5
clearly shows that a linear correlation exists between log P
values and the ratio of potencies against parasite and affinities
against PLM II, thus indicating that PLMs are the main target
of these compounds in the parasite. These results indicate that
a good balance between inhibitory activity and lipophilicity is
required in order to achieve efficacy in the cell.
The log P data indicate that for the compounds described here
this rule is true for all but one (9). Compound 9 might undergo
biotransformation, reducing its lipophilicity and thus interfering
with cell bioavailability. We have previously shown that the
breakdown of Lys-Xaa releases fragments much less potent than
the parent compounds.15
The difference of the order of magnitude between enzymatic
Ki and cell potency (IC50) ranged between 10- and 400-fold. In
light of the recent paper by Liu et al.,14 it appears that
bioavailability is not a limitation for killing cultured parasites,
since pepstatin A was able to enter the food vacuole. Then it is
possible that the potency of the compounds described is
determined not only by their bioavailability but rather by the
possibility that they inhibit not only plasmepsins but also other
proteolytic enzymes (such as the falcipains).
Figure 5. Correlation between lipophilicity (ALogP) and biological
activity. A linear correlation (r ) 0.86) between lipophilicity and
activity ratio was obtained for all compounds but one (9). The log P
values were calculated using Accelrys DS ViewerPro 5.0 property
calculator (trial version) and are indicated as ALogP.
(Table 1), indicating a good selectivity for the plasmodial
enzyme. Similarly, no antiproliferative activity was found when
human skin fibroblasts were cultured in the presence of the
compounds at concentrations as high as 100 µM. These results
support for a negligible cytotoxic effect on mammalian cells at
concentrations necessary to kill the parasite (below 1 µM).
Discussion
The PQ-statine compounds examined in this study represent
the evolution of the first series of statine-based PLM II inhibitors
designed by following the double-drug approach as devised by
Romeo et al.15 In agreement with previous results, it is confirmed
that with this approach we have synthesized PLM II inhibitors
that are much more active on P. falciparum than 1 (PS777621)
and other transition state analogue inhibitors previously
reported.9-11
Considering the redundancy of PLMs,12,13 it is therefore
necessary to design compounds able to inhibit two or more in
this enzyme family in order to develop drugs active on P.
falciparum. Some of the synthesized compounds, designed as
PLM II inhibitors, are also strong inhibitors of PLM I, which
is, in view of the recent literature, a prerequisite for developing
new antimalarial agents.13,14 In this regard, work is in progress
to demonstrate the ability of PQ-statine inhibitors to block PLM
IV and the orthologues from P. malariae, P. oVale, and P. ViVax
(manuscript in preparation).
By comparison of the PLM I inhibitory activity of the
reference compound 1 with the effect exerted by compounds
2-10, it is evident that most of them showed a remarkable
improvement of activity. In particular the presence of the
dipeptide influences the affinity for PLM I to a greater extent
than PLM II with a preference for the Ala-Ala dipeptide (5),
which shows an 80-fold improvement compared to compound
3, which lacks the dipeptide linker.
It is noteworthy that most of the tested compounds possess
an anti-PLM II activity much greater than compound 1. With
the exclusion of compounds 7 and 8, any change to the peptide
chain linking the PQ group to the 4,4′-oxybisbenzoic ring or
the substitution of PQ with a propyl ester group did not bear
heavy changes of PLM II Ki values (Table 1), indicating that
PQ is not essential for PLM II inhibition. From modeling studies
it looks like most of the conformations show solvent-exposed
PQ and lack of interactions of the dipeptide linker with the
enzyme. If this is true, no significant score differences will be
The most promising inhibitors of P. falciparum growth (2,
6, 10) showed only negligible or no cytoxicity when tested on
human fibroblasts cell line, indicating that these compounds
interfere with mechanisms unique for the parasite; this observa-
tion was confirmed by the high selectivity of these compounds
for PLMs in comparison to human cathepsin D.
The compounds under study, which have been designed on
the basis of the double-drug approach, contain PQ, which is a
drug active against liver-stage malaria parasites. Thus, com-
pounds 2-8 containing PQ could also exert an inhibitory effect
on this stage in addition to the intraerythrocytic effect. The in
vivo studies should elucidate this hypothesis.

Plasmepsins I and II Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7445
[S]Vmax
In conclusion, the evidence that some of the inhibitors
reported possess very good efficacy against P. falciparum
growth and a great selectivity in comparison to other inhibitors
reported in the literature (0.1 vs 2-20 µM)19-21 makes these
novel compounds very promising for further development as
antimalarial agents.
V )
[I]
Ki
[S] + Km 1 +
(
)
of the single substrate-single inhibitor (competitive) program in
the Enzyme Kinetic Module 1.0 of SigmaPlot 2000 (version 6.10)
(SYSTAT Software Inc.).
Experimental Section
PLM II Assay. Pro-PLM II was activated by addition of one-
tenth volume of 100 mM sodium acetate buffer, pH 4.7, followed
by incubation at 37 °C for 90 min. The enzyme activity of PLM II
was evaluated spectrophotometrically at 300 nm by following the
cleavage of the chromogenic substrate Lys-Glu-Phe-Val-Phe-NPhe-
Ala-Leu-Lys (where NPhe is p-nitrophenylalanine), as described.23
Inhibitors were tested at 1-100 nM dissolved in DMSO (final
concentration of 1% of the sample volume). IC50 values were
obtained using Graph Pad Prism 4. The Ki values for PLM II were
subsequently calculated by using
Expression of Recombinant PLMs I and II. PLM I preparation
will be described in full (Liu et al., manuscript in preparation).
Briefly, expression of the recombinant proPfPLM I was induced
at 37 °C in the BL21 Star (DE3) pLysS E. coli using 1 mM IPTG.
After 3 h of induction, cell pellets were harvested and inclusion
bodies were extracted and purified according to the procedure
previously described.22 Inclusion body materials were solubilized
in 6 M urea, 50 mM sodium phosphate, pH 8.5, and 500 mM
sodium chloride. Protein was refolded by dialysis against 20 mM
Tris-HCl, pH 8.0, and further purified by anion exchange and size
exclusion chromatography.
IC50
Ki )
[S]
1 +
Km
PLM II was prepared according to the procedure previously
described,23 with slight modifications. Briefly, pET-3a expression
plasmid containing PLM II precursor gene was transformed into
BL21-(DE3) pLysS E. coli. A single colony was used to inoculate
100 mL of LB medium. The culture was grown overnight at 30
°C, and an amount of 60 mL was used to inoculate 3 L of fresh
LB. Cells were grown until the OD at 600 nm reached 0.4. They
were then induced with IPTG at a final concentration of 1 mM.
After a further 3 h of incubation, cells were harvested by
centrifugation and resuspended in 100 mM Tris-HCl, pH 8,
containing 0.2% Triton. After the addition of lysozyme (1 mg/mL),
resuspended cells were incubated at 37 °C for 30 min. The volume
was sonicated. Then solid urea was added to reach a final
concentration of 500 mM and the solution was stirred at room
temperature for 30 min. Inclusion bodies were harvested by
centrifugation at 16000g for 10 min and then resuspended in the
same buffer, stirred for 30 min, and spun as before. Inclusion bodies
were solubilized in 6 M urea, 100 mM Tris-HCl, pH 8.5, and 32
mM â-mercaptoethanol. Protein was refolded by dialysis against
10 L of 10 mM Tris-HCl, pH 8.5. Protein concentration was
determined according to the method of Bradford.24 Protein was
diluted to a final concentration of 0.5 mg/mL in 50% glycerol and
stored at -20 °C.
and a Km value determined according to Michaelis-Menten. Results
are expressed as the mean ( standard deviation of at least two
separate experiments performed in triplicate.
Cathepsin D Assay. Human liver cathepsin D was purchased
from Sigma-Aldrich, Milan, Italy. The enzyme activity was
measured as described for PLM II by incubating 0.714 U of protein
with 27 µg of substrate in 100 mM sodium acetate buffer, pH 4.7.
Final incubation volume was 0.6 mL. The compounds were tested
against cathepsin D at concentrations 100-fold higher than that
required to inhibit PLM II by 50% (IC50).
Parasite Growth. The CQ-sensitive (D10) and the CQ-resistant
(W2) strains of P. falciparum were sustained in vitro as described
by Trager and Jensen.25 Parasites were maintained at 5% hematocrit
(human type A-positive red blood cells) in RPMI 1640 (Gibco BRL,
NaHCO3 24 mM) medium with the addition of 10% heat-inactivated
A-positive human plasma, 20 mM Hepes (Euroclone), and 2 mM
glutammine (Euroclone). All cultures were maintained at 37 °C in
a standard gas mixture consisting of 1% O2, 5% CO2, 94% N2.
Drug Susceptibility Assay on P. falciparum. Compounds were
dissolved in either water (chloroquine) or DMSO and then diluted
with a medium to achieve the required concentrations (final DMSO
concentration of <1%, which is nontoxic to the parasite). Drugs
were placed in 96-well flat-bottom microplates (COSTAR), and
serial dilutions were made. Asynchronous cultures with parasitemia
of 1-1.5% and 1% final hematocrit were aliquoted into the plates
and incubated for 72 h at 37 °C. Parasite growth was determined
spectrophotometrically (OD650) by measuring the activity of the
parasite lactate dehydrogenase (LDH) according to a modified
version of Makler’s method in control and drug-treated cultures.26
Antiplasmodial activity is expressed as the 50% inhibitory con-
centrations (IC50). Each IC50 value is the mean ( standard deviation
of at least three separate experiments performed in duplicate.
Hemoglobin Degradation Assay by PLM II in the Presence
of PQ-Statine Compounds. The ability of the most active
compounds to prevent the hemoglobin degradation by PLM II was
assayed by SDS-PAGE.27 Briefly, hemoglobin (10 µg) was
incubated with preactivated PLM II (200 ng) at 37 °C, pH 4.7, in
a final digest volume of 16 µL of 100 mM sodium acetate buffer
and in the presence of the vehicle or the PLM II inhibitor for 10,
30, and 60 min. The reaction was stopped by the addition of SDS
loading dye. Samples were denatured by boiling for 5 min and
loaded onto 18% polyacrylamide gel.
Enzyme Inhibition Assays. PLM I Assay. For a tight binding
(Ki ) 50 pM to 10 nM) competitive inhibitor, the initial rates (AU/
s) of enzymatic cleavage of a chromogenic peptide substrate (Lys-
Pro-Ile-Leu-Phe-Nph-Arg-Leu) of known concentration (µM) in the
presence of different concentrations (nM) of inhibitors were
measured using a Cary 50 spectrophotometer (Varian).22 The Ki
value was determined by fitting the initial cleavage velocities and
related inhibitor concentrations into the competitive tight binding
inhibitor equation (J. F. Morrison, 1969):
0.5Vmax/[E]
V )
{[E] - [I] - Kiap
+
K
m + 1
{
}
[S]
([E] - [I] - K ap)2 + 4[E]Kiap
}
x
i
where Kiap ) Ki([S]/Km + 1), with the Enzfitter 1.05 program.
For a non-tight-binding (Ki ) 50 nM to 10 µM) competitive
inhibitor, the initial rates (AU/s) of enzymatic cleavage of at least
six different concentrations of a chromogenic peptide substrate (Lys-
Pro-Ile-Leu-Phe-Nph-Arg-Leu) in the presence of at least two
different concentrations of inhibitors were measured and the Ki value
was determined by fitting the initial hydrolysis rates and related
substrate and inhibitor concentrations into the equation
Cytotoxicity Assay. Cytotoxicity was evaluated in human
fibroblasts from skin biopsies. Fibroblasts (8 × 104 per well) were
grown in 24-well plates with DMEM containing 10% fetal calf
serum, 1% penicillin/streptomycin, and 1% L-glutamine as previ-
ously described.28 Cell proliferation was followed by the MTT (3-

7446 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Dell’Agli et al.
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test29
in the absence or in the presence of the tested compounds (up to
100 µM).
(3S,4S)-4-(tert-Butoxycarbonylamino)-3-hydroxy-6-methyl-
heptanoic Acid Ethyl Ester (BocStaOEt) (11). Anhydrous THF
(24 mL) and LDA (2 N, 28.3 mL, 56.6 mmol) were cooled under
nitrogen atmosphere at -80 °C. Ethyl acetate (5.6 mL, 56.6 mmol)
was then added followed by a 0 °C solution of anhydrous ZnBr2
(12.7 g, 56.6 mmol) in anhydrous THF (24 mL). A -78 °C solution
of Boc-leucinal17 (1.81 g, 8.4 mmol) in anhydrous THF (6 mL)
was then added, and the mixture was stirred at -78 °C for 10 min
and finally allowed to reach room temperature overnight. After the
reaction mixture was cooled at 0 °C, 25 mL of a solution of aqueous
saturated NH4Cl and acetic acid (9:1) was added and extracted with
cooled ethyl ether (four times). The organic phase was washed with
1 N HCl (2 times) and with brine, dried over sodium sulfate, filtered,
and concentrated under vacuum. The brownish oil was purified by
silica gel chromatography (cyclohexane/ethyl acetate (90:10, 85:
15, 70:30) to give 11 as a white solid (253 mg, 0.84 mmol, 10%).
1H NMR (CDCl3, 300 MHz) δ: 0.82-1.02 (9H, m), 1.02-1.74
(12H, m), 2.50 (2H, m), 3.60 (1H, bm), 4.00 (1H, bm), 4.15 (2H,
q), 4.73 (1H, bd). Anal. (C15H29NO5) C, H, N.
(2S,3S)-1-((3S,4S)-1-(Butylamino)-3-hydroxy-6-methyl-1-oxo-
heptan-4-ylamino)-3-methyl-1-oxopentan-2-ylcarbamic Acid tert-
Butyl Ester (BocIleStaNHC4H9) (12). A solution of 2 N NaOH
(0.84 mmol, 0.42 mL) was added dropwise to a solution of Boc-
StaOEt (253 mg, 0.84 mmol) in dioxane/water (3:1, 6 mL) cooled
to 0 °C. The mixture was stirred at room temperature for 1.5 h.
After completion, the reaction mixture was cooled to 0 °C and
cooled 2 N HCl was added until acidic pH was attained. Dioxane
was removed by evaporation, and the aqueous phase was extracted
with dichloromethane (three times). The organic phases were dried
and concentrated, and the product was reacted with butylamine (61.4
mg, 0.84 mmol) under general procedure A. The reaction mixture
was extracted with ethyl acetate, dried, and concentrated to give a
crude product (195 mg), which was used without any further
purification in the next reaction.
Modeling of PLM II with PQ-Statine Inhibitors. The 3D
structural models of compound 6, one of the most promising
compounds (Table 1), were constructed from pepstatin A using the
X-ray crystal coordinates of pepstatin A in complex with plasmepsin
II (PDB entry 1XDH) as a starting point. The Val residue in P2 of
pepstatin A was changed in Ile and substituted with 4,4′-oxybis-
benzoic acid. The C-terminal residues AlaSta of pepstatin A were
removed, and the n-butylamine chain was added. This molecule
was minimized within the active site allowing all atoms to move,
while the enzyme was kept fixed. The LeuAlaPQ part of the
molecule was separately built and minimized in an extended
conformation. The two parts of 6 were then joined and minimized
in the active site as previously described. Conformers of 6 were
manually generated by rotating the bonds of the 4,4′-oxybisbenzoic
ring system while leaving the LeuAlaPQ part of the molecule fixed.
Each conformer was minimized within the PLM II allowing all
atoms of 6 to move, while the enzyme was kept fixed. The
minimizations were performed in Accelrys DS ViewerPro 5.0
Conformer (trial version) with the steepest descent. All minimiza-
tions converged before 10 000 iterations.
General Methods. NMR spectra were recorded on a Varian 300
MHz instrument using CDCl3, CD3OD, or DMSO-d6 as solvents.
TMS was used as reference. Peaks were assigned with 2D COSY
experiments and are consistent with the proposed structures. Melting
points were determined with a Bu¨chi apparatus and are uncorrected.
TLC was carried out on Merck precoated 60 F254 plates using
UV light and dipping with ethanol/phosphomolybdic acid (10%)
or a solution of 0.5% ninhydrin in ethanol for visualization. Flash
column chromatography was performed using silica gel 60 (0.040-
0.063 mm, Merck). Organic phases were dried over anhydrous
sodium sulfate. Concentrations were performed under diminished
pressure (1-2 kPa) at a bath temperature of 40 °C. High-resolution
mass spectra (ESI) were recorded on a Fourier transform ion
cyclotron resonance mass spectrometer APEX II and Xmass
software (Bruker Daltonics). Mass spectra (ESI, positive ions) were
recorded on a LCG Advantage (Thermo Finnigan).
The crude product was reacted under general procedure B,
followed by reaction under general procedure A with BocIleOH
(194 mg, 0.84 mmol) to give after crystallization with ethyl acetate
product 12 (257 mg, 0.58 mmol, 32%). Rf ) 0.4 (cycloexane/ethyl
1
acetate, 1:1). Mp 174-175 °C. H NMR (CDCl3, 300 MHz) δ:
0.80-1.04 (15H, m), 1.26-1.40 (4H, m), 1.43 (9H, s), 1.41-1.68
(5H, m), 1.91 (1H, m), 2.20 (1H, d, J ) 3.67 Hz), 2.25 (1H, d, J
) 3.73 Hz), 3.17-2.29 (2H, m), 3.91 (2H, m), 3.98 (1H, m), 4.90
(1H, d, J ) 7.12 Hz), 6.36 (1H, bm), 6.41 (1H, d J ) 9.65 Hz).
Anal. (C23H45N3O5) C, H, N.
(3S,4S)-4-((2S,3S)-2-Amino-3-methylpentanamido)-N-butyl-
3-hydroxy-6-methylheptanamide (IleStaNHC4H9 HCl) (13).
Product 12 (150 mg, 0.34 mmol) was reacted under general
procedure B to give 13 as a white solid purified by digestion with
ethyl ether. The product was used in the next reaction without any
further purification or characterization.
General Synthetic Procedures. Procedure A. Coupling. The
carboxylic acid (1.1 equiv) and the amine (1.0 equiv) are dissolved
under nitrogen in an appropriate solvent (DMF, dichloromethane,
or acetonitrile) (5 mL/mmol). N-Methylmorpholine was then added
followed by hydroxybenzotriazole solution (HOBt) (1.5 equiv). The
resulting solution was cooled at 0 °C, and O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) was
added (1.1 equiv). The mixture was then stirred at room temperature
overnight. Dichloromethane was then added, and the organic phase
was washed with citric acid (5%), saturated NaHCO3, water, and
brine, dried, filtered, and concentrated.
General Synthetic Procedures. Procedure B. Removal of the
Boc Group (HCl). After cooling the Boc-protected compound at
0 °C, 4 N HCl in dioxane was added (1 mL/100 mg of compound).
The mixture was stirred at room temperature for 30 min. After this
time the mixture was concentrated and the residue washed three
times with ethyl ether and used in the next step without any further
purification or characterization.
General Synthetic Procedures. Procedure C. Removal of the
Boc Group (TFA). To the Boc-protected compound was added
dropwise at room temperature a 1:1 solution of TFA/dichlo-
romethane (4 mL/mmol). After being stirred for 40-50 min, the
mixture was concentrated and washed three times with ethyl ether
and filtered, and the solid obtained was used in the next step without
any further purification or characterization.
4-(4-(Benzyloxycarbonyl)phenoxy)benzoic Acid (14). 4,4′-
Oxybisbenzoic acid (2.00 g, 7.74 mmol) was dissolved at room
temperature in DMF (20 mL), and TEA was added (1.02 g, 10.1
mmol). Benzyl bromide (1.14 g, 10.1 mmol) was then added
dropwise, and the mixture was heated at 60 °C under stirring for 3
h. The mixture was then cooled at room temperature and extracted
with ethyl acetate (3 times). The organic phase washed with water
and brine, dried, and concentrated.
Product 14 was obtained as a white solid (631 mg, 2.44 mmol,
32%) after purification of the crude reaction mixture by silica gel
flash chromatography (CH2Cl2/MeOH, 95:5) followed by digestion
with ethyl ether. Rf ) 0.45 (CH2Cl2/CH3OH, 90:10). 1H NMR
(CDCl3/CD3OD, 95:5, 300 MHz) δ: 5.31 (2H, s), 7.02 (4H, m),
7.38 (5H, m), 8.02 (4H, m).
General Synthetic Procedures. Procedure D. Removal of the
Cbz Group by Catalytic Hydrogenation. The Cbz-protected
compound was dissolved in methanol (10 mL/mmol), and then Pd/C
(80 mg/mmol) added. When the reaction was completed (30-40
min), the mixture was filtered and washed with methanol and the
residue was concentrated and used in the next step without any
further purification or characterization.
4-(4-(((2S,3S)-1-((3S,4S)-1-(Butylamino)-3-hydroxy-6-methyl-
1-oxoheptan-4-ylamino)-3-methyl-1-oxopentan-2-yl)carbamoyl)-
phenoxy)benzoic Acid Benzyl Ester (15). By reaction under
general procedure A of amine 13 (131 mg, 0.49 mmol) and acid
14 (172 mg, 0.49 mmol), product 15 was obtained (325 mg, 0.48
mmol) as a white solid and used in the next reaction without
purification. Rf ) 0.5 (CH2Cl2/CH3OH, 95:5). Mp 173 °C. 1H NMR

Plasmepsins I and II Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7447
(CDCl3, 300 MHz) δ: 0.81-1.00 (12H, m), 1.03 (3H, d, J ) 6.66
Hz), 1.12-1.74 (9H, m), 2.03 (1H, m), 2.29 (1H, dd, J ) 14.9, 4.0
Hz), 2.40 (1H, dd, J ) 14.7, 9.1 Hz), 3.24 (2H, m), 3.92 (1H, m),
4.02 (1H, m), 4.48 (1H, t), 5.36 (2H, s), 6.45 (1H, m), 6.52 (1H, d,
J ) 9.3 Hz), 6.78 (1H, d, J ) 8.2 Hz), 6.95-7.14 (4H, m), 7.32-
7.54 (5H, m), 7.80 (2H, d), 8.08 (2H, d, J ) 8.6 Hz). MS (M +
Na)+: 696.5.
carbamic Acid tert-Butyl Ester (4′-N-[Cbz-alanyl(NE-Boc-lysyl)]-
primaquine) (21). Compound 21 was prepared according to general
procedure A by reacting 4′-N-(Nꢀ-Boc-lysyl)primaquine6 (149 mg,
0.306 mmol) with Cbz-alanine (68 mg, 0.306 mmol). The product
was obtained (113 mg, 0.16 mmol, 46%) as a yellow amorphous
solid after purification by silica gel gradient chromatography (CH2-
1
Cl2/CH3OH, 99:1 to 98:2). Rf ) 0.4 (CH2Cl2/CH3OH, 95:5). H
NMR (CDCl3, 300 MHz) δ: 0.83-2.26 (25H, m), 2.89-3.30 (3H,
bm), 3.33-3.75 (2H, bm), 3.95 (3H, s), 4.32 (1H, bm), 4.59 (1H,
bm), 4.85 (1H, bm), 5.08 (2H, s), 6.57 (2H, s), 7.32 (8H, m), 7.69
(1H, m), 8.53 (2H, m). MS (M + Na)+: 715.5.
tert-Butyl (S)-1-(4-(6-Methoxyquinolin-8-ylamino)pentylamino)-
1-oxopropan-2-ylcarbamate (4′-N-(Boc-alanyl)primaquine) (16).
Compound 16 was prepared according to general procedure A, by
reacting primaquine diphosphate (179 mg, 0.5 mmol) with Boc-
alanine (95 mg, 0.5 mmol). The product was obtained (171 mg,
0.4 mmol, 79%) as a brownish amorphous solid and used in the
next reaction without further purification. Rf ) 0.5 (CH2Cl2/CH3-
{(S)-5-((S)-2-Benzyloxycarbonylamino-4-methylpentanoy-
lamino)-5-[4-(6-methoxyquinolin-8-ylamino)pentylcarbamoyl]-
pentyl}carbamic Acid tert-Butyl Ester (4′-N-[Cbz-leucyl(NE-Boc-
lysyl)]primaquine) (22). Compound 22 was prepared according
to general procedure A by reacting 4′-N-(Nꢀ-Boc-lysyl)PQ6 (97 mg,
0.2 mmol) with Cbz-leucine (53 mg, 0.2 mmol). The product was
obtained (102 mg, 0.14 mmol, 69%) as a yellow amorphous solid
after purification by silica gel gradient chromatography (CH2Cl2/
CH3OH, 99:1 to 98:2). Rf ) 0.5 (CH2Cl2/CH3OH, 95:5). 1H NMR
(CDCl3, 300 MHz) δ: 0.88 (6H, m), 1.00-1.78 (25 H, m), 2.89-
3.30 (3H, bm), 3.33-3.75 (2H, bm), 3.95 (3H, s), 4.33 (1H, bm),
4.57 (1H, bm), 4.82 (1H, bm), 5.08 (2H, s), 6.57 (2H, s), 7.32 (9H,
m), 8.53 (2H, m). MS (M + Na)+: 757.6.
1
OH, 95:5). H NMR (CDCl3, 300 MHz) δ: 1.06-2.25 (19H, m),
3.27 (2H, bm), 3.62 (1H, m), 3.95 (3H, s), 4.27 (1H, bs), 5.45 (1H,
bm), 6.55 (2H, m), 7.26 (3 H, m), 7.69 (1H, bd), 8.57 (1H, bd).
MS (M + Na)+: 453.2
(S)-1-(4-(6-Methoxyquinolin-8-ylamino)pentylamino)-4-meth-
yl-1-oxopentan-2-ylcarbamic Acid tert-Butyl Ester (4′-N-(Boc-
leucyl)primaquine) (17). Compound 17 was prepared according
to general procedure A by reacting primaquine diphosphate (300
mg, 0.84 mmol) with Boc-leucine (209 mg, 0.84 mmol). The
product was obtained (330 mg, 0.698 mmol, 83%) as a yellow
amorphous solid and used in the next reaction without further
(3S,4S)-4-((2S,3S)-2-(4-(4-((S)-((S)-1-(6-(tert-Butoxycarbam-
oyl)-1-(4-(6-methoxyquinolin-8-ylamino)pentylamino)-1-oxohexan-
2-ylamino)-1-oxopropan-2-yl)carbamoyl)phenoxy)benzamido)-
3-methylpentanamido)-3-hydroxy-6-methylheptanoic Acid Butyl-
amide (23). Compound 23 was obtained by reacting a mixture of
4′-N-[Cbz-alanyl(Nꢀ-Boc-lysyl)]PQ (21) (69 mg, 0.1 mmol) and
benzyl ester 15 (67 mg, 0.1 mmol) according to general procedure
D followed by general procedure A. The product was obtained (44
mg, 0.039 mmol, 39%) as a yellow solid after crystallization from
1
purification. Rf ) 0.55 (CH2Cl2/CH3OH, 95:5). H NMR (CDCl3,
300 MHz) δ: 0.92 (6H, m), 1.25 (3H, m), 1.41 (9H, m), 1.54-
1.82 (7H, bm), 3.27 (2H, bm), 3.61 (1H, bm), 3.89 (3H, s), 4.08
(1H, bm), 4.99 (1H, bs), 6.32 (2H, d), 7.34 (1H, bs), 7.97 (1H, bs),
8.53 (1H, d). MS (M + Na)+: 495.4.
((S)-1-{(S)-1-[4-(6-Methoxyquinolin-8-ylamino)pentylcarba-
moyl]ethylcarbamoyl}ethyl)carbamic Acid Benzyl Ester (4′-N-
(Cbz-alanylalanyl)primaquine) (18). Compound 18 was obtained
by reacting compound 16 (93 mg, 0.217 mmol) according to general
procedure B, followed by general procedure A with Cbz-alanine
(48 mg, 0.217 mmol). The product was obtained (86 mg, 0.161
mmol, 74%) as a green amorphous solid and purified by digestion
with ether. Rf ) 0.3 (CH2Cl2/CH3OH, 95:5). 1H NMR (CDCl3, 300
MHz) δ: 1.02-1.42 (7H, m), 1.44-1.74 (6H, bs), 3.21 (2H, bm),
3.56 (1H, bm), 3.83 (3H, s), 4.11 (1H, bm), 4.35 (1H, bm), 5.01
(2H, s), 5.25 (1H, bs), 5.98 (1H, bs), 6.26 (2H, d), 6.60 (1H, bs),
7.26 (7H, m), 7.88 (1H, bd), 8.46 (1H, m). MS (M + Na)+: 558.2.
((S)-1-{(S)-1-[4-(6-Methoxyquinolin-8-ylamino)pentylcarba-
moyl]ethylcarbamoyl}-3-methylbutyl)carbamic Acid Benzyl Es-
ter (4′-N-(Cbz-leucylalanyl)primaquine) (19). Compound 19 was
obtained by reacting compound 16 (56 mg, 0.13 mmol) according
to general procedure B followed by general procedure A with Cbz-
leucine (35 mg, 0.13 mmol). The product was obtained (37 mg,
0.066 mmol, 51%) as a green amorphous solid after purification
by silica gel gradient chromatography (CH2Cl2/CH3OH, 99:1 to 98:
2). Rf ) 0.4 (CH2Cl2/CH3OH, 95:5). 1H NMR (CDCl3, 300 MHz)
δ: 0.84 (6H, m), 1.13-1.35 (6H, m), 1.48-1.80 (7H, bm), 3.20
(2H, bm), 3.56 (1H, bm), 3.83 (3H, s), 4.08 (1H, bm), 4.34 (1H,
bm), 5.01 (2H, s), 5.14 (1H, bs), 5.96 (1H, bs), 6.25 (2H, d), 6.58
(1H, bs), 7.26 (7H, m), 7.87 (1H, bd), 8.46 (1H, m). MS (M +
Na)+: 600.4.
1
ethyl acetate. Rf ) 0.5 (CH2Cl2/CH3OH, 90:10). Mp 190 °C. H
NMR (CDCl3/CD3OD, 95:5, 300 MHz) δ: 0.65-1.04 (15H, m),
1.06-1.82 (34H, m), 1.91 (1H, bm), 2.20 (2H, d), 2.90 (2H, bm),
3.07-3.26 (4H, m), 3.57 (1H, bm), 3.84 (1H, s), 3.74-3.89 (2H,
m), 4.22-4.29 (1H, bm), 4.36 (1H, t), 4.51-4.61 (1H, bm), 6.23-
6.35 (2H, d), 6.99 (4H, m), 7.79 (5H, t), 8.41-8.51 (2H, bm). MS
(M + Na)+: 1146.5.
(3S,4S)-N-Butyl-3-hydroxy-6-methyl-4-((2S,3S)-3-methyl-2-(2-
(naphthalen-2-yloxy)acetamido)pentanamido)heptanamide (1).
Product 1 was obtained by reacting amine 13 (34.7 mg, 0.25 mmol)
with 2-(naphthalen-2-yloxy)acetic acid (50 mg, 0.25 mmol) ac-
cording to general procedure A and purified by crystallization from
ethyl acetate to give a white solid (61 mg, 0.12 mmol, 47%). Rf )
1
0.5 (CH2Cl2/CH3OH, 95:5). Mp 179 °C. H NMR (CDCl3, 300
MHz) δ: 0.79-1.19 (15H, m), 1.22-1.83 (9H, m), 1.97 (1H, bm),
2.26 (2H, m), 3.24 (2H, m), 3.88-4.05 (2H, bm), 4.33 (1H, m),
4.65 (2H, s), 6.39 (1H, bd), 7.26 (3H, m), 7.47 (2H, m), 7.78 (2H,
m). HRMS calcd for C30H45N3O5Na (M + Na)+: 550.3251.
Found: 550.3242.
(3S,4S)-4-((2S,3S)-2-(4-(4-((4-(6-Methoxyquinolin-8-ylamino)-
pentyl)carbamoyl)phenoxy)benzamido)-3-methylpentanamido)-
3-hydroxy-6-methylheptanoic Acid Butylamide (3). Compound
3 was obtained by reacting benzyl ester 15 (128 mg, 0.19 mmol)
according to general procedure D followed by general procedure
A with primaquine diphosphate (74 mg, 0.16 mmol). The product
was obtained (92 mg, 0.11 mmol, 69%) as a yellow solid after
silica gel gradient chromatography (CH2Cl2/CH3OH, 99:1 to 95:5)
followed by digestion in ether. Rf ) 0.38 (CH2Cl2/CH3OH, 95:5).
(S)-1-((S)-1-(4-(6-Methoxyquinolin-8-ylamino)pentylamino)-
4-methyl-1-oxopentan-2-ylamino)-4-methyl-1-oxopentan-2-yl-
carbamic Acid Benzyl Ester (4′-N-(Cbz-leucylleucyl)primaquine)
(20). Compound 20 was obtained by reacting compound 17 (130
mg, 0.275 mmol) according to general procedure B followed by
general procedure A with Cbz-leucine (73 mg, 0.275 mmol). The
product was obtained (130 mg, 0.21 mmol, 76%) as a yellow
amorphous solid after purification by silica gel gradient chroma-
tography (CH2Cl2/CH3OH, 99:1 to 98:2). Rf ) 0.4 (CH2Cl2/CH3-
1
Mp 167 °C (dec). H NMR (DMSO-d6, 300 MHz) δ: 0.73-1.01
(15H, m), 1.01-1.43 (10H, m), 1.43-1.77 (6H, bm), 1.92 (1H,
bm), 2.07 (2H, m), 3.00 (2H, m), 3.28 (2H, m), 3.63 (1H, m), 3.76-
3.89 (5H, m), 4.28 (1H, t), 4.90 (1H, d), 6.14 (1H, d), 6.26 (1H,
m), 6.46 (1H, m), 7.04-7.13 (4H, m), 7.41 (1H, q), 7.49 (1H, d),
7.68 (1H, t), 7.81-7.97 (4H, m), 8.06 (1H, dd), 8.33 (1H, d), 8.46
(1H, t), 8.51 (1H, m). HRMS calcd for C47H64N6O7Na (M +
Na)+: 847.4729. Found: 847.4706.
1
OH, 95:5). H NMR (CDCl3, 300 MHz) δ: 0.90 (12H, s), 1.28
(3H, m), 1.39-1.82 (10H, bm), 3.23 (2H, bm), 3.63 (1H, bd), 3.90
(3H, s), 4.18 (1H, bm), 4.45 (1H, bm), 5.06 (2H, m), 6.24-6.61
(2H, bm), 7.30 (7H, m), 8.53 (1H, s). MS (M + Na)+: 624.2
{(S)-5-((S)-2-Benzyloxycarbonylaminopropionylamino)-5-[4-
(6-methoxyquinolin-8-ylamino)pentylcarbamoyl]pentyl}-
(3S,4S)-4-((2S,3S)-2-(4-(4-((S)-((S)-1-(6-Amino-1-(4-(6-meth-
oxyquinolin-8-ylamino)pentylamino)-1-oxohexan-2-ylamino)-1-

7448 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Dell’Agli et al.
oxopropan-2-yl)carbamoyl)phenoxy)benzamido)-3-methylpen-
tanamido)-3-hydroxy-6-methylheptanoic Acid Butylamide (4).
Compound 23 (30 mg, 0.027 mmol) was reacted under general
procedure C. The resulting salt was dissolved in methanol, and H3-
PO4 (0.054 mmol) was added. The mixture was dried, and product
4 (30 mg, 0.025 mmol, 91%) was obtained as an orange solid after
crystallization from methanol/ether. 1H NMR (DMSO-d6, 300 MHz)
δ: 0.70-1.01 (15H, m), 1.97-1.76 (25H, m), 1.92 (1H, bm), 2.08
(2H, m), 2.72 (2H, s), 2.91-3.16 (4H, m), 3.60 (1H, bm), 3.75-
3.90 (5H, m), 4.17 (1H, m), 4.29 (1H, t), 4.41 (1H, t), 6.08 (1H,
d), 6.24 (1H, m), 6.45 (1H, d), 7.09 (5H, d), 7.43 (2H, m), 7.67
(1H, t), 7.94 (7H, m), 8.33 (1H, m), 8.51 (1H, m). HRMS calcd
for C56H81N9O9Na (M + Na)+: 1046.6050. Found: 1046.6059.
(3S,4S)-4-((2S,3S)-2-(4-(4-((S)-((S)-1-(1-(4-(6-Methoxyquino-
lin-8-ylamino)pentylamino)-1-oxopropan-2-ylamino)-1-oxopro-
pan-2-yl)carbamoyl)phenoxy)benzamido)-3-methylpentanamido)-
3-hydroxy-6-methylheptanoic Acid Butylamide (5). Compound
5 was obtained by reacting a mixture of 4′-N-(Cbz-alanylalanyl)-
PQ (18) (35 mg, 0.065 mmol) and benzyl ester 15 (44 mg, 0.065
mmol) according to general procedure D followed by general
procedure A. The product was obtained (32 mg, 0.033 mmol, 50%)
as a yellow solid after crystallization from ethyl acetate. Rf ) 0.65
digestion in ether. Rf ) 0.5 (CH2Cl2/CH3OH, 90:10). Mp 211 °C.
1H NMR (CDCl3, 300 MHz) δ: 0.72-1.06 (27H, m), 1.09-1.99
(22H, m), 1.91 (1H, bs), 2.21 (2H, m), 3.05-3.43 (4H, m), 3.66
(1H, bd), 3.78-3.95 (2H, m), 3.91 (3H, s), 4.34-4.53 (2H, m),
4.67 (1H, bt), 6.53 (2H, s), 6.98 (4H, m), 7.80 (5H, m), 8.57 (2H,
m). HRMS calcd for C59H86N8O9Na (M + Na)+: 1073.6410.
Found: 1073.6400.
4-(4-(((S)-1-(4-(6-Methoxyquinolin-8-ylamino)pentylamino)-
4-methyl-1-oxopentan-2-yl)carbamoyl)phenoxy)benzoic Acid Ben-
zyl Ester (24). Compound 24 was obtained by reacting compound
17 (125 mg, 0.264 mmol) according to general procedure B
followed by general procedure A with acid 14 (92 mg, 0.264 mmol).
The product was obtained (142 mg, 0.2 mmol, 78%) as a yellow
amorphous solid after purification by silica gel chromatography
(CH2Cl2/CH3OH, 98:2). Rf ) 0.5 (CH2Cl2/CH3OH, 95:5). 1H NMR
(CDCl3, 300 MHz) δ: 0.95 (6H, m), 1.26 (3H, m), 1.55-1.80 (7H,
bm), 3.23 (2H, bm), 3.60 (1H, bm), 3.88 (3H, s), 4.63 (1H, bs),
5.36 (2H, s), 5.98 (1H, bs), 6.27 (1H, bd), 6.72 (1H, bs), 7.00 (5H,
m), 7.43 (7H, m), 7.78 (3H, m), 8.05 (2H,m), 8.51 (1H, m). MS
(M + Na)+: 725.1.
(3S,4S)-4-((2S,3S)-2-(4-(4-((S)-(1-(4-(6-Methoxyquinolin-8-
ylamino)pentylamino)-4-methyl-1-oxopentan-2-yl)carbamoyl)-
phenoxy)benzamido)-3-methylpentanamido)-3-hydroxy-6-meth-
ylheptanoic Acid Butylamide (2). Compound 2 was obtained by
reacting benzyl ester 24 (62 mg, 0.09 mmol) according to general
procedure D followed by general procedure A with amine 13 (24.0
mg, 0.09 mmol). The product was obtained (24 mg, 0.025 mmol,
28%) as a yellow solid after silica gel gradient chromatography
(CH2Cl2/CH3OH, 99:1 to 95:5) followed by digestion in ethyl ether.
1
(CH2Cl2/CH3OH, 90:10). Mp 194 °C. H NMR (CDCl3/CD3OD,
95:5, 300 MHz) δ: 0.63-1.00 (15H, m), 1.03-2.04 (23H, m), 2.24
(2H, m), 3.16-3.23 (4H, m), 3.52 (1H, m), 3.88 (3H, s), 3.77-
3.95 (2H, m), 4.38 (1H, bd), 4.50 (1H, m), 4.65 (1H, m), 6.51 (2H,
s), 6.89 (4H, m), 7.75 (5H, m), 8.49 (1H, d), 8.60 (1H, dd). HRMS
calcd for C53H74N8O9Na (M + Na)+: 989.5471. Found: 989.5451.
(3S,4S)-4-((2S,3S)-2-(4-(4-((S)-((S)-1-(1-(4-(6-Methoxyquino-
lin-8-ylamino)pentylamino)-1-oxopropan-2-ylamino)-4-methyl-
1-oxopentan-2-yl)carbamoyl)phenoxy)benzamido)-3-methylpen-
tanamido)-3-hydroxy-6-methylheptanoic Acid Butylamide (6).
Compound 6 was obtained by reacting a mixture of 4′-N-(Cbz-
leucylalanyl)PQ (19) (29 mg, 0.052 mmol) and benzyl ester 15
(35 mg, 0.052 mmol) according to general procedure D followed
by general procedure A. The product was obtained (20 mg, 0.02
mmol, 39%) as a yellow solid after crystallization from ethyl acetate.
1
Rf ) 0.6 (CH2Cl2/CH3OH, 95:5). Mp 193 °C. H NMR (CDCl3/
CD3OD, 95:5, 300 MHz) δ: 0.73-1.00 (21H, m), 1.08-1.96 (20H,
m), 2.18 (2H, bd), 3.12 (2H, m), 3.60 (2H, bm), 3.87 (3H, s), 3.76-
3.90 (3H, m), 4.35 (1H, m), 4.66 (1H, bm), 6.50 (2H, bs), 6.98
(4H, m), 7.74 (5H, m), 8.45-8.59 (2H, bm). HRMS calcd for
C53H75N7O8Na (M + Na)+: 960.5569. Found: 960.5558.
(S)-2-Benzyloxycarbonylamino-6-tert-butoxycarbonylamino-
hexanoic Acid Allyl Ester (Cbz-(NE-Boc-lysine allyl ester) (25).
Cbz-lysine allyl ester18 (1.25 g, 3.50 mmol) was dissolved in
dichloromethane (25 mL). TEA (0.53 mL, 3.50 mmol) was then
added dropwise under stirring, followed by di-tert-butyl dicarbonate
(764 mg, 3.50 mmol). After being stirred for 12 h at room
temperature, the reaction mixture was washed with HCl (1 M, ×3),
NaHCO3 (saturated, ×3), H2O (×3), and brine, dried, and concen-
trated to give 24, which was used in the next reaction without
purification (1.50 g, 2.60 mmol). Rf ) 0.8 (CH2Cl2/MeOH, 95: 5).
1H NMR (CDCl3, 300 MHz) δ: 1.42 (9H, s), 1.48 (2H, bm), 1.60-
1.76 (2H, bm), 1.77-1.93 (2H, bm), 3.10 (2H, t), 4.37 (1H, bt),
4.64 (2H, m), 5.10 (2H, s), 5.29 (2H, m), 5.35 (1H, s), 5.87 (1H,
bm), 7.53 (5H, m). MS (M + Na)+: 443.3.
1
Rf ) 0.5 (CH2Cl2/CH3OH, 90:10). Mp 204 °C. H NMR (CDCl3/
CD3OD, 95:5, 300 MHz) δ: 0.67-1.05 (21H, m), 1.07-1.76 (22H,
m), 1.88 (1H, bm), 2.20 (2H, d), 3.06-3.26 (4H, m), 3.55 (1H,
bm), 3.84 (3H, s), 3.72-3.95 (2H, m), 4.22-4.41 (2H, m), 4.59
(1H, bm), 6.24 (2H, s), 7.00 (4H, m), 7.79 (5H, m), 8.44 (2H, bd).
HRMS calcd for C56H80N8O9Na (M + Na)+: 1031.5940. Found:
1031.5923.
(3S,4S)-4-((2S,3S)-2-(4-(4-((S)-((S)-1-(6-(tert-Butoxycarbam-
oyl)-1-(4-(6-methoxyquinolin-8-ylamino)pentylamino)-1-oxohexan-
2-ylamino)-4-methyl-1-oxopentan-2-yl)carbamoyl)phenoxy)-
benzamido)-3-methylpentanamido)-3-hydroxy-6-methyl-
heptanoic Acid Butylamide (7). Compound 7 was obtained by
reacting a mixture of 4′-N-[Cbz-leucyl(Nꢀ-Boc-lysyl)]primaquine
22 (41 mg, 0.056 mmol) and benzyl ester 15 (38 mg, 0.056 mmol)
according to general procedure D followed by general procedure
A. The product was obtained (17 mg, 0.015 mmol, 26%) as a yellow
solid after silica gel gradient chromatography (CH2Cl2/CH3OH, 99:1
to 96:4) followed by digestion in ethyl ether. Rf ) 0.4 (CH2Cl2/
(S)-2-((S)-2-Benzyloxycarbonylamino-4-methylpentanoylamino)-
6-tert-butoxycarbonylaminohexanoic Acid Propyl Ester (Cbz-
leucyl-NE-Boc-lysine Propyl Ester) (26). Compound 26 was
obtained by reacting compound 25 (500 mg, 1.20 mmol) according
to general procedure D followed by general procedure A with Cbz-
leucine (1.18 mmol, 313 mg). The product was obtained (500 mg,
1.15 mmol) as a white solid and was used in the next reaction
1
CH3OH, 90:10). Mp 203 °C. H NMR (CDCl3/CD3OD 95:5, 300
MHz) δ: 0.72-1.03 (21H, m), 1.06-1.96 (35H, m), 2.20 (2H, d),
3.03-3.29 (6H, m), 3.56 (1H, m), 3.89 (3H, s), 3.80-3.93 (2H,
m), 4.35-4.37 (2H, m), 4.63 (1H, m), 6.52 (2H, m), 6.99 (4H, m),
7.75-7.91 (5H, m), 8.40-8.44 (1H, bs), 8.57 (1H, m). HRMS calcd
for C64H95N9O11Na (M + Na)+: 1188.7043. Found: 1188.7026.
(3S,4S)-4-((2S,3S)-2-(4-(4-((S)-((S)-1-(1-(4-(6-Methoxyquino-
lin-8-ylamino)pentylamino)-4-methyl-1-oxopentan-2-ylamino)-
4-methyl-1-oxopentan-2S-yl)carbamoyl)phenoxy)benzamido)-3-
methylpentanamido)-3-hydroxy-6-methylheptanoic Acid Butyl-
amide (8). Compound 8 was obtained by reacting a mixture of
4′-N-(Cbz-leucylleucyl)primaquine 20 (47 mg, 0.076 mmol) and
benzyl ester 15 (51 mg, 0.076 mmol) according to general procedure
D followed by general procedure A. The product was obtained (14
mg, 0.013 mmol, 17%) as a yellow solid after silica gel gradient
chromatography (CH2Cl2/CH3OH, 99:1 to 96:4) followed by
1
without purification. Rf ) 0.6 (CH2Cl2/MeOH, 95:5). H NMR
(CDCl3, 300 MHz) δ: 0.94 (9H, m), 1.21-1.36 (2H, bm), 1.43
(9H, s), 1.49-1.90 (9H, bm), 3.05 (2H, bt), 4.09 (2H, t), 4.20 (1H,
bt), 4.57 (1H, bt), 5.11 (2H, s), 7.34 (5H, m). MS (M + Na)+:
558.6.
4-{4-[(S)-1-((S)-5-tert-Butoxycarbonylamino-1-propoxycarbo-
nylpentylcarbamoyl)-3-methylbutylcarbamoyl]phenoxy}-
benzoic Acid Benzyl Ester (27). Product 27 was obtained from
26 (500 mg, 1.15 mmol) according to general procedure D followed
by general procedure A with acid 14 and purified by silica gel flash
chromatography (cyclohexane/ethyl acetate, 7:3) (380 mg, 0.52
1
mmol, 45%). Rf ) 0.5 (CH2Cl2/MeOH, 97: 3). H NMR (CDCl3,
300 MHz) δ: 0.93 (7H, m), 1.35-1.52 (13H, bm), 1.63-1.88 (9H,
bm), 3.02 (2H, t), 4.11 (2H, t), 4.58 (1H, bt), 4.70 (1H, bt), 5.36

Plasmepsins I and II Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7449
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Biochemistry 2003, 42, 8459-8464.
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(2H, s), 7.06 (4H, m), 7.40 (5H, m), 7.84 (2H, m), 8.07 (2H, m).
MS (M + Na)+: 754.2.
(S)-2-(2S-(4-(4-(((2S,3S)-1-((3S,4S)-1-(Butylamino)-3-hydroxy-
6-methyl-1-oxoheptan-4-ylamino)-3-methyl-1-oxopentan-2-yl)-
carbamoyl)phenoxy)benzamido)propanamido)-6-(tert-butoxy-
carbonyl)hexanoic Acid Propyl Ester (10). Compound 10 was
obtained by reacting benzyl ester 27 (280 mg, 0.38 mmol) according
to general procedure D followed by general procedure A with amine
13 (88.0 mg, 0.38 mmol). The product was obtained (230 mg, 0.24
mmol, 63%) as a white solid after crystallization from ether/ethyl
acetate (1:1). Rf ) 0.25 (CH2Cl2/MeOH, 95: 5). 1H NMR (CDCl3/
CD3OD, 95:5, 300 MHz) δ: 0.79-0.98 (24H, m), 1.15-1.68 (32H,
bm), 1.79 (1H, m), 1.92 (1H, m), 2.29-2.53 (2H, bm), 2.97 (2H,
t), 3.23 (2H, t), 3.83 (2H, bm), 4.04 (2H, t), 4.35-4.46 (2H, m),
4.63 (1H, t), 7.02 (4H, d), 7.80 (4H, d). HRMS calcd for
C52H82N6O11Na (M + Na)+: 989.5934. Found: 989.5923.
(S)-2-(2S-(4-(4-(((2S,3S)-1-((3S,4S)-1-(Butylamino)-3-hydroxy-
6-methyl-1-oxoheptan-4-ylamino)-3-methyl-1-oxopentan-2-yl)-
carbamoyl)phenoxy)benzamido)propanamido)-6-aminohexano-
ic Acid Propyl Ester (9). Product 10 (40.0 mg, 0.04 mmol) was
reacted under general procedure B to give compound 9 as a white
solid purified by digestion with ethyl ether (35 mg, 0.04 mmol,
96%): mp 154 °C. 1H NMR (DMSO-d6, 300 MHz) δ: 0.60-0.99
(24H, m), 1.21-1.71 (20H, bm), 1.93 (1H, m), 2.08 (2H, m), 2.75
(2H, t), 3.04 (2H, t), 3.82 (2H, m), 3.98 (2H, t), 4.27 (2H, m), 4.54
(1H, m), 7.01 (4H, d), 7.96 (4H, d). HRMS calcd for C47H74N6O9-
Na (M + Na)+: 889.54095. Found: 889.54006. HRMS calcd for
C47H75N6O9 (M + H)+: 867.5590. Found: 867.5572.
Acknowledgment. The authors thank Dr. C. Berry (Cardiff
University) for donating cDNA for proplasmepsin II and Prof.
R. Maffei Facino for the mass spectrometry facility. This work
was supported by Grants FIRST 2004 and 2005, University of
Milan.
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Supporting Information Available: HPLC purity measure-
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http://pubs.acs.org.
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JM061033D