Design and synthesis of potent inhibitors of the malaria aspartyl proteases plasmepsin I and II. Use of solid-phase synthesis to explore novel statine motifs

Article abstract of DOI:10.1021/jm031106i

Picomolar to low nanomolar inhibitors of the two aspartic proteases plasmepsin (Plm) I and II, from the malaria parasite Plasmodium falciparum, have been identified from sets of libraries containing novel statine-like templates modified at the amino and c

Full text of DOI:10.1021/jm031106i

J. Med. Chem. 2004, 47, 3353-3366
3353
Design and Synthesis of Potent Inhibitors of the Malaria Aspartyl Proteases
Plasmepsin I and II. Use of Solid-Phase Synthesis to Explore Novel Statine
Motifs
Per-Ola Johansson,† Yantao Chen,† Anna Karin Belfrage,† Michael J. Blackman,‡ Ingemar Kvarnstro¨m,†
Katarina Jansson,§ Lotta Vrang,§ Elizabeth Hamelink,§ Anders Hallberg,| Åsa Rosenquist,*,†,§ and
Bertil Samuelsson*,§,
Department of Chemistry, Linko¨ping University, S-581 83 Linko¨ping, Sweden, Division of Parasitology, National Institute for
Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK, Medivir AB, Lunastigen 7, S-141 44 Huddinge, Sweden,
Department of Medicinal Chemistry, Uppsala University, BMC, Box 574, S-751 23 Uppsala, Sweden, and Department of
Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
Received November 19, 2003
Picomolar to low nanomolar inhibitors of the two aspartic proteases plasmepsin (Plm) I and
II, from the malaria parasite Plasmodium falciparum, have been identified from sets of libraries
containing novel statine-like templates modified at the amino and carboxy terminus. The
syntheses of the novel statine templates were carried out in solution phase using efficient
synthetic routes and resulting in excellent stereochemical control. The most promising statine
template was attached to solid support and diversified by use of parallel synthesis. The products
were evaluated for their Plm I and II inhibitory activity as well as their selectivity over cathepsin
D. Selected inhibitors were, in addition, evaluated for their inhibition of parasite growth in
cultured infected human red blood cells. The most potent inhibitor in this report, compound
16, displays Ki values of 0.5 and 2.2 nM for Plm I and II, respectively. Inhibitor 16 is also
effective in attenuating parasite growth in red blood cells showing 51% inhibition at a
concentration of 5 µM. Several inhibitors have been identified that exhibit Ki values between
0.5 and 74 nM for both Plm I and II. Some of these inhibitors also show excellent selectivity
vs cathepsin D.
Introduction
consuming up to 80% of the hemoglobin as a source of
nutrients for growth and development. Hemoglobin
degradation takes place in an acidic food vacuole of the
parasite and many of the current antimalarial drugs
appear to disrupt important vacuolar functions.4a The
food vacuole contains aspartic, cysteine, and metallo
proteases, which are all considered to play a role in the
process of hemoglobin degradation. At least 10 genes
encoding aspartic proteases have been identified in the
P. falciparum genome, potentially complicating the
picture of target selection and target redundancy.5 Four
of the aspartic proteases have been localized to the
acidic food vacuole of the parasite, namely plasmepsin
I, II, IV, and HAP, a histo-aspartic protease.1e,f,6 The
parasite cysteine proteases falcipain 1-3 and the as-
partic proteases plasmepsin I and II (Plm I and II) have
been studied in detail, and inhibitors of these falcipains
and plasmepsins have shown efficacy in cell and animal
models of malaria, indicating that these enzymes may
be suitable molecular targets for drug discovery.7
The two plasmepsins I and II show a high degree of
sequence homology (73%), suggesting that both enzymes
can be inhibited by similar compounds. However, they
also show structural similarity with human cathepsin
D, making selectivity an important factor in inhibitor
design.3,8 Several structural motifs have been pursued
in the design and synthesis of Plm I and II inhibitors
mimicking the tetrahedral intermediate formed during
the aspartyl protease catalysis.3,4,8-10 These motifs
include hydroxyethylamines,3,10a,b C2-symmetric dipep-
tide mimetics,8,10e,f and reversed-statines.10c,d
Malaria is considered as one of the most serious
infectious diseases in the world, affecting approximately
500 million people. It is estimated that the annual
mortality from malaria is 2 million, and that more than
40% of the world’s population is at risk of infection.1
The disease is spread by the Anopheles mosquito, mostly
found in the tropical regions of the world, although not
all species of the mosquito transmit malaria.2 There are
four major species of the malaria parasite, i.e. Plasmo-
dium falciparum, P. vivax, P. malariae, and P. ovale,1a
of which P. falciparum is responsible for more than 95%
of malaria-related morbidity and mortality. Increasingly
P. falciparum is becoming resistant to existing thera-
pies, including e.g. chloroquine, mefloquine, and sulfa-
doxime/pyrimethamine, and there are strains of P.
falciparum reported to be resistant to all known anti-
malarial therapies with potentially devastating conse-
quences in particular for the developing world. This has
been recognized as a major health concern, highlighting
the urgent need for new treatment paradigms and for
new efficacious drugs to combat this disease.1a,3
In the erythrocytic stage of the parasite’s life cycle
the parasite invades the red blood cells of its host
* Corresponding authors. Phone: +46 8 54683100. Fax: +46 8
54683199. E-mail: bertil.samuelsson@medivir.se; asa.rosenquist@
medivir.se.
† Linko¨ping University.
‡ National Institute for Medical Research.
§ Medivir AB.
| Uppsala University.
Stockholm University.
10.1021/jm031106i CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/26/2004

3354 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Johansson et al.
Table 1
Figure 1. Two potent plasmepsin inhibitors encompassing a
modified statine (A) and a hydroxyethylamine (B) motif.
Figure 2. Synthetic approach toward the statine mimetic
plasmepsin inhibitors.
The crystal structure of Plm II in complex with
pepstatin A has been reported. Pepstatin A is a highly
potent peptidic inhibitor of Plm II showing a Ki value
of 0.006 nM, suggesting that the statine core may well
serve as a useful starting point for the design of new
and selective antimalarial compounds.4a This strategy
has previously been successfully employed resulting in
the development of potent plasmepsin I and II inhib-
itors,4,9b-d e.g. inhibitor A4 which shows Ki values of 16
nM and 0.56 nM for Plm I and II, respectively, and show
in vivo activity, albeit low, in a parasite growth assay11
exhibiting 54% inhibition at a concentration of 20 µM
(Figure 1). The selectivity for this inhibitor toward
cathepsin D is, however, moderate displaying a Ki value
of 21 nM against cathepsin D. A potent inhibitor,
compound B,3 incorporating the hydroxyethylamine
template is also exemplified in Figure 1.
Previous studies have shown that Plm II has a
continuous S1-S3 crevice that potentially could accom-
modate large P1 substituents.3,4a We have in this study
focused on P1 modifications in the phenyl statine core
of inhibitor A, i.e. compounds C, D, and E shown in
Figure 2, as a useful starting point toward novel and
improved inhibitors of plasmepsin I and II. The homo-
logue of compound A extending the P1 substituent with
one carbon (compound C, Figure 2, compound 14,
Scheme 4 and Table 1) was initially prepared and
assayed. This inhibitor was less potent against both Plm
I and II than the reference inhibitor A, displaying Ki
values of 27 nM and 46 nM respectively for Plm I and
II (Table 1).
a Synthesized in solution and the yield is over three steps. b ND
) not determined.
but can lead to simplified synthesis and provide in-
creased potency against desired proteases.10e,12 With this
in mind, the benzyloxy statine inhibitor 15 was synthe-
sized (compound D, Figure 2, Scheme 4 and Table 1)
which delivered an increase in potency compared to
compound 14 with Ki values of 5.4 nM and 11 nM for
Previous work from our laboratories has shown that
insertion of an oxygen atom in the appropriate position
of a peptidomimetic or dipeptidomimetic structure not
only introduces a new useful center for diversification

Malaria Aspartyl Protease Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3355
Scheme 1a
Scheme 2a
a Reagents and conditions: (i) DIAD, PPh3, CHCl3, reflux; (ii)
benzylmagnesium chloride, THF; (iii) PPh3, DIAD, DPPA, THF;
(iv) HOAc/H2O 1:1, 115 °C; (v) NaIO4, KMnO4; (vi) 1 M NaOH.
a Reagents and conditions: (i) Bu2SnO, toluene, reflux; (ii)
tetrabutylammonium bromide, BnBr, toluene 90 °C; (iii) tetrabu-
tylammonium bromide, pBrBnBr, toluene, 90 °C; (iv) PPh3, DIAD,
DPPA, THF; (v) HOAc/H2O 1:1, 115 °C; (vi) NaIO4, KMnO4; (vii)
1 M NaOH; (viii) AcCl, MeOH.
Plm I and II, respectively. Further refinement in the
aromatic portion of the P1 position resulted in the
synthesis of inhibitor 16 having a p-bromobenzyloxy P1
statine substituent (compounds E, Figure 2, Table 1),
which delivered very impressive Ki values of 0.5 nM and
2.2 nM, respectively for Plm I and II and moreover
exerted an encouraging inhibition of parasite growth of
51% at 5 µM concentration. The cathepsin D selectivity
was however moderate (Ki value of 4.9 nM) but viewed
as amenable to further optimization. On the basis of
these encouraging results, the p-bromobenzyloxy P1
statine template was selected as a model and was
diversified in the P and P′ positions. The synthesis of
this building block was straightforward starting from
D-glucose, as depicted in Scheme 2. The substitutions
of the amino and carboxy terminus were carried out on
solid support (Figure 2, Scheme 5) furnishing the
inhibitors 16-34 (Table 1).
The compounds synthesized, 14-34, were evaluated
for their Plm I and II inhibitory activity as well as for
their selectivity over cathepsin D. Selected compounds
were tested for inhibition of parasite growth in culture.
The most promising inhibitors identified show Ki values
in the 0.5-74 nM range for both Plm I and II, and with
2-1100-fold selectivity over cathepsin D. The best
inhibitor, 16, exhibits 51% inhibition of parasite growth
at 5 µM concentration.
yield.18 Initially diethyl azodicarboxylate (DEAD) was
employed in this reaction; however, due to proble-
matic purifications and unsatisfying yields the related
DIAD was employed which resulted in excellent yield
of product 4. Subsequently the isopropylidene group of
4 was hydrolyzed using 50% aqueous acetic acid at 115
°C affording the corresponding diol which was thereafter
oxidized with sodium periodate in the presence of a
catalytic amount of potassium permanganate to gener-
ate a mixture of carboxylic acid 5 and its corresponding
formic ester. Hydrolysis of the mixture using 1 M
aqueous sodium hydroxide produced compound 5 in 64%
yield over the three steps.13
For the synthesis of the two azido benzyloxy scaffolds
10 and 11 (Scheme 2), of the generic structures D and
E (Figure 2), similar synthetic routes as used for the
synthesis 5 were employed (Scheme 1). Selective alky-
lation of the primary hydroxyl group in compound 1 was
performed by reacting 1 with dibutyltin oxide in toluene
followed by benzyl bromide and tetrabutylammonium
bromide furnishing the corresponding 6-O-benzylated
compound 6 in 94% yield.19 Compound 7 was obtained
in ∼100% yield using the same procedure but employing
p-bromobenzyl bromide as alkylating agent. Compound
6 and 7 were then transformed to their corresponding
azides 8 and 9 (Scheme 2) in quantitative yields. The
benzyloxy derivative 10 was obtained in 60% yield over
the three steps from 8, according to the synthesis of 5.
The p-bromobenzyloxy scaffold 11 was delivered from
9 also according to the same procedure as used in the
synthesis of 5 with the exception that the crude acid
product was directly transformed into the corresponding
methyl ester by treatment with acidic methanol which
yielded 11 in 39% yield over the four steps (Scheme 2).
Silva et al.4 have reported that statine-like inhibitors
incorporating the (S)-3-methyl-2-[(1-pyridin-2-ylmetha-
noyl)amino]butyric acid (B1) in P-side and H-Ala-Leu-
NH220 (A1) in the P′-side (Scheme 3) are potent inhibi-
tors of Plm II. These amino acid derivatives were
therefore used as reference substituents for the new
statine-like inhibitors.
Results and Discussion
Chemistry. For the synthesis of compounds C (Fig-
ure 2) the pivotal intermediate (5)13 was used (Scheme
1). Starting from diacetone D-glucose, 3-deoxy-1,2-O-
isopropylidene-D-glucose (1) was synthesized in an over-
all yield of 68% over three steps according to literature
procedure.14 Reacting 1 with diisopropyl azodicarboxy-
late (DIAD) and triphenylphosphine in refluxing chlo-
roform15 delivered epoxide (2)13,16 in 69% yield. Regio-
selective opening of the epoxide (2) at the sterically least
hindered site with benzylmagnesium chloride in dry
THF gave compound 3 in ∼100% yield.13,17 Subsequent
treatment of 3 under Mitsunobu-like conditions using
triphenylphosphine, DIAD, and diphenylphosphoryl
azide (DPPA) in THF provided the corresponding azide
(4) with inversion of configuration in quantitative

3356 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Johansson et al.
Scheme 3a
Scheme 5a
a Reagents and conditions: (i) H-Val-OtBu‚HCl, HATU, DIEA,
DMF; (ii) TFA, Et3SiH CH2Cl2; (iii) H-Leu-NH2, EDC, HOAt,
Proton Sponge, DMF; (iv) H2/Pd-C, EtOH.
a Reagents and conditions: (i) 11, PPTS, 1,2-dichloroethane; (ii)
LiOH, THF/MeOH/H2O; (iii) R1NH2, PyBOP, NMM DMF; (iv)
SnCl2, thiophenol, TEA, THF; (v) R2COOH PyBOP, HOBt, DIEA,
DMF; (vi) TFA, CH2Cl2, EtOH.
Scheme 4a
to give the support-bound azidoalkyl ester II with a
loading efficiency/yield of 86% and a practical loading
level of 0.71 mmol/g (Scheme 5). The loading level of
the resin was estimated from the mass balance of
purified recovered scaffold 11 obtained after cleavage
from the solid support. The support-bound ester II was
hydrolyzed with lithium hydroxide in THF-MeOH-
H2O and the resulting carboxylic acid was coupled to a
selection of amines using (benzotriazol-1-yloxy)tripyr-
rolidino phosphonium hexafluoro phosphate (PyBOP)
and N-methylmorpholine (NMM) in DMF to introduce
the R1 substituents in III. Trials to employ potassium
trimethylsilanolate in the hydrolysis of the support-
bound ester were initially made but resulted in our
hands in poor or no formation of final compounds.
Reduction of the azide group in III with tin(II) chloride,
thiophenol, and triethylamine in THF24 provided the
corresponding support-bound amines which were then
coupled with selected carboxylic acids using PyBOP,
1-hydroxybenzotriazole (HOBt), and diisopropylethyl-
amine (DIEA) in DMF to introduce the R2 substituents
in IV. Finally, treatment of IV with TFA, dichloro-
methane and ethanol (99.5%) (2:2:1)10c gave the desired
target compounds (V) in solution (Scheme 5).
When inhibitors having the carboxylic acid B1 as the
R2 group were synthesized (Figure 3) the valine moiety
of B1 was partly racemized, due to the basic coupling
conditions on solid support, giving a diastereomeric
mixture of products in ratios ranging from 1.8:1 to 1.1:1
in favor of the desired isomer B1 vs the unfavored B2
diastereomer. The diastereomeric pairs were separated
by column chromatography and both isomers were
tested for their plasmepsin inhibitory properties. The
yields of products in Figure 3 ranged between 8 and 23%
based upon the loading of the hydroxyalkyl ester scaffold
11 to the solid-support. These modest yields can in part
be attributed to the formation of diastereomers.
a Reagents and conditions: (i) A1, HATU, DIEA, DMF; (ii) PPh3,
MeOH, H2O; (iii) B1, HATU, DIEA, DMF.
For the synthesis of B1, 2-picolinic acid was coupled
with H-Val-OtBu‚HCl using O-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate
(HATU) and DIEA in DMF (Scheme 3). Subsequent
hydrolysis of the tert.-butyl ester using TFA and trieth-
ylsilane in dichloromethane21 furnished B1 in 86% yield.
For the synthesis of A1, Z-Ala-OH was coupled with
H-Leu-NH2 using standard coupling conditions, i.e.,
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hy-
drochloride (EDC), 1-hydroxy-7-azabenzotriazole (HOAt),
and (1,8-bis(N,N-dimethylamino)naphthalene (Proton
Sponge) in DMF. Catalytic hydrogenation over 10%
palladium on active carbon afforded A1 in quantitative
yield over the two steps.
Scheme 4 depicts the solution-phase synthesis of
inhibitors 14 and 15 from scaffolds 5 and 10. Couplings
of 5 and 10 with A1 using HATU and DIEA in DMF
gave compounds 12 and 13 in 99% and 63% yields,
respectively. Reduction of the azide groups with triphen-
ylphosphine in methanol containing a few drops of water
and subsequent coupling of the resulting amines with
carboxylic acid B1 employing HATU and DIEA in DMF
yielded compounds 14 and 15 in 34% and 43%, respec-
tively.
For scaffold 11 containing the p-bromobenzyloxy
substituent a solid-phase combinatorial synthetic route
was employed to obtain the target molecules. The azido
hydroxyalkyl ester scaffold 11 was attached to a dihy-
dropyran functionalized polystyrene support (loading:
1.17 mmol/g)22 using pyridinium p-toluenesulfonate
(PPTS) as acid catalyst in 1,2-dichloroethane at 82 °C23
In Figure 4 a library with R1 fixed to the amine A1 is
shown. These products were obtained in overall yields
ranging from 10 to 42%.
The most promising small molecule substituents R1
and R2 from the previous two libraries were then
combined in a new library to investigate the possibilities
of identifying potent and low molecular weight inhibi-

Malaria Aspartyl Protease Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3357
of the enzyme was fixed. The non-hydrogen heavy atoms
in the P2, P2′, and P3′ substituents of the inhibitors
were also kept fixed during the minimizations while the
N-terminus 2-picolinic acid, the P1 substituent and the
C-terminal amide were allowed to move.
The minimizations were performed with MINIMIZE
in SYBYL 6.9, with the conjugate gradient method
allowing each run for 10 000 iterations. The Tripos force
field and Gasteiger-Huckel charges were used during
the minimizations. All the minimizations converged
before 10 000 iterations.
Modeling of Inhibitor A. The hydrophobic P1 side
chain is positioned in the S1 pocket and extends slightly
into the S3 pocket. The phenyl ring has close contacts
with Tyr77, Ile123, Ile 32, and Phe111. The P1 phenyl
also has intramolecular close stacking contacts with
the 2-picolinic amide ring, which is positioned in the
outer parts of the S3 pocket. The 2-picolinic amide has
close contacts with Thr114 and Met15. The amide
carbonyl has lost the H-bond that the corresponding
carbonyl in pepstatin A makes with Ser218 main chain
NH while the H-bond to Ser79 side chain OH is still
there (Figure 6).
Modeling of Inhibitor 14. The P1 methylbenzyl
substituent in structure 14 is less flexible than the
methylbenzyloxy groups of structures 15 and 16. This
results in unfavorable steric interactions with either
Phe111 or Gly216, dependent on the orientation of the
phenyl ring.
Modeling of Inhibitors 15 and 16. The compounds
15 and 16 gave very similar results in the minimiza-
tions. The P1 side chain reaches through the S1 and
deep into the S3 pocket as predicted. The methyl-O-
methyl ether linkage has close contacts with Gly216,
Tyr77, Ile123, and Ile 32. The phenyl ring has close
contacts with Ile123, Ile32, Phe 111, Thr114, Phe120,
and Met15. The phenyl ring has a favorable intramo-
lecular stacking interaction with the 2-picolinic amide
ring (Figure 7).
It is thus evident from modeling that the methyl-
O-methyl ether linkage in structures 15 and 16 gives
flexibility to the P1 substituent so that it can readily
accommodate the phenyl in a suitable position in the
continuous S1-S3 subsite. There are several beneficial
hydrophobic close contacts of the P1 that contribute to
the over all good binding properties of compounds 15
and 16. From modeling there is no obvious advantage
of the p-bromo substituent in inhibitor 16. The clear loss
of activity of inhibitor 14 as compared to inhibitors A,
15, and 16 is likely due to the inflexibility of the ethyl
linkage in 14, forcing the phenyl group into a less
advantageous positions in the S1-S3 subsite resulting
in steric interactions with enzyme residues.
Figure 3. A library of compounds where different amines are
explored as R1 groups. R2 is fixed to B1 or B2.
tors (Figure 5). The overall yields of the products in
Figure 5 were in the range of 22-40%.
Biological Data and Structure-Activity
Relationships
All products were screened against both Plm I and II
and inhibitors with Ki values below 60 nM are shown
in Table 1 together with reference compound A.4 The
two most potent small molecule inhibitors, compounds
33 and 34, are also included. Table 1 also show the total
synthesis yields of the inhibitors, Ki values for the
inhibition of human cathepsin D, and for selected
compounds, the percentage inhibition of parasite growth
in infected red blood cells (RBC) at an inhibitor concen-
tration of 5 µM.
Plasmepsin I and II share significant homology with
human cathepsin D,5b an aspartic peptidase with a wide
tissue distribution predominantly located in the intra-
cellular lysosomal compartments. Knockout mice lack-
ing functional cathepsin D have a limited life span25
indicating that high selectivity will be of critical impor-
tance in the development of anti-malarial plasmepsin
inhibitor drugs.
From examination of the X-ray crystal structures of
pepstatin A bound to plasmepsin II (PDB entry 1sme),
it is evident that the S1 pocket extends into the S3
pocket occupied by valine in the P3 position of pepstatin
A. To deliver more potent and selective drug-like plas-
mepsin I and II inhibitors, we have now extended the
P1 substituent in the statine core to bridge from the S1
pocket into the S3 subsite.
3-D structural models of compounds A, 14, 15, and
16 (Table 1) were constructed from pepstatin A using
the X-ray crystal coordinates of pepstatin A in complex
with plasmepsin II (PDB entry 1sme) as a starting
point. The capped Val N-terminus in pepstatin A was
replaced by 2-picolinic acid, the statine i-butyl side chain
was replaced by in turn a methylphenyl (A), methyl-
benzyl (14), methylbenzyloxy (15), and p-bromometh-
ylbenzyloxy (16), and finally the C-terminal was re-
placed with H-Ala-Leu-NH2.
Compounds 14-24 all incorporate the picolinic amide-
valine capping group R2 (B1). Previously published data
indicate that Plm II shows preference for â-branched
carbon in the inhibitor P2 position.9b From molecular
modeling we predict that the picolinic acid capping
group only occupies the edge of the S3 pocket of Plm II,
making it likely that the S1-S3 pocket should be
favorably positioned to accommodate larger P1 groups
than those previously explored for these type of inhibi-
tors. Compounds 14-24 are potent inhibitors of both
Plm I and II, indicating that this structural fragment
These inhibitors were minimized in complex with Plm
II. The residues closest to the inhibitor, within 5 Å, were
allowed to move during the minimizations while the rest

3358 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Johansson et al.
Figure 4. R2 carboxylic acid library. R1 is fixed to A1. aThe amine functionality was Boc protected during the coupling reaction.
The Boc group was removed during the acidic cleavage in the final step.
epimerization during the solid phase synthesis). It is
approximately 10 times less potent than 16, demon-
strating the importance of (S) configuration in this
position. However, an intriguing observation is that
although compound 25 is about 10 times less potent
than compound 16, it is almost an equipotent inhibitor
of parasite growth (50% at 5 µM concentration).
For compounds 26-32 having H-Ala-Leu-NH2 as R1
substituent, the picolinic amide-valine capping group R2
(B1) is replaced with smaller carboxylic acid derivatives
furnishing more druglike inhibitor compounds. Within
this series, inhibitor 27 having 2,4,6-trifluorobenzoic
acid as R2 substituent exhibits surprisingly good Ki
values of 3.4 nM and 10 nM for Plm I and II, respec-
Figure 5. Target compounds combining the most promising
tively. This inhibitor also shows good selectivity toward
small R1 and R2 groups.
cathepsin D (Ki > 4000 nM). High selectivity toward
cathepsin D is also seen for inhibitors 27, 33, and 34.
indeed extends into the S3 subsite of the plasmepsins
and also gives rise to favorable interactions and a good
It is apparent that cathepsin D selective inhibitors can
be designed by reframing form utilizing the S3 binding
fit in the active site. Moreover, we have found that
pockets and by use of appropriate arylbenzoic acids or
larger groups in the P1 position of these inhibitors
bulky alkyl acids as capping groups for the N-terminus
preferentially show increased affinity toward Plm I over
of the statine-like core. From examination of the X-ray
Plm II. While inhibitor 15 and 16 show modest selectiv-
ity toward cathepsin D with Ki values of 41 nM and 4.9
nM, respectively, inhibitor 14 shows promising selectiv-
ity with a Ki value of 980 nM. The most potent of the
crystal structures of cathepsin D and Plamepsin II, both
in complex with pepstatin A, it can be seen that amino
acid Ile 290 in plasmepsin II has a larger amino acid
Met 307 in the corresponding position in cathepsin D
and that Leu 292 in proximity to the S2 pocket in
plasmepsin II has the amino acid Met 309 in the
corresponding position in cathepsin D. Moreover, the
p-fluoro atom in compound 27 is likely pointing toward
the sulfur atom of Met 307 making unfavorable interac-
tions. This supports our observation that the S2 pocket
in cathepsin D is smaller than that in plasmepsin II
and that this can be used to advantage in the design of
cathepsin D selective plasmepsin inhibitors.
Compounds 33 and 34 represent the two most potent
of the small molecule inhibitors. They both show modest
but promising activities against both Plm I and II, and
inhibitor 33 inhibits parasite growth with 29% inhibi-
tion at 5 µM.
inhibitors incorporating the picolinic acid-valine capping
group R2 (B1) is compound 16, having the H-Ala-Leu-
NH2 (A1) motif as the R1 substituent, with Ki values of
0.5 nM and 2.2 nM for Plm I and II, respectively.
Interestingly, compound 16 is also the most efficient
inhibitor of parasite growth in vitro showing 51%
inhibition at 5 µM concentration. In attempts to reduce
size and peptidic characters while maintaining potency
of the inhibitors the two amide groups, H-Ala-Leu-NH2,
on the P′-side were replaced by various alkyl groups.
From the exemplar inhibitors 17, 18, 20, 21, 22, and
24 it can be seen that this approach holds promise with
inhibitors exhibiting Ki values for Plm I below 10 nM.
Compound 25 is the diastereomer of 16 having the (R)
stereochemistry in the P2 valine moiety (a result of

Malaria Aspartyl Protease Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3359
Figure 6. The active site of plasmepsin II (crystal structure PDB entry 1sme) is shown with inhibitor A modeled (yellow) to the
left and with pepstatin A (orange) to the right.
Figure 7. Inhibitor 16 (magenta) to the left is modeled into plasmepsin II (crystal structure PDB entry 1sme). The
p-bromomethylbenzyloxy P1 group reaches through the S1 and into the S3 subsite defined by Met15, Ile32, and Phe120. Pepstatin
A (orange) is shown to the right as a comparison.
Conclusion
Experimental Section
Enzyme Inhibition Measurements. Pro-plasmepsin II
was a generous gift from Helena Danielson (Department of
Biochemistry, Uppsala University, Uppsala, Sweden). The
expression and purification of plasmepsin I will be pub-
lished elsewhere.26 Human liver cathepsin D was purchased
from Sigma-Aldrich, Sweden. The activities of plasmepsin I
(Plm I), plasmepsin II (Plm II) and cathepsin D was mea-
sured essentially as described previously,3 using a total
reaction volume of 100 µL. The concentration of pro-Plm II
was 3 nM, the amount of Plm I was adjusted to give sim-
ilar catalytic activity and 50 ng/mL pro-cathepsin D was used.
The pro-sequence of Plm II was cleaved off by preincu-
bation in assay reaction buffer (100 mM sodium acetate buf-
fer (pH 4.5), 10% glycerol, and 0.01% Tween 20) at room
temperature for 40 min, and cathepsin D was activated by
incubation in the same reaction buffer at 37 °C for 20 min.
The reaction was initiated by the addition of 3 µM subs-
trate (DABCYL-Glu-Arg-Nle-Phe-Leu-Ser-Phe-Pro-EDANS,
AnaSpec Inc, San Jose, CA), and hydrolysis was recorded as
the increase in fluorescence intensity over a 10 min time
period, during which the fluorescence increased linearly with
time.
Efficient synthetic routes with excellent stereochem-
ical control have been developed for the key intermedi-
ates 5, 10, and 11 (Schemes 1 and 2). Subsequently,
several directed compound libraries have been prepared
utilizing solid-phase chemistry to diversify the promis-
ing p-bromobenzyloxy scaffold 11. Highly potent inhibi-
tors of Plm I and II have been identified, and several of
these inhibitors show excellent cathepsin D selectivity.
The X-ray crystal structure of cathepsin D and plasme-
psin II and SAR analysis indicates that the observed
cathepsin D selectivity is mainly attributed to the P3
and P2 substituents of the inhibitors. Moreover, it has
been shown that the P1 substituent is important for
achieving high Plm I inhibition with potency being
favored by larger P1 groups. The most potent inhibitor
in this report, compound 16, displays Ki values of 0.5
and 2.2 nM for Plm I and II, respectively. Inhibitor 16
is also effective in attenuating parasite growth in red
blood cells, showing 51% inhibition at 5 µM concentra-
tion.
Stock solutions of inhibitors in DMSO were serially diluted
in DMSO and added directly before addition of substrate,
giving a final DMSO concentration of 1%.
IC50 values were obtained by assuming competitive inhibi-
tion and fitting a Langmuir isotherm (vi/vo ) 1/(1 + [I]/IC50))
to the dose response data (Grafit), where vi and vo are the
initial velocities for the inhibited and uninhibited reaction,
respectively, and [I] is the inhibitor concentration.27 The Ki
Several inhibitors, i.e., 15, 17, 18, 20, 21, 22, 24, 25,
and 27, exhibit Ki values for Plm I below 10 nM. Two
small molecule inhibitors, compounds 33 and 34, show
promising activities against both Plm I and II where
inhibitor 33 inhibits parasite growth in red blood cells
with 29% inhibition at a concentration of 5 µM.

3360 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Johansson et al.
was subsequently calculated by using Ki ) IC50/(1 + [S]/Km)28
and a Km value determined according to Michaelis-Menten.
Plasmodium falciparum Growth Inhibition Assay.
Culture and synchronization of the asexual blood stages of
Plasmodium falciparum (clone 3D7) was performed as previ-
ously described.29 Assays evaluating the effects of compounds
on parasite growth were performed using a modification of the
[3H]hypoxanthine incorporation assay described by Chulay et
al.11 Briefly, a highly synchronous culture containing early
trophozoite stage parasites at ∼1% haematocrit and 1%
parasitaemia was supplemented with [3H]hypoxanthine (Am-
ersham Biotech) to 10 µCi ml-1, and then 50 µL aliquots were
dispensed into wells of flat-bottomed 96-well microtiter plates.
Wells were supplemented with an equal volume of medium
containing various concentrations of test compound (1-5 µM
final) or DMSO only (maximum final concentration 1% v/v).
Plates were transferred to gassed boxes and cultured at 37 °C
for 30 h to allow parasite development through to mature
schizont stage. Cultures were then harvested onto glass fiber
filters (Filtermat A, Wallac, Turku, Finland) using a cell
harvester. Filters were wetted with scintillation cocktail and
bound radioactivity quantified in a â-counter. Control cultures
containing established growth-inhibitory compounds or with-
out parasites were included in each experiment. The amount
of radioactivity in each sample was expressed relative to that
in the control wells containing DMSO only. Four independent
experiments were performed for each concentration of each
test compound.
General Methods. NMR-spectra were recorded on a Varian
300 MHz instrument using CDCl3 and CD3OD as solvents.
TMS was used as reference. Optical rotations were measured
using a Perkin- Elmer 141 polarimeter. TLC was carried out
on Merck precoated 60 F254 plates using UV-light and charring
with ethanol/sulfuric acid/p-anisaldehyde/acetic acid 90:3:2:
1, and 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 magnesium sulfate. Concentrations were performed
under diminished pressure (1-2 kPa) at a bath temperature
of 40 °C. MALDI-TOF-spectra were recorded on a Voyager-
DE STR Biospectrometry Workstation using R-cyano-4-hy-
droxycinnamic acid as a matrix and reference. HPLC was
performed on a preparative C-18 column.
removed and a solution of PyBOP in DMF was added to the
resin followed by the amine in DMF and NMM in DMF to give
total concentrations of 0.4 M in respect to PyBOP and amine
and 0.8 M in respect to NMM. The slurry was shaken at room-
temperature overnight before the solution was drained, and
the resin was washed with DMF (5×), methanol (3×), and
dichloromethane (5×).
Reduction of the Azide and Coupling of the Carboxy-
lic Acids (IV). The resin was washed with dry THF (2×), after
which it was allowed to swell in dry THF for 30 min. Solutions
of tin(II) chloride, thiophenol, and triethylamine in dry THF
were added to give the total concentrations of 0.2, 0.8, and
1.0 M in the reagents, respectively. The mixture was shaken
at room temperature for 3 h and 15 minutes. Thereafter it was
dried and washed with THF/H2O (2:1) (2×), DMF (2×), and
dichloromethane (3×).
The resin was washed twice with DMF and was allowed to
swell in DMF for 30 min before the DMF was drained and a
solution of the carboxylic acid, PyBOP, HOBt, and DIEA (0.2,
0.2, 0.2, 0.6 M, respectively in DMF) that had been stirred for
5 min was added. The slurry was shaken overnight after which
the resin was isolated by filtration and washed with DMF (3×),
dichloromethane (3×), and methanol (3×).
Cleavage of the Target Molecules from the Resin (V).
A solution of TFA, dichloromethane and ethanol (99.5%) (2:
2:1) was added to the resin, and the slurry was shaken at room
temperature for 90 min. The solvents were collected, and the
resin was washed and shaken for 5 min with dichloromethane,
methanol, and dichloromethane, respectively. The solvents
were pooled, evaporated, and coevaporated with toluene.
Synthetic Procedures. 3-Deoxy-1,2-O-isopropylidene-
D-glucose (1). Compound 1 was synthesized in 68% yield over
three steps according to ref 14.
5,6-Anhydro-3-deoxy-1,2-O-isopropylidene-D-glucose (2).
To the diol 1 (2.51 g, 12.3 mmol) dissolved in chloroform (200
mL) were added triphenylphosphine (3.87 g, 14.8 mmol) and
diisopropyl azodicarboxylate (DIAD) (2.90 mL, 15.0 mmol), and
the mixture was refluxed overnight. The solvent was evapo-
rated and purification by flash column chromatography (toluene/
ethyl acetate 15:1) yielded 2 (1.57 g, 69%) as a colorless oil. 2:
Analytical data in accordance with ref 16.
6-Benzyl-3,6-dideoxy-1,2-O-isopropylidene-D-glucose (3).
Compound 2 (0.73 g, 3.90 mmol) was dissolved in dry tetrahy-
drofuran (70 mL) and the temperature was lowered to 0 °C.
Benzylmagnesium chloride (1.3 M in THF) (24.0 mL, 31.2
mmol) was added and the mixture was allowed to attain room
temperature and was then stirred overnight. The reaction was
quenched by the addition of water and the THF was evapo-
rated. The water phase was extracted three times with ethyl
acetate and the combined organic phases were dried, filtered
and concentrated. The crude product was purified by flash
column chromatography (gradient elution from toluene/ethyl
acetate 15:1 to 2:1) to give 3 (1.08 g, 100%) as yellow-white
solid. 3: [R]22D -6.9 (c 0.6, CHCl3); 1H NMR (CD3OD, 300 MHz)
δ 1.28 (s, 3H), 1.44 (s, 3H), 1.53-1.69 (m, 1H), 1.70-1.86 (m,
2H), 1.99 (dd, J ) 4.5, 13.3 Hz, 1H), 2.58-2.72 (m, 1H), 2.79-
2.92 (m, 1H), 3.63-3.72 (m, 1H), 4.03-4.14 (m, 1H), 4.64-
Target Molecules. All target molecules depicted in Figures
3-5 were purified by flash column chromatography or HPLC,
identified by NMR and MALDI-TOF, and lyophilized from
dioxane before the biological testing.
General Synthetic Procedures. Procedure A. Solid-
Phase Reactions (used in the synthesis of compounds
16-34):
Preparation of the DHP-resin (I). The DHP resin was
prepared according to ref 23.
Preparation of the Scaffold Loaded Resin (II). To the
scaffold (4) (3.01 g, 8.40 mmol) dissolved in dry 1,2-dichloro-
ethane was added the DHP-resin (1) (2.44 g, 1.17 mmol/g
loading, 2.85 mmol). The slurry was allowed to swell in room
temperature for 30 min before PPTS (2.11 g, 8.40 mmol)
dissolved in dry 1,2-dichloroethane was added, and the tem-
perature was raised to 82 °C. The mixture was shaken for 48
h, and the resin was dried and washed with DMF (4×),
dichloromethane (5×), and methanol (2×). The loaded resin
was dried on a high vacuum pump for 24 h to afford II (0.71
mmol/g loading, 86% loading efficiency) as a brown powder.
Hydrolysis of the Methyl Ester and Coupling of the
Amines (III). The scaffold loaded resin (II) was washed twice
with THF and was then allowed to swell in THF (6 mL) for 30
min. To the slurry was added a solution of lithium hydroxide
(1 M in methanol/water (7:1), 2.11 mL), and the resin was
shaken in room temperature for 72 h. The resin was isolated
by filtration and washed with methanol/water (3:1) (3×),
methanol (3×), THF (3×), THF/HOAc (8:1) (2×, rapidly), THF
(3×), and methanol (3×).
4.71 (m, 1H), 5.75 (d, J ) 3.6 Hz, 1H), 7.10-7.29 (m, 5H); 13
C
NMR (CD3OD, 75.5 MHz) δ 26.5, 27.2, 32.8, 34.0, 36.2, 71.6,
81.6, 82.4, 106.5, 111.9, 126.6, 129.2, 129.3, 143.1. Anal.
(C16H22O4) C, H.
5-Azido-6-benzyl-1,2-O-isopropylidene-3,5,6-trideoxy-
L-idose (4). Compound 3 (1.06 g, 3.81 mmol) and triphen-
ylphosphine (1.51 g, 5.75 mmol) were dissolved in dry THF
(14 mL). Thereafter the mixture was cooled to -15 °C, and
diisopropyl azodicarboxylate (DIAD) (1.90 mL, 9.65 mmol) was
added. The mixture was stirred for 10 min at -15 °C, the
temperature was raised to 0 °C, and diphenylphosphoryl azide
(DPPA) (1.25 mL, 5.78 mmol) was added. After stirring for 30
min at 0 °C, the reaction mixture was allowed to attain room
temperature and was stirred overnight. The solvent was
evaporated and the crude material was purified by flash
column chromatography (toluene) yielding the azide (4) (1.06
g, 92%). 4: [R]22D -67.8 (c 0.7, CHCl3); 1H NMR (CD3OD, 300
The resin was then washed twice with DMF and was then
allowed to swell in DMF for 30 min. Thereafter the DMF was

Malaria Aspartyl Protease Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3361
MHz) δ 1.28 (s, 3H), 1.45 (s, 3H), 1.62-1.74 (m, 1H), 1.76-
1.86 (m, 2H), 2.00 (dd, J ) 4.5, 13.3 Hz, 1H), 2.62-2.73 (m,
1H), 2.75-2.86 (m, 1H), 3.08-3.17 (m, 1H), 4.15-4.24 (m, 1H),
4.68-4.74 (m, 1H), 5.77 (d, J ) 3.6 Hz, 1H), 7.13-7.30 (m,
5H); 13C NMR (CD3OD, 75.5 MHz) δ 26.4, 27.1, 33.2, 33.7, 36.7,
64.9, 81.6, 82.0, 106.8, 112.4, 127.1, 129.4, 129.5, 142.3. Anal.
(C16H21N3O3) C, H, N.
(3S,4S)-4-Azido-3-hydroxy-6-phenylhexanoic Acid (5).
Compound 4 (1.04 g, 3.43 mmol) was dissolved in acetic acid/
water (1:1, 26 mL) and was allowed to reflux for 1 h. Thereafter
the solvent was evaporated, the crude product was dissolved
in tert-butyl alcohol/water (2:3, 25 mL), and sodium periodate
(3.68 g, 17.2 mmol) was added. After the mixture was stirred
at room temperature for 15 min, potassium permanganate
(0.076 g, 0.48 mmol) was added and the mixture was stirred
at room temperature for an additional 1 h. The reaction
mixture was extracted three times with chloroform after which
the organic phases were pooled, washed once with brine, dried,
filtered, and concentrated.
for 30 min at 0 °C, the reaction mixture was allowed to attain
room temperature and was stirred overnight. The solvent was
evaporated, and the crude product was purified by flash
column chromatography (toluene/ethyl acetate 24:1) yielding
1
the azide (8) (4.35 g, 100%). 8: [R]22 -5.3 (c 0.9, CHCl3); H
D
NMR (CDCl3, 300 MHz): δ 1.30 (s, 3H), 1.49 (s, 3H), 1.79-
1.92 (m, 1H), 2.04 (dd, J ) 4.7, 13.5 Hz, 1H), 3.48-3.56 (m,
1H), 3.67-3.75 (m, 2H), 4.27-4.35 (m, 1H), 4.57 (s, 2H), 4.68-
4.75 (m, 1H), 5.79 (d, J ) 3.6 Hz, 1H), 726-7.38 (m, 5H). 13C
NMR (CDCl3, 75.5 MHz): δ 26.2, 26.8, 35.4, 62.6, 70.5, 73.6,
77.4, 80.3, 105.5, 111.5, 127.7, 127.9, 128.5, 137.6. Anal.
(C16H21N3O4) C, H, N.
5-Azido-6-(4-bromobenzyloxy)-3,5-dideoxy-1,2-O-iso-
propylidene-L-idose (9). Compound 9 was synthesized in
100% yield from 7 according to the method for the preparation
of compound 8. 9: [R]22D -2.6 (c 0.5, CHCl3); 1H NMR (CDCl3,
300 MHz): δ 1.30 (s, 3H), 1.49 (s, 3H), 1.82-1.93 (m, 1H), 2.04
(dd, J ) 4.7, 13.5 Hz, 1H), 3.49-3.57 (m, 1H), 3.66-3.75 (m,
2H), 4.30 (dt, J ) 4.4, 10.4 Hz, 1H), 4.57 (s, 2H), 4.71 (dd,
J ) 4.1, 8.2 Hz, 1H), 5.79 (d, J ) 3.6 Hz, 1H), 7.20 (d, J ) 8.5
Hz, 2H), 7.47 (d, J ) 8.5 Hz, 2H). 13C NMR (CDCl3, 75.5
MHz): δ 26.4, 27.0, 34.7, 35.6, 62.8, 70.8, 72.7, 77.5, 80.4,
105.7, 121.9, 129.5, 129.7, 131.7, 131.8, 137.0. Anal. (C16H20-
BrN3O4) C, H, N.
(3S,4S)-4-Azido-5-benzyloxy-3-hydroxypentanoic Acid
(10). Compound 8 (4.30 g, 13.5 mmol) was dissolved in acetic
acid/water (1:1, 100 mL), and the mixture was heated to reflux
for 1 h. Thereafter the solvent was evaporated crude product
was dissolved in tert-butyl alcohol/water (2:3, 100 mL), and
sodium periodate (14.40 g, 67.3 mmol) was added. The reaction
mixture was allowed to stir at room temperature for 15 min
before potassium permanganate (0.300 g, 1.90 mmol) was
added, and the mixture was stirred for an additional 1 h. The
reaction mixture was extracted three times with chloroform
after which the organic phases were pooled, washed once with
brine, dried, filtered, and concentrated.
The crude material was dissolved in water/dioxane 3:1 (40
mL), and the mixture was cooled to 0 °C. Sodium hydroxide
(0.55 g, 13.7 mmol) in water (19 mL) was added dropwise at 0
°C after which the mixture was stirred in room temperature
for 1 h. The water phase was washed twice with diethyl ether
and was then acidified with 6 M hydrochloric acid to pH 1-2.
The acidified water phase was extracted three times with
chloroform, and the combined organic phases were evaporated.
The crude material was purified by flash column chromatog-
raphy (toluene/ethyl acetate 2:1 + 1% HOAc) to give 5 (0.54
1
g, 64%) as yellow oil. 5: [R]22 -25.8 (c 0.7, MeOH); H NMR
D
(CD3OD, 300 MHz) δ 1.88-1.99 (m, 2H), 2.52 (d, J ) 6.9 Hz,
2H), 2.64-2.83 (m, 2H), 3.13-3.22 (m, 1H), 4.03-4.13 (m, 1H),
7.11-7.25 (m, 5H); 13C NMR (CD3OD, 75.5 MHz) δ 33.2, 33.4,
39.9, 66.2, 71.4, 127.0, 129.5, 142.4, 174.9. Anal. (C12H15N3O3)
C, H, N.
6-Benzyloxy-3-deoxy-1,2-O-isopropylidene-D-glucose (6).
Compound 1 (3.00 g, 14.7 mmol) and dibutyl tin oxide (4.84 g,
19.4 mmol) was dissolved in toluene (90 mL). The mixture was
refluxed for 5 h after which the temperature was lowered to
90 °C and tetrabutylammonium bromide (TBAB) (6.27 g, 19.4
mmol) and benzyl bromide (2.21 mL, 18.6 mmol) were added.
The mixture was allowed to stir at 90 °C overnight, and
thereafter the solvent was evaporated and the crude product
was purified by flash column chromatography (toluene/ethyl
The crude material was dissolved in water (110 mL), and
the mixture was cooled to 0 °C. Sodium hydroxide (2.16 g, 54.0
mmol) in water (67 mL) was added dropwise at 0 °C after
which the mixture was stirred in room temperature for 1 h.
The water phase was washed twice with diethyl ether and was
then acidified with 6 M hydrochloric acid to pH 1-2. The
acidified water phase was extracted three times with chloro-
form, and the combined organic phases were evaporated.
Purification by flash column chromatography (toluene/ethyl
acetate 2:1 + 1% HOAc) provided 10 (2.13 g, 60%) as a colorless
acetate 2:1) to give 6 (4.06 g, 94%) as a colorless solid. 6: [R]22
D
-10.2 (c 0.6, CHCl3); 1H NMR (CDCl3 300 MHz): δ 1.32 (s,
3H), 1.50 (s, 3H), 1.78-1.91 (m, 1H), 2.06 (dd, J ) 4.4, 13.5
Hz, 1H), 3.48 (dd, J ) 6.3, 9.9 Hz, 1H), 3.59 (dd, J ) 3.9, 9.9
Hz, 1H), 3.96-4.03 (m, 1H), 4.18-4.28 (m, 1H), 4.54 (d, J )
12.1 Hz, 1H), 4.58 (d, J ) 12.1 Hz, 1H), 4.73 (app. t, J ) 3.9
Hz, 1H), 5.79 (d, J ) 3.9 Hz, 1H), 7.27-7.38 (m, 5H). 13C NMR
(CDCl3, 75.5 MHz): δ 26.2, 26.8, 33.9, 71.0, 71.3, 73.5, 78.4,
80.6, 105.4, 111.3, 127.8, 127.9, 128.5, 137.8. Anal. (C16H22O5)
C, H.
solid. 10: [R]22 8.2 (c 1.3, CHCl3); 1H NMR (CDCl3, 300
D
MHz): δ 2.55 (dd, J ) 4.4, 16.5 Hz, 1H), 2.67 (dd, J ) 8.2,
16.5 Hz, 1H), 3.48-3.56 (m, 1H), 3.69-3.79 (m, 2H), 4.15-
4.24 (m, 1H), 4.55 (s, 2H), 7.27-7.38 (m, 5H). 13C NMR (CDCl3,
75.5 MHz): δ 38.3, 63.7, 68.2, 70.2, 73.7, 127.8, 128.0, 128.6,
137.2, 177.0. Anal. (C12H15N3O4‚0.20 HOAc) C, H, N.
(3S,4S)-4-Azido-5-(4-bromobenzyloxy)-3-hydroxypen-
tanoic Acid Methyl Ester (11). Compound 11 was synthe-
sized from 9 in 39% yield according to the method for the
preparation of compound 10 except that the crude carboxylic
acid obtained in the last step was dissolved in methanol (100
mL) at 0 °C, and acetyl chloride (9 mL) was added in order to
form the methyl ester. The solution was stirred for 20 min at
0 °C and for an additional 48 h at room temperature. The
solvent was evaporated and coevaporated with toluene. Puri-
fication by flash column chromatography (toluene/ethyl acetate
4:1) provided 11 as a slightly yellow solid. 11: [R]22D -11.5 (c
6-(4-Bromobenzyloxy)-3-deoxy-1,2-O-isopropylidene-
D-glucose (7). Compound 7 was prepared in 100% yield
(colorless solid) according to the method for the preparation
of compound 6 using 4-bromobenzyl bromide. 7: [R]22 -8.4
D
(c 0.8, CHCl3); 1H NMR (CDCl3 300 MHz): δ 1.32 (s, 3H), 1.50
(s, 3H), 1.73-1.85 (m, 1H), 2.06 (dd, J ) 4.4, 13.5 Hz, 1H),
3.46 (dd, J ) 6.6, 9.6 Hz, 1H), 3.57 (dd, J ) 3.9, 9.9 Hz, 1H),
3.89-3.97 (m, 1H), 4.16-4.25 (m, 1H), 4.49 (s, 2H), 4.72 (t, J
) 4.3 Hz, 1H), 5.78 (d, J ) 3.9 Hz, 1H), 7.19 (d, J ) 8.3 Hz,
2H), 7.46 (d, J ) 8.3 Hz, 2H).13C NMR (CDCl3, 75.5 MHz): δ
25.8, 26.5, 33.5, 70.5, 72.0, 78.1, 78.2, 79.7, 105.0, 110.8, 121.2,
129.0, 129.0, 131.1, 131.1, 136.7. Anal. (C16H21BrO5) C, H.
1
1.0, CHCl3); H NMR (CDCl3, 300 MHz): δ 2.53 (dd, J ) 4.4,
16.5 Hz, 1H), 2.63 (dd, J ) 8.4, 16.5 Hz, 1H), 3.19-3.27 (b,
1H), 3.50-3.58 (m, 1H), 3.69 (s, 3H), 3.72-3.80 (m, 1H), 4.13-
4.22 (m, 1H), 4.51 (s, 2H), 7.19 (d, J ) 8.2 Hz, 2H), 7.46 (d, J
) 8.2 Hz, 2H). 13C NMR (CDCl3, 75.5 MHz): δ 38.3, 51.9, 64.0,
68.1, 70.4, 72.7, 121.7, 125.3, 129.3, 129.6, 131.6, 136.6, 172.3.
Anal. (C13H16BrN3O4) C, H, N.
5-Azido-6-benzyloxy-3,5-dideoxy-1,2-O-isopropylidene-
L-idose (8). Compound 6 (4.01 g, 13.6 mmol) and triphen-
ylphosphine (4.65 g, 17.7 mmol) were dissolved in dry THF
(50 mL). Thereafter the mixture was cooled to -15 °C, and
diisopropyl azodicarboxylate (DIAD) (5.70 mL, 29.0 mmol) was
added. The mixture was stirred for 10 min at -15 °C, the
temperature was raised to 0 °C, and diphenylphosphoryl azide
(DPPA) (3.93 mL, 18.5 mmol) was added. After being stirred
(S)-3-Methyl-2-[(1-pyridin-2-ylmethanoyl)amino]bu-
tyric Acid (B1). 2-Picolinic acid (0.332 g, 2.70 mmol), H-Val-
OtBu×HCl (0.566 g, 2.70 mmol), and diisopropylethylamine
(DIEA) (1.05 g, 8.10 mmol) were dissolved in DMF (5 mL),

3362 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Johansson et al.
and the temperature was lowered to 0 °C. Thereafter O-(7-
azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluo-
rophosphate (HATU) (1.03 g, 2.70 mmol) was added, and the
reaction mixture was stirred for 0.5 h at 0 °C and then for an
additional 1 h at room temperature. The solvent was evapo-
rated, the crude mixture was extracted with ethyl acetate and
washed twice with brine, and the organic phase was dried,
filtered, and concentrated. The crude material was filtered
through a short silica plug and was eluated with toluene/ethyl
acetate 2:1, and the solvents were evaporated. The remainder
was dissolved in dichloromethane (13 mL). Triethylsilane
(0.785 g, 6.75 mmol) and trifluoroacetic acid (6.5 mL) were
added, and the reaction mixture was allowed to stir in room
temperature for 3 h. The solvents were evaporated and
coevaporated with toluene, and the crude material was purified
by flash column chromatography (toluene/ethyl acetate 2:1 +
1% acetic acid) to give compound B1 (0.516 g, 86%) as a white
powder. B1: [R]22D 38.5 (c 1.1, MeOH); 1H NMR (CD3OD, 300
MHz) δ 1.02 (d, J ) 6.9 Hz, 6H), 2.25-2.32 (m, 1H), 4.56 (d,
J ) 5.2 Hz, 1H), 7.57 (ddd, J ) 1.1, 4.7, 7.7 Hz, 1H), 7.97 (ddd,
J ) 1.7, 7.7, 8.0 Hz, 1H), 8.10 (d, J ) 8.0 Hz, 1H), 8.65 (d, J
) 4.7 Hz, 1H); 13C NMR (CD3OD, 75.5 MHz) δ 18.1, 19.6, 32.4,
58.8, 123.2, 128.0, 138.9, 149.8, 150.4, 166.2, 174.3. Anal.
(C11H14N2O3) C, H, N.
amide (13). Compound 13 was synthesized in 63% yield from
10 according to the method for the preparation of compound
12. 13: [R]22 -31.2 (c 0.7, MeOH); 1H NMR (CD3OD, 300
D
MHz) δ 0.90 (d, J ) 6.3 Hz, 3H), 0.94 (d, J ) 6.0 Hz, 3H), 1.36
(d, J ) 7.1 Hz, 3H), 1.56-1.74 (m, 3H), 2.50 (app. d, J ) 6.6
Hz, 2H), 3.01 (app. q, J ) 7.4 Hz, 1H), 3.57-3.66 (m, 1H),
3.69-3.78 (m, 2H), 4.10-4.18 (m, 1H), 4.32-4.41 (m, 1H), 4.54
(d, J ) 12.1 Hz, 1H), 4.59 (d, J ) 12.1 Hz, 1H), 7.24-7.39 (m,
5H); 13C NMR (CD3OD, 75.5 MHz) δ 17.7, 21.8, 23.5, 25.9, 41.1,
41.8, 50.8, 52.8, 66.1, 69.4, 71.3, 74.2,128.7, 128.8, 129.3, 129.4,
139.2, 173.5, 175.0, 177.4. Anal. (C21H32N6O5) C, H, N.
Pyridine-2-carboxylic Acid ((S)-1-{(1S,2S)-3-[(S)-1-((S)-
1-Carbamoyl-3-methylbutylcarbamoyl)ethylcarbamoyl]-
2-hydroxy-1-phenethylpropylcarbamoyl}-2-methylpro-
pyl)amide (14). Compound 12 (150 mg, 0.347 mmol) was
dissolved in 18 mL of MeOH. Triphenylphosphine (134 mg,
0.511 mmol) and three drops of water were added, and the
solution was stirred at room-temperature overnight. The
solvent was evaporated, and the crude material was run
through a short silica column using ethyl acetate/methanol 9:1
+ 1% TEA as eluent. The crude amine was dissolved in DMF
(10 mL) after which B1 (100 mg, 0.451 mmol) and DIEA (181
µL, 1.04 mmol) were added, and the temperature was lowered
to 0 °C. HATU (172 mg, 0.451 mmol) was added, and the
reaction was stirred for 30 min at 0 °C and then for an
additional 3 h at room temperature. The solvent was evapo-
rated, and the residual was extracted with chloroform and
washed with brine. The organic phase was dried over magne-
sium sulfate, filtered, and concentrated. The crude product was
purified by flash column chromatography (chloroform/ethanol
13:1 + 1.5% methanol saturated with NH3) yielding 14 (72
(S)-2-((S)-2-Aminopropanoylamino)-4-methylpentano-
ic Acid Amide (A1). (Benzyloxy)carbonyl-protected alanine
(Z-Ala-OH) (0.200 g, 0.90 mmol), leucine amide (H-Leu-NH2)
(0.123 g, 0.94 mmol), 1-hydroxy-7-azabenzotriazole (HOAt)
(0.122 g, 0.90 mmol), and (1,8-bis(N,N-dimethylamino)naph-
thalene (Proton sponge) (0.192 g, 0.90 mmol) were dissolved
in DMF (2 mL). The reaction mixture was cooled to 0 °C, and
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlo-
ride (EDC) (0.189 g, 0.99 mmol) was added. The mixture was
stirred at 0 °C for 2 h and then at room-temperature overnight.
The solvent was removed after which the crude mixture was
extracted with chloroform and washed with brine. The solvent
was evaporated, and the crude product was dissolved in
ethanol (95%, 15 mL). Palladium (10%) on active carbon (∼25
mg) and hydrogen (atmospheric pressure, with flushing) were
added, and the mixture was stirred for 90 min. The suspension
was filtered trough Celite followed by evaporation of the
ethanol, and the crude product was purified by flash column
chromatography (ethyl acetate/methanol 9:1 + 1% TEA)
mg, 34%) as a slightly yellow solid. 14: [R]22 -33.9 (c 0.3,
D
CHCl3); 1H NMR (CD3OD, 300 MHz) δ 0.89 (d, J ) 6.0 Hz,
3H), 0.93 (d, J ) 6.1 Hz, 3H), 1.09 (d, J ) 4.4 Hz, 3H), 1.11 (d,
J ) 4.4 Hz, 3H), 1.41 (d, J ) 7.4 Hz, 3H), 1.65-1.83 (m, 3H),
1.84-1.98 (m, 2H), 2.26-2.37 (m, 1H), 2.40 (app. d, J ) 7.1
Hz, 2H), 2.50-2.70 (m, 2H), 3.89-3.99 (m, 1H), 4.09-4.17 (m,
1H), 4.19 (q, J ) 7.4 Hz, 1H), 4.37-4.46 (m, 2H), 7.08-7.23
(m, 5H), 7.55-7.63 (m, 1H), 7.94-8.02 (m, 1H), 8.15 (d, J )
7.7 Hz, 1H), 8.67 (d, J ) 4.7 Hz, 1H); 13C NMR (CD3OD, 75.5
MHz) δ 17.6, 18.9, 20.3, 21.5, 23.6, 26.1, 32.1, 33.8, 34.9, 40.9,
41.0, 52.0, 53.0, 53.8, 61.2, 71.1, 123.3, 126.8, 128.1, 129.3,
129.4, 139.0, 143.1, 149.9, 150.5, 166.8, 173.8, 174.4, 175.7,
177.8. Anal. (C32H46N6O6) C, H, N.
providing A1 (181 mg, 100%) as a white powder. A1: [R]22
D
-10.2 (c 0.5, MeOH); 1H NMR (CD3OD, 300 MHz) δ 0.94 (d, J
) 6.3 Hz, 3H), 0.97 (d, J ) 6.3 Hz, 3H), 1.30 (d, J ) 6.9 Hz,
3H), 1.50-1.78 (m, 3H), 3.46 (q, J ) 6.9 Hz, 1H), 4.41 (dd, J
) 6.3, 8.5 Hz, 1H); 13C NMR (CD3OD, 75.5 MHz) δ 21.4, 22.0,
23.5, 25.9, 42.3, 51.5, 52.6, 177.5, 178.1. Anal. (C9H19N3O2‚
0.47MeOH) C, H, N.
Pyridine-2-carboxylic Acid ((S)-1-{(1S,2S)-1-Benzy-
loxymethyl-3-[(S)-1-((S)-1-carbamoyl-3-methylbutylcar-
bamoyl)ethylcarbamoyl]-2-hydroxypropylcarbamoyl}-2-
methylpropyl)amide15. Compound 15 was synthesized in
43% yield from 13 according to the method for the preparation
of compound 14. 15: [R]22 -20.4 (c 0.2, CHCl3); 1H NMR
D
(3S,4S)-4-Azido-3-hydroxy-6-phenylhexanoic Acid [(S)-
1-((S)-1-Carbamoyl-3-methylbutylcarbamoyl)ethyl]-
amide (12). Compound 5 (100 mg, 0.401 mmol) was dissolved
in DMF (3 mL) after which A1 (122 mg, 0.606 mmol) and DIEA
(210 µL, 1.21 mmol) were added, and the temperature was
lowered to 0 °C. HATU (161 mg, 0.423 mmol) was added, and
reaction was stirred for 30 min at 0 °C and then at room
temperature for 90 min. The solvent was evaporated, and the
residual was extracted with EtOAc and washed with brine (3
×). The aqueous phase was extracted with EtOAc, and the
combined organic phases were dried over magnesium sulfate,
filtered, and concentrated. The crude product was purified by
flash column chromatography (ethyl acetate/methanol 4:1 +
1% TEA) yielding 12 (172 mg, 99%) as a colorless solid. 12:
[R]22D -51.6 (c 0.4, MeOH); 1H NMR (CD3OD, 300 MHz) δ 0.91
(d, J ) 6.0 Hz, 3H), 0.95 (d, J ) 6.3 Hz, 3H), 1.36 (d, J ) 7.1
Hz, 3H), 1.55-1.75 (m, 3H), 1.86-1.99 (m, 2H), 2.43-2.55 (m,
2H), 2.66-2.83 (m, 2H), 3.18-3.27 (m, 1H), 4.04-4.12 (m, 1H),
4.29-4.43 (m, 2H), 7.14-7.31 (m, 5H); 13C NMR (CD3OD, 75.5
MHz) δ 17.7, 21.8, 23.5, 25.9, 33.3, 33.4, 41.2, 41.8, 50.8, 52.8,
66.6, 71.9, 127.1, 129.4, 129.5, 142.5, 173.8, 175.0 177.4. Anal.
(C21H32N6O4) C, H, N.
(CD3OD, 300 MHz) δ 0.87 (d, J ) 6.3 Hz, 3H), 0.90 (d, J ) 6.0
Hz, 3H), 1.03 (d, J ) 6.9 Hz, 3H), 1.05 (d, J ) 6.9 Hz, 3H),
1.59-1.82 (m, 3H), 2.22-2.35 (m, 1H), 2.42 (app. d, J ) 7.4
Hz, 2H), 3.63 (app. d, J ) 6.3 Hz, 2H), 4.14-4.30 (m, 3H),
4.35-4.51 (m, 4H), 7.16-7.32 (m, 5H), 7.54-7.61 (m, 1H),
7.93-8.03 (m, 1H), 8.13 (d, J ) 8.0 Hz, 1H), 8.66 (d, J ) 4.7
Hz, 1H); 13C NMR (CD3OD, 75.5 MHz) δ 17.6, 18.7, 20.1, 21.5,
23.6, 26.1, 32.2, 41.0, 41.1, 52.0, 53.1, 53.2, 61.1, 68.8, 71.1,
74.0, 123.3, 128.1, 128.5, 128.8, 129.2, 138.9, 139.5, 149.9,
150.4, 166.7, 173.9, 174.2, 175.7, 177.8. Anal. (C32H46N6O7) C,
H, N.
Pyridine-2-carboxylic Acid ((S)-1-{(1S,2S)-1-(4-Bro-
mobenzyloxymethyl)-3-[(S)-1-((S)-1-carbamoyl-3-methyl-
butylcarbamoyl)ethylcarbamoyl]-2-hydroxypropylcar-
bamoyl}-2-methylpropyl)amide (16). Synthesized according
to Procedure A. Purification by column chromatography using
chloroform/ethanol 13:1 + 1.5% methanol saturated with NH3
gave 16 in 20% yield. 16: 1H NMR (CD3OD, 300 MHz) δ 0.87
(d, J ) 6.3 Hz, 3H), 0.90 (d, J ) 6.1 Hz, 3H), 1.03 (d, J ) 4.4
Hz, 3H), 1.05 (d, J ) 4.4 Hz, 3H), 1.41 (d, J ) 7.4 Hz, 3H),
1.59-1.82 (m, 3H), 2.21-2.33 (m, 1H), 2.43 (app. d, J ) 7.4
Hz, 2H), 3.63 (app. d, J ) 6.9 Hz, 2H), 4.13-2.28 (m, 3H),
4.36-4.49 (m, 4H), 7.17 (d, J ) 8.5 Hz, 2H), 7.33 (d, J ) 8.5
Hz, 2H), 7.54-7.62 (m, 1H), 7.92-8.03 (m, 1H), 8.12 (d, J )
(3S,4S)-4-Azido-5-benzyloxy-3-hydroxypentanoic Acid
[(S)-1-((S)-1-Carbamoyl-3-methylbutylcarbamoyl)ethyl]-

Malaria Aspartyl Protease Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3363
7.7 Hz, 1H), 8.65 (d, J ) 4.7 Hz, 1H); HRMS calcd for C32H45-
BrN6O7Na (M + Na)+: 727.2431. Found: 727.2402.
1H), 8.62-8.69 (m, 1H); HRMS calcd for C31H39BrN5O5 (M +
H)+: 640.2136. Found: 640.2148.
Pyridine-2-carboxylic Acid {(S)-1-[(1S,2S)-1-(4-bro-
mobenzyloxymethyl)-2-hydroxy-3-(3-phenylpropylcar-
bamoyl)propylcarbamoyl]-2-methylpropyl}amide (17).
Synthesized according to Procedure A. Purification by column
chromatography was performed using chloroform + 1.5%
methanol saturated with NH3 giving 17 in 21% yield. 17: 1H
NMR (CD3OD, 300 MHz) δ 1.02 (d, J ) 6.9 Hz, 3H), 1.06 (d,
J ) 6.6 Hz, 3H), 1.76-1.89 (m, 2H), 2.19-2.31 (m, 1H), 2.33
(app. d, J ) 7.2 Hz, 2H), 2.65 (t, J ) 7.7 Hz, 2H), 3.21 (t, J )
7.0 Hz, 2H) 3.51-3.69 (m, 2H), 4.11-4.19 (m, 1H), 4.20-4.29
(m, 1H), 4.40-4.53 (m, 3H), 7.10-7.29 (m, 7H), 7.33 (d, J )
8.5 Hz, 2H), 7.52-7.61 (m, 1H), 7.91-8.01 (m, 1H), 8.09 (d, J
) 8.0 Hz, 1H), 8.65 (d, J ) 4.7 Hz, 1H); HRMS calcd for C32H39-
BrN4O5Na (M + Na)+: 661.2002. Found: 661.2003.
Pyridine-2-carboxylic Acid {(S)-1-[(1S,2S)-1-(4-Bro-
mobenzyloxymethyl)-3-(cyclohexylmethylcarbamoyl)-2-
hydroxypropylcarbamoyl]-2-methylpropyl}amide (18).
Synthesized according to Procedure A. Purification by column
chromatography was performed using chloroform + 1.5%
methanol saturated with NH3 giving 18 in 15% yield. 18: 1H
NMR (CDCl3, 300 MHz) δ 0.84-1.00 (m, 2H), 1.03 (d, J ) 7.1
Hz, 3H), 1.06 (d, J ) 7.2 Hz, 3H), 1.10-1.35 (m, 3H), 1.37-
1.53 (m, 1H), 1.59-1.79 (m, 6H), 2.22 (dd, J ) 4.1, 14.8 Hz,
1H), 2.32-2.49 (m, 2H), 3.09 (t, J ) 6.3 Hz, 2H), 3.61 (app. d,
J ) 5.5 Hz, 2H), 4.01-4.12 (m, 1H), 4.26-4.34 (m, 1H), 4.36-
4.50 (m, 3H), 6.14-6.24 (b, 1H), 6.55-6.64 (b, 1H), 7.13 (d, J
) 8.2 Hz, 2H), 7.40 (d, J ) 8.2 Hz, 2H), 7.43-7.52 (m, 1H),
7.82-7.91 (m, 1H), 8.15 (d, J ) 7.7 Hz, 1H), 8.48-8.54 (b, 1H),
8.59 (d, J ) 4.1 Hz, 1H); HRMS calcd for C30H41BrN4O5Na (M
+ Na)+: 639.2158. Found: 639.2178.
Pyridine-2-carboxylic Acid {(S)-1-[(1S,2S)-1-(4-Bro-
mobenzyloxymethyl)-2-hydroxy-3-(3-methoxypropylcar-
bamoyl)propylcarbamoyl]-2-methylpropyl}amide (19).
Synthesized according to Procedure A. Purification by col-
umn chromatography was performed using chloroform/ethanol
40:1 + 1% triethylamine giving 19 in 15% yield. 19: 1H NMR
(CD3OD, 300 MHz) δ 1.01 (d, J ) 6.8 Hz, 3H), 1.05 (d, J ) 6.9
Hz, 3H), 1.69-1.81 (m, 2H), 2.18-2.30 (m, 1H), 2.32 (app. d,
J ) 7.1 Hz, 2H), 3.25 (t, J ) 6.9 Hz, 2H), 3.30 (s, 3H), 3.43 (t,
J ) 6.3 Hz, 2H), 3.51-3.68 (m, 2H), 4.08-4.18 (m, 1H), 4.09-
4.25 (m, 1H), 4.39-4.53 (m, 3H), 7.19 (d, J ) 8.2 Hz, 2H), 7.34
(d, J ) 8.2 Hz, 2H), 7.52-7.61 (m, 1H), 7.92-8-02 (m, 1H),
8.09 (d, J ) 7.7 Hz, 1H), 8.65 (d, J ) 4.7 Hz, 1H); HRMS calcd
for C27H37BrN4O6Na (M + Na)+: 615.1794. Found: 615.1775.
Pyridine-2-carboxylic Acid {(S)-1-[(1S,2S)-1-(4-Bromo-
benzyloxymethyl)-2-hydroxy-3-[2-(3-methoxyphenyl)eth-
ylcarbamoyl]propylcarbamoyl}-2-methylpropyl)amide
(20). Synthesized according to Procedure A. Purification by
column chromatography was performed using chloroform +
1.5% methanol saturated with NH3 giving 20 in 21% yield.
20: 1H NMR (CD3OD, 300 MHz) δ 1.01 (d, J ) 6.6 Hz, 3H),
1.04 (d, J ) 6.9 Hz, 3H), 2.17-2.28 (m, 1H), 2.31 (app. d, J )
6.9 Hz, 2H), 2.78 (t, J ) 7.3 Hz, 2H), 3.41 (t, J ) 7.6 Hz, 2H),
3.51-3.67 (m, 2H), 3.77 (s, 3H), 4.09-4.17 (m, 1H), 4.18-4.27
(m, 1H), 4.40-4.50 (m, 3H), 6.71-6.83 (m, 3H), 7.13-7.22 (m,
1H), 7.19 (d, overlapped, 2H), 7.34 (d, J ) 8.5 Hz, 2H), 7.54-
7.60 (m, 1H), 7.92-8.01 (m, 1H), 8.08 (d, J ) 8.0 Hz, 1H), 8.65
(d, J ) 4.7 Hz, 1H); HRMS calcd for C32H39BrN4O6Na (M +
Na)+: 677.1951. Found: 677.1925.
Pyridine-2-carboxylic Acid {(S)-1-[(1S,2S)-1-(4-Bromo-
benzyloxymethyl)-3-cyclohexylcarbamoyl-2-hydroxypro-
pylcarbamoyl]-2-methylpropyl}amide (22). Synthesized
according to Procedure A. Purification by column chromatog-
raphy was performed using chloroform + 1% methanol satu-
rated with NH3 giving 22 in 20% yield. 22: 1H NMR (CD3OD,
300 MHz) δ 1.02 (d, J ) 6.9 Hz, 3H), 1.05 (d, J ) 6.9 Hz, 3H),
1.12-1.49 (m, 6H), 1.54-1.95 (m, 4H), 2.19-2.37 (m, 3H),
3.50-3.71 (m, 3H), 4.09-4.17 (m, 1H), 4.18-4.27 (m, 1H),
4.39-4.54 (m, 3H), 7.19 (d, J ) 8.5 Hz, 2H), 7.34 (d, J ) 8.5
Hz, 2H), 7.52-7.61 (m, 1H), 7.92-8.02 (m, 1H), 8.19 (d, J )
8.0 Hz, 1H), 8.66 (d, J ) 4.1 Hz, 1H); HRMS calcd for C29H39-
BrN4O5Na (M + Na)+: 625.2002. Found: 625.2014.
Pyridine-2-carboxylic Acid {(S)-1-[(1S,2S)-1-(4-Bro-
mobenzyloxymethyl)-3-(4-cyanobenzylcarbamoyl)-2-hy-
droxypropylcarbamoyl]-2-methylpropyl}amide (23). Syn-
thesized according to Procedure A. Purification was performed
by HPLC using methanol/H2O 4:1 + 0.1% TFA yielding 23 in
19%. 23: 1H NMR (CD3OD, 300 MHz) δ 1.00 (d, J ) 6.6 Hz,
3H), 1.01 (d, J ) 6.9 Hz, 3H), 2.11-2.24 (m, 1H), 2.41-2.54
(m, 2H), 3.53-3.69 (m, 2H), 4.14-4.24 (m, 1H), 4.26-4.35
(m, 1H), 4.40 (s, overlapped, 2H), 4.40-4.46 (m, overlapped,
1H), 4.47-4.53 (m, 2H), 7.26 (d, J ) 8.5 Hz, 2H), 7.42 (d,
J ) 8.4 Hz, 2H), 7.47 (d, J ) 8.5 Hz, 2H), 7.51-7.62 (m,
overlapped, 1H), 7.59 (d, overlapped, J ) 8.4 Hz, 2H), 7.89-
7.97 (m, 1H), 8.00 (d, J ) 8.0 Hz, 1H), 8.65 (d, J ) 4.4 Hz,
1H); HRMS calcd for C31H34BrN5O5Na (M + Na)+: 658.1641.
Found: 658.1642.
Pyridine-2-carboxylic Acid {(S)-1-[(1S,2S)-1-(4-Bro-
mobenzyloxymethyl)-3-butylcarbamoyl-2-hydroxypro-
pylcarbamoyl]-2-methylpropyl}amide (24). Synthesized
according to Procedure A. Purification by column chromatog-
raphy was performed using chloroform + 1.5% methanol
saturated with NH3 giving 24 in 19% yield. 24: 1H NMR (CD3-
OD, 300 MHz): δ 0.92 (t, J ) 7.3 Hz, 3H), 1.02 (d, J ) 6.6 Hz,
3H), 1.05 (d, J ) 6.9 Hz, 3H), 1.29-1.42 (m, 2H), 1.43-1.56
(m, 2H), 2.19-2.30 (m, 1H), 2.33 (app. d, J ) 7.4 Hz, 2H), 3.18
(t, J ) 7.0 Hz, 2H), 3.51-3.68 (m, 2H), 4.10-4.20 (m, 1H),
4.21-4.29 (m, 1H), 4.38-4.52 (m, 3H), 7.18 (d, J ) 8.5 Hz,
2H), 7.33 (d, J ) 8.5 Hz, 2H), 7.52-7.59 (m, 1H), 7.93-8.00
(m, 1H), 8.09 (d, J ) 7.7 Hz, 1H), 8.65 (d, J ) 4.1 Hz, 1H);
HRMS calcd for C27H37BrN4O5Na (M + Na)+: 599.1845.
Found: 599.1851.
Pyridine-2-carboxylic Acid ((R)-1-{(1S,2S)-1-(4-Bro-
mobenzyloxymethyl)-3-[(S)-1-((S)-1-carbamoyl-3-methyl-
butylcarbamoyl)ethylcarbamoyl]-2-hydroxypropylcar-
bamoyl}-2-methylpropyl)amide (25). Synthesized according
to Procedure A. Purification by column chromatography was
performed using chloroform/ethanol 13:1 + 1.5% methanol
saturated with NH3 giving 25 in 18% yield. 25: 1H NMR
(CD3OD, 300 MHz): δ 0.83 (d, J ) 6.3 Hz, 3H), 0.90 (d, J )
6.0 Hz, 3H), 0.96 (d, J ) 6.6 Hz, 3H), 1.01 (d, J ) 6.9 Hz, 3H),
1.17 (d, J ) 7.1 Hz, 3H), 1.59-1.85 (m, 3H), 2.10-2.29 (m,
1H), 2.47 (dd, J ) 5.5, 14.8 Hz, 1H), 2.64 (dd, J ) 9.3, 14.8
Hz, 1H), 3.59-3.73 (m, 3H), 4.04 (app. q, J ) 7.1 Hz, 1H),
4.18-4.32 (m, 2H), 4.33-4.52 (m, 4H), 7.24 (d, J ) 8.5 Hz,
2H), 7.46 (d, J ) 8.5 Hz, 2H), 7.54-7.62 (m, 1H), 7.94-8.03
(m, 1H), 8.12 (d, J ) 8.0 Hz, 1H), 8.64 (d, J ) 4.7 Hz, 1H);
HRMS calcd for C32H45BrN6O7Na (M + Na)+: 727.2431.
Found: 727.2437.
Pyridine-2-carboxylic Acid {(S)-1-[(1S,2S)-1-(4-Bro-
mobenzyloxymethyl)-2-hydroxy-3-(2-phenylaminoethyl-
carbamoyl)propylcarbamoyl]-2-methylpropyl}amide (21).
Synthesized according to Procedure A. Purification by column
chromatography was performed using chloroform + 1.5%
methanol saturated with NH3 giving 21 in 19% yield. 21: 1H
NMR (CD3OD, 300 MHz) δ 1.02 (d, J ) 6.9 Hz, 3H), 1.05 (d,
J ) 6.9 Hz, 3H), 2.19-2.30 (m, 1H), 2.31-2.37 (m, 2H), 3.25
(t, J ) 6.0 Hz, 2H), 3.32-3.51 (m, 2H), 3.53-3.68 (m, 2H),
4.11-4.19 (m, 1H), 4.20-4.27 (m, 1H), 4.38-4.49 (m, 3H), 6.60
(t, J ) 7.3 Hz, 1H), 6.66 (d, J ) 8.1 Hz, 2H), 7.08 (dd, J ) 7.3,
8.1 Hz, 2H), 7.18 (d, J ) 8.5 Hz, 2H), 7.34 (d, J ) 8.5 Hz, 2H),
7.52-7.60 (m, 1H), 7.91-8.00 (m, 1H), 8.09 (d, J ) 7.7 Hz,
N-{(1S,2S)-1-(4-Bromobenzyloxymethyl)-3-[(S)-1-((S)-
1-carbamoyl-3-methylbutylcarbamoyl)ethylcarbamoyl]-
2-hydroxypropyl}-3,4-dichlorobenzamide (26). Synthe-
sized according to Procedure A. Purification by column
chromatography was performed using ethyl acetate/methanol
9:1 + 1% triethylamine providing 26 in 42% yield. 26: 1H NMR
(CD3OD, 300 MHz): δ 0.89 (d, J ) 6.3 Hz, 6H), 1.37 (d, J )
7.4 Hz, 3H), 1.55-1.68 (m, 1H), 1.69-1.86 (m, 2H), 2.48 (dd,
overlapped, 1H), 2.53 (dd, overlapped, 1H), 3.75 (app. d, J )
7.4 Hz, 2H), 4.12-4.20 (m, 1H), 4.32-4.44 (m, 3H), 4.47 (d, J
) 12.2 Hz, 1H), 4.52 (d, J ) 12.2 Hz, 1H), 7.22 (d, J ) 8.5 Hz,
2H), 7.42 (d, J ) 8.5 Hz, 2H), 7.63 (d, J ) 8.4 Hz, 1H), 7.80 (d,

3364 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Johansson et al.
J ) 8.4 Hz, 1H), 8.06 (s, 1H); HRMS calcd for C28H35BrCl2N4O6-
Na (M + Na)+: 695.1015. Found: 695.1025.
(S)-2,3-Dihydro-1H-indole-2-carboxylic Acid {(1S,2S)-
1-(4-Bromobenzyloxymethyl)-3-[(S)-1-((S)-1-carbamoyl-
3-methylbutylcarbamoyl)ethylcarbamoyl]-2-hydroxy-
propyl}amide (32). Synthesized according to Procedure A.
Purification by column chromatography was performed using
chloroform/ethanol 9:1 + 2% methanol saturated with NH3
providing 32 in 31% yield. 32: 1H NMR (CD3OD, 300 MHz) δ
0.89 (d, J ) 6.6 Hz, 3H), 0.92 (d, J ) 6.3 Hz, 3H), 1.38 (d, J )
7.1 Hz, 3H), 1.54-1.78 (m, 3H), 2.47 (app. d, J ) 7.4 Hz, 2H),
2.97 (dd, J ) 8.5, 16.2 Hz, 1H), 3.46 (dd, J ) 10.9, 16.2 Hz,
1H), 3.58 (app d, J ) 6.6 Hz, 2H), 4.10-4.21 (m, 2H), 4.22-
4.32 (m, 1H), 4.33-4.46 (m, 4H), 6.71-6.78 (m, 2H), 7.01-
7.08 (m, 2H), 7.10 (d, J ) 8.5 Hz, 2H), 7.30 (d, J ) 8.5 Hz,
2H); HRMS calcd for C30H40BrN5O6Na (M + Na)+: 668.2060.
Found: 668.2059.
N-{(1S,2S)-1-(4-Bromobenzyloxymethyl)-2-hydroxy-3-
[2-(3-methoxyphenyl)ethylcarbamoyl]propyl}-2,4,6-tri-
fluorobenzamide (33). Synthesized according to Procedure
A. Purification by column chromatography was performed
using toluene/ethyl acetate 2:1 providing 33 in 24% yield. 33:
1H NMR (CDCl3, 300 MHz) δ 2.27 (dd, J ) 3.6, 15.1 Hz, 1H),
2.43 (dd, J ) 9.6, 15.1 Hz, 1H), 2.78 (t, J ) 7.0 Hz, 2H), 3.44-
3.57 (m, 2H), 3.69 (app. d, J ) 5.5 Hz, 2H), 3.78 (s, 3H), 4.19-
4.28 (m, 1H), 4.32-4.39 (m, 1H), 4.50 (app. s, 2H), 5.98-6.06
(b, 1H), 6.49-6.55 (b, 1H), 6.68-6.80 (m, 4H), 7.15-7.24 (m,
2H), 7.19 (d, overlapped, 2H), 7.46 (d, J ) 8.3 Hz, 2H); HRMS
calcd for C28H28BrF3N2O5Na (M + Na)+: 631.1031. Found:
631.1039.
N-{(1S,2S)-1-(4-Bromobenzyloxymethyl)-3-[(S)-1-((S)-
1-carbamoyl-3-methylbutylcarbamoyl)ethylcarbamoyl]-
2-hydroxypropyl}-2,4,6-trifluorobenzamide (27). Synthe-
sized according to Procedure A. Purification by column
chromatography was performed using ethyl acetate/methanol
9:1 + 1% triethylamine providing 27 in 29% yield. 27: 1H NMR
(CD3OD, 300 MHz) δ 0.84 (d, J ) 6.6 Hz, 3H), 0.85 (d, J ) 5.8
Hz, 3H), 1.37 (d, J ) 7.4 Hz, 3H), 1.51-1.77 (m, 3H), 2.52 (dd,
J ) 6.9, 15.1 Hz, 1H), 2.63 (dd, J ) 7.4, 15.1 Hz, 1H), 3.64-
3.77 (m, 2H), 4.21 (app. q, J ) 7.3 Hz, 1H), 4.30-4.46 (m, 3H),
4.49 (d, J ) 15.9 Hz, 1H), 4.54 (d, J ) 15.9 Hz, 1H), 6.95 (d,
J ) 7.7 Hz, 1H), 6.98 (d, J ) 7.7 Hz, 1H), 7.28 (d, J ) 8.5 Hz,
2H), 7.47 (d, J ) 8.5 Hz, 2H); HRMS calcd for C28H34BrF3N4O6-
Na (M + Na)+: 681.1512. Found: 681.1513.
(3S,4S)-5-(4-Bromobenzyloxy)-4-diphenylacetylami-
no-3-hydroxypentanoic Acid [(S)-1-((S)-1-Carbamoyl-3-
methylbutylcarbamoyl)ethyl]amide (28). Synthesized ac-
cording to Procedure A. Purification by column chromatogra-
phy was performed using ethyl acetate/methanol 9:1 + 1%
triethylamine providing 28 in 27% yield. 28: 1H NMR (CD3-
OD, 300 MHz) δ 0.78 (d, J ) 6.1 Hz, 3H), 0.83 (d, J ) 6.0 Hz,
3H), 1.29 (d, J ) 7.4 Hz, 3H), 1.54-1.71 (m, 3H), 2.38 (app. d,
J ) 7.4 Hz, 2H), 3.63 (d, J ) 6.6 Hz, 2H), 4.20-4.30 (m, 2H),
4.31-4.50 (m, 4H), 5.11 (s, 1H), 7.17 (d, J ) 8.5 Hz, 2H), 7.19-
7.38 (m, 10H), 7.42 (d, J ) 8.5 Hz, 2H); HRMS calcd for C35H43-
BrN4O6Na (M + Na)+: 717.2264. Found: 717.2267.
(3S,4S)-5-(4-Bromobenzyloxy)-4-(3,3-diphenylpropio-
nylamino)-3-hydroxypentanoic Acid [(S)-1-((S)-1-Car-
bamoyl-3-methylbutylcarbamoyl)ethyl]amide (29). Syn-
thesized according to Procedure A. Purification by column
chromatography was performed using ethyl acetate/methanol
9:1 + 1% triethylamine providing 29 in 25% yield. 29: 1H NMR
(CD3OD, 300 MHz) δ 0.90 (d, J ) 6.6 Hz, 3H), 0.95 (d, J ) 6.3
Hz, 3H), 1.34 (d, J ) 7.1 Hz, 3H), 1.55-1.79 (m, 3H), 2.05 (dd,
J ) 7.4, 14.8 Hz, 1H), 2.14 (dd, J ) 7.1, 14.8 Hz, 1H), 2.99
(dd, J ) 8.3, 14.2 Hz, 1H), 3.06 (dd, J ) 8.0, 14.2 Hz, 1H),
3.32 (dd, overlapped, 1H), 3.42 (dd, J ) 6.6, 9.9 Hz, 1H), 3.96-
4.05 (m, 1H), 4.10-4.22 (m, 2H), 4.32-4.43 (m, 3H), 4.54 (app.
t, J ) 8.1 Hz, 1H), 7.09-7.34 (m, 12 H), 7.47 (d, J ) 8.5 Hz,
2H); HRMS calcd for C36H45BrN4O6Na (M + Na)+: 731.2420.
Found: 731.2433.
(3S,4S)-5-(4-Bromobenzyloxy)-3-hydroxy-4-(2-thiophen-
2-ylacetylamino)pentanoic Acid [(S)-1-((S)-1-Carbamoyl-
3-methylbutylcarbamoyl)ethyl]amide (30). Synthesized
according to Procedure A. Purification by column chromatog-
raphy was performed using ethyl acetate/methanol 9:1 + 1%
triethylamine providing 30 in 29% yield. 30: 1H NMR
(CD3OD, 300 MHz) δ 0.83 (d, J ) 6.3 Hz, 3H), 0.84 (d, J ) 6.3
Hz, 3H), 1.36 (d, J ) 7.4 Hz, 3H), 1.53-1.74 (m, 3H), 2.41 (dd,
J ) 6.9, 14.8 Hz, 1H), 2.48 (dd, J ) 7.7, 14.8 Hz, 1H), 3.62
(app. d, J ) 6.3 Hz, 2H), 3.75-3.88 (m, 2H), 4.10-4.20 (m,
2H), 4.22-4.30 (m, 1H), 4.31-4.38 (m, 1H), 4.41 (d, J ) 12.0
Hz, 1H), 4.47 (d, J ) 12.0 Hz, 1H), 6.89-6.97 (m, 2H), 7.19 (d,
J ) 8.5 Hz, 2H), 7.25 (dd, J ) 1.4, 4.9 Hz, 1H), 7.44 (d, J )
8.5 Hz, 2H); HRMS calcd for C27H37BrN4O6SNa (M + Na)+:
647.1515. Found: 647.1487.
Benzo[1,3]dioxole-5-carboxylic Acid {(1S,2S)-1-(4-Bro-
mobenzyloxymethyl)-3-[(S)-1-((S)-1-carbamoyl-3-methyl-
butylcarbamoyl)ethylcarbamoyl]-2-hydroxypropyl}-
amide (31). Synthesized according to Procedure A. Purification
by column chromatography was performed using chloroform/
ethanol 13:1 + 1.5% methanol saturated with NH3 providing
31 in 10% yield. 31: 1H NMR (CD3OD, 300 MHz) δ 0.89 (d, J
) 6.6 Hz, 3H), 0.90 (d, J ) 6.6 Hz, 3H), 1.37 (d, J ) 7.2 Hz,
3H), 1.55-1.68 (m, 1H), 1.69-1.83 (m, 2H), 2.47 (dd, J ) 6.6,
15.0 Hz, 1H), 2.54 (dd, J ) 8.2, 15.0 Hz, 1H), 3.73 (app. d, J )
6.9 Hz, 2H), 4.16 (app. q, J ) 7.3 Hz, 1H), 4.33-4.44 (m, 3H),
4.47 (d, J ) 12.4 Hz, 1H), 4.52 (d, J ) 12.4 Hz, 1H), 6.04 (s,
2H), 6.89 (d, J ) 8.3 Hz, 1H), 7.24 (d, J ) 8.3 Hz, 2H), 7.38 (d,
J ) 1.7 Hz, 1H), 7.43 (d, J ) 8.2 Hz, 2H), 7.48 (dd, J ) 1.7,
8.3 Hz, 1H); HRMS calcd for C29H37BrN4O8Na (M + Na)+:
671.1693. Found: 671.1702.
N-[(1S,2S)-1-(4-Bromobenzyloxymethyl)-3-cyclohex-
ylcarbamoyl-2-hydroxypropyl]-2,4,6-trifluorobenz-
amide (34). Synthesized according to Procedure A. Purifica-
tion by column chromatography was performed using chloro-
form giving 34 in 27% yield. 34: 1H NMR (CDCl3, 300 MHz)
δ 1.04-1.21 (m, 3H), 1.23-1.43 (m, 2H), 1.54-1.78 (m, 3H),
1.81-1.98 (m, 2H), 2.28 (dd, J ) 3.6, 15.1 Hz, 1H), 2.45 (dd, J
) 9.6, 15.1 Hz, 1H), 3.65-3.80 (m, 3H), 4.22-4.31 (m, 1H),
4.32-4.41 (m, 1H), 4.50 (app. s, 2H), 5.90-5.99 (b, 1H), 6.49-
6.57 (b, 1H), 6.71 (d, J ) 8.1 Hz, 1H), 6.74 (d, J ) 8.1 Hz, 1H),
7.20 (d, J ) 8.4 Hz, 2H), 7.46 (d, J ) 8.4 Hz, 2H); HRMS calcd
for C25H28BrF3N2O4Na (M + Na)+: 579.1082. Found: 579.1089.
Acknowledgment. We gratefully thank Medivir
AB, the Swedish Foundation for Strategic Research
(SSF), and Knut and Alice Wallenberg’s Foundation for
financial support, and Medivir AB for performing the
biological testings.
Supporting Information Available: Combustion analy-
ses; HPLC purity measurements. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) (a) Murray, M. C.; Perkins, M. E. Chemotherapy of Malaria.
Annu. Rep. Med. Chem. 1996, 31, 141-150. (b) Radding, J. A.
Development of Anti-Malarial Inhibitors of Hemoglobinases.
Annu. Rep. Med. Chem. 1999, 34, 159-168. (c) Breman, J. G.
The Ears of the Hippopotamus: Manifestations, Determinants,
and Estimates of the Malaria Burden. Am. J. Trop. Med. Hyg.
2001, 64, 1-11. (d) Guerin, P. J.; Olliaro, P.; Nosten, F.; Druilhe,
P.; Laxminarayan, R.; Binka, F.; Kilama, W. L.; Ford, N.; White,
N. J. Lancet Infect. Dis. 2002, 2, 564-573. (e) Asojo, O. A.;
Gulnik, S. V.; Afonina, E.; Yu, B.; Ellman, J. A.; Haque, T. S.;
Silva, A. M. Novel Uncomplexed and Complexed Structures of
Plasmepsin II, an Aspartic Protease from Plasmodium falci-
parum. J. Mol. Biol. 2003, 327, 173-181. (f) Wyatt, D. M.; Berry,
C. Activity and inhibition of plasmepsin IV, a new aspartic
proteinase from the malaria parasite, Plasmodium falciparum.
FEBS Lett. 2002, 513, 159-162.
(2) Francis, S. E.; Sullivan, D. J., Jr.; Goldberg, D. E. Hemoglobin
Metabolism in the Malaria Parasite Plasmodium Falciparum.
Annu. Rev. Microbiol. 1997, 51, 97-123.
(3) Haque, T. S.; Skillman, A. G.; Lee, C. E.; Habashita, H.;
Gluzman, I. Y.; Ewing, T. J. A.; Goldberg, D. E.; Kuntz, I. D.;
Ellman, J. A. Potent, Low-Molecular-Weight Non-Peptide In-
hibitors of Malarial Aspartyl Protease Plasmepsin II. J. Med.
Chem. 1999, 42, 1428-1440.

Malaria Aspartyl Protease Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3365
(4) (a) Silva, A. M.; Lee, A. Y.; Gulnik, S. V.; Majer, P.; Collins, J.;
Bhat, T. N., Collins, P. J.,; Cachau, R. E.; Luker, K. E.; Gluzman,
I. Y.; Francis, S. E.; Oksman, A.; Goldberg, D. E.; Erickson, J.
W. Structure and Inhibition of Plasmepsin II, a Hemoglobin-
Degrading Enzyme from Plasmodium Falciparum. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 10034-10039. (b) Majer, P.; Collins,
J.; Gulnik, S.; Erickson, J.; Pan, W.; Eissenstat, M. A. World
Patent WO 97/30072.
(5) (a) Eggelson, K. K.; Duffin, K. L.; Goldberg, D. E. Identification
and Characterization of Falcilysin, a Metallopeptidase Involved
in Hemoglobin Catabolism within the Malaria Parasite Plas-
modium Falciparum. J. Biol. Chem. 1999, 274, 32411-32417.
(b) Coombs, G. H.; Goldberg, D. E.; Klemba, M.; Berry, C.; Kay,
J.; Mottram, J. C. Aspartic Proteases of Plasmodium Falciparum
and other Parasitic Protozoa as Drug Targets. Trends Parasitol.
2001, 17, 532-537.
(6) (a) Banerjee, R.; Francis, S. E.; Goldberg, D. E. Food vacuole
plasmepsins are processed at a conserved site by an acidic
convertase activity in Plasmodium falciparum. Mol. Biochem.
Parsitol. 2003, 129, 157-165. (b) Nezami, A.; Freire, E. The
integration of genomic and structural information in the devel-
opment of high affinity plasmepsin inhibitors. Int. J. Parasitol.
2002, 32, 1669-1676. (c) Nezami, A.; Kimura, T.; Hidaka, K.;
Kiso, A.; Liu, J.; Kiso, Y.; Goldberg, D. E.; Freire, E. High-
Affinity Inhibition of a Family of Plasmodium falciparum
Proteases by a Designed Adaptive Inhibitor. Biochemistry 2003,
42, 8459-8464.
(7) (a) Francis, S. E.; Gluzman, I. Y.; Oksman, A.; Knickerbocker,
A.; Mueller, R.; Bryant, M.; Sherman, D. R.; Rusell, D. G.;
Goldberg, D. E. Molecular Characterization and Inhibition of a
Plasmodium Falciparum Aspartic Hemoglobinase. Embo. J.
1994, 13, 306-317. (b) Rosenthal, P.; Olson, J. E.; Lee, K.;
Palmer, J. T. Antimalarial Effects of Vinyl Sulfone Cysteine
Proteinase Inhibitors. Antimicrob. Agents Chemother. 1996, 40,
1600-1603. (c) Rosenthal, P. J. Plasmodium Falciparum: Ef-
fects of Proteinase Inhibitors on Globin Hydrolysis by Cultured
Malaria Parasites. Exp. Parasitol. 1995, 80, 272-281. (d)
Rosenthal, P. J.; McKerow, J. H.; Rasnick, D.; Leech, J. H.
Plasmodium Falciparum: Inhibitors of Lysosomal Cysteine
Proteinases Inhibit a Trophozite Proteinase and Block Parasite
Development. Mol. Biochem. Parasitol. 1989, 35, 177-184. (e)
Rosenthal, P. J.; Wollish, W. S.; Palmer, J. T.; Rasnick, D.
Antimalarial Effects of Peptide Inhibitors of a Plasmodium
Falciparum Cysteine Proteinase. J. Clin. Invest. 1991, 88, 1467-
1472.
Plasmepsin I and Plasmepsin II Inhibitors with Activity in
Plasmodium falciparum-Infected Cultured Human Erythrocytes.
J. Med. Chem. 2003, 46, 734-746. (b) No¨teberg, D.; Schaal, W.;
Hamelink, E.; Vrang, L.; Larhed, M. High-Speed Optimization
of Inhibitors of the Malarial Proteases Plasmepsin I and II. J.
Comb. Chem. 2003, 5, 456-464. (c) Dahlgren, A.; Kvarnstro¨m,
I.; Vrang, L.; Hamelink, E.; Hallberg, A.; Rosenquist, Å.;
Samuelsson, B. Solid-Phase Library Synthesis of Reversed-
Statine Type Inhibitors of the Malarial Aspartyl Proteases
Plasmepsin I and II. Bioorg. Med. Chem. 2003, 11, 827-841.
(d) Dahlgren, A.; Kvarnstro¨m, I.; Vrang, L.; Hamelink, E.;
Hallberg, A.; Rosenquist, Å.; Samuelsson, B. New Inhibitors of
the Malaria Aspartyl Proteases Plasmepsin I and II. Bioorg.
Med. Chem. 2003, 11, 3423-3437. (e) Oscarsson, K.; Oscarson,
S.; Vrang, L.; Hamelink, E.; Hallberg, A.; Samuelsson, B. New
Potent C2-Symmetric Malaria Plasmepsin I and II Inhibitors.
Bioorg. Med Chem. 2003, 11, 1235-1246. (f) Ersmark, K.;
Feierberg, I.; Bjelic, S.; Hulte´n, J.; Samuelsson, B.; Åqvist, J.;
Hallberg, A. C2-Symmetric Inhibitors of Plasmodium falciparum
Plasmepsin II: Synthesis and Theoretical Predictions. Bioorg.
Med. Chem. 2003, 11, 3723-3733.
(11) Chulay, J. D.; Diggs, C. L. Plasmodium falciparum: assessment
of in vitro growth by [3H]hypoxanthine incorporation. Exp.
Parasitol. 1983, 55, 138-146.
(12) See for example: (a) Alterman, M.; Bjo¨rsne, M.; Mu¨hlman, A.;
Classon, B.; Kvarnstro¨m, I.; Danielson, H.; Markgren, P.-O.,
Nillroth, U.; Unge, T.; Hallberg, A.; Samuelsson, B. Design and
Synthesis of New Potent C2-Symmetric HIV-1 Protease Inhibi-
tors. Use of L-Mannaric Acid as a Peptidomimetic Scaffold. J.
Med. Chem. 1998, 41, 3782-3792. (b) Pyring, D.; Lindberg, J.;
Rosenquist, Å.; Zuccarello, G.; Kvarnstro¨m, I.; Zhang, H.; Vrang,
L.; Unge, T.; Classon, B.; Hallberg, A.; Samuelsson, B. Design
and Synthesis of Potent C2-Symmetric Diol-Based HIV-1 Pro-
tease Inhibitors: Effects of Fluoro Substitution. J. Med. Chem.
2001, 44, 3083-3091.
(13) Yanagisawa, H.; Kanazaki, T.; Nishi, T. Synthesis of Statine and
Its Analogues. Chemistry Lett. 1989, 687-690.
(14) (a) Arslan, T.; Abraham, A. T.; Hecht, S. M. Structurally Altered
Substrates for DNA Topoisomerase I. Effects of Inclusion of a
Single 3′-Deoxynucleotide within the Scissile Strand. Nucleosides
Nucleotides 1998, 17 (1-3), 515-530. (b) Rasmussen, J. R.;
Slinger, C. J.; Kordish, R. J.; Newman-Evans, D. D. Synthesis
of Deoxy Sugars. Deoxygenation by Treatment with N,N′-
Thiocarbonyldiimidazole/Tri-n-butylstannane. J. Org. Chem.
1981, 46, 4843-4846. (c) Rondot, B.; Durand, T.; Girard, J. P.;
Rossi, J. C.; Schio, L.; Khanapure, S. P.; Rokach, J. A Free
Radical Route to Syn Lactones and other Prostanoid Intermedi-
ates in Isoprostaglandin Synthesis. Tetrahedron Lett. 1993, 34,
8245-8248.
(8) Ersmark, K.; Feierberg, I.; Bjelic, S.; Hamelink, E.; Hackett, F.;
Blackman, M. J.; Hulte´n, J.; Samuelsson, B.; Åqvist, J.; Hallberg,
A. Potent Inhibitors of the Plasmodium falciparum Enzymes
Plasmepsin I and II Devoid of Cathepsin D Inhibitory Activity.
J. Med. Chem. 2004, 47, 110-122.
(9) (a) Moon, R. P.; Tyas, L.; Certa, U.; Rupp, K.; Bur, D.; Jacquet,
C.; Matile, H.; Loetscher, H.; Grueninger-Leitch, F.; Kay, J.;
Dunn, B. M.; Berry, C.; Ridley, R. G. Expression and Charac-
terisation of Plasmepsin I from Plasmodium falciparum. Eur.
J. Biochem. 1997, 244, 552-560. (b) Carroll, C. D.; Patel, H.;
Johnson, T. O.; Guo, T.; Orlowski, M.; He, Z.-M.; Cavallaro, C.
L.; Guo, J.; Oksman, A.; Gluzman, I. Y.; Connelly, J.; Chelsky,
D.; Goldberg, D. E.; Dolle, R. E. Identification of Potent Inhibi-
tors of Plasmodium falciparum Plasmepsin II from an Encoded
Statine Combinatorial Library. Bioorg. Med. Chem. Lett. 1998,
8, 2315-2320. (c) Carroll, C. D.; Johnson, T. O.; Tao, S.; Lauri,
G.; Orlowski, M.; Gluzman, I. Y.; Goldberg, D. E.; Dolle, R. E.
Evaluation of a Structure-Based Statine Cyclic Diamino Amide
Encoded Combinatorial Library Against Plasmepsin II and
Cathepsin D. Bioorg. Med. Chem. Lett. 1998, 8, 3203-3206. (d)
Dolle, R. E.; Guo, J.; O’Brien, L.; Jin, Y.; Piznik, M.; Bowman,
K. J.; Li, W.; Egan, W. J.; Cavallaro, C. L.; Roughton, A. L.; Zhao,
Q.; Reader, J. C.; Orlowski, M.; Jacob-Samuel, B.; Carroll, C. D.
A Statistical-Based Approach to Assessing the Fidelity of Com-
binatorial Libraries Encoded with Electrophoric Molecular Tags.
Development and Application of Tag Decode-Assisted Single
Bead LC/MS Analysis. J. Comb. Chem. 2000, 2, 716-731. (e)
Jiang, S.; Prigge, S. T.; Wei, L.; Gao, Y.-E.; Hudson, T. H.;
Gerena, L.; Dame, J. B.; Kyle, D. E. New Class of Small
Nonpeptidyl Compounds Blocks Plasmodium falciparum Devel-
opment In Vitro by Inhibiting Plasmepsins. Antimicrob. Agents
Chemother. 2001, 45, 2577-2584. (f) Nezami, A.; Luque, I.;
Kimura, T.; Kiso, Y.; Freire, E. Identification and Characteriza-
tion of Allophenylnorstatine-Based Inhibitors of Plasmepsin II,
an Antimalarial Target. Biochemistry 2002, 41, 2273-2280. (g)
Asojo, O. A.; Afonina, E.; Gulnik, S. V.; Yu, B.; Erickson, J. W.;
Randad, R.; Medjahed, D.; Silva, A. M. Structures of Ser205
Mutant Plasmepsin II from Plasmodium falciparum at 1.8 Å in
Complex with the Inhibitors rs367 and rs370. Acta Crystallogr.
2002, D58, 2001-2008.
(15) Robinson, P. L.; Barry, C. N.; Bass, S. W.; Jarvis, S. E.; Evans,
S. A., Jr. Regioselective Cyclodehydration of Chiral Diols with
Diethoxytriphenylphosphorane, Triphenylphosphine-Tetrachlo-
romethane-Potassium Carbonate, and Triphenylphosphine-Di-
ethyl Azodicarboxylate Reagents. A Comparative Study. J. Org.
Chem. 1983, 48, 5396-5398.
(16) Rauter, A. P.; Figueiredo, J.; Ismael, M.; Canda, T.; Font, J.;
Figueredo, M. Efficient synthesis of R,â-unsaturated γ-lactones
linked to sugars. Tetrahedron: Asymmetry, 2001, 12, 1131-
1146.
(17) Brånalt, J.; Kvarnstro¨m, I.; Svensson, S. C. T.; Classon, B.;
Samuelsson, B. A New Synthesis of 4-Thiofuranosides via
Regioselective Opening of an Episulfide with Allylmagnesium
Bromide. J. Org. Chem. 1994, 59, 4430-4432.
(18) Zuccarello, G.; Abderrahim, B.; Kvarnstro¨m, I.; Niklasson, G.;
Svensson, S. C. T.; Brisander, M.; Danielsson, H.; Nillroth, U.;
Karle´n, A.; Hallberg, A.; Classon, B.; Samuelsson, B. HIV-1
Protease Inhibitors Based on Acyclic Carbohydrates. J. Org.
Chem. 1998, 63, 4898-4906.
(19) Mu¨ller, M.; Huchel, U.; Geyer, A.; Schmidt, R. R. Efficient
Intramolecular Glycosylation Supported by a Rigid Spacer. J.
Org. Chem. 1999, 64, 6190-6201.
(20) Polglase, W. J.; Smith, E. L. Some Peptides and Peptide
Derivatives Containing Leucine and Alanine. J. Am. Chem. Soc.
1949, 71, 3081-3084.
(21) Mehta, A.; Jaouhari, R.; Benson, T. J.; Douglas, K. T. Improved
Efficiency and Selectivity in Peptide Synthesis: Use of Trieth-
ylsilane as a Carbocation Scavenger in Deprotection of tert-Butyl
Esters and t-Butoxycarbonyl-Protected Sites. Tetrahedron Lett.
1992, 33, 5441-5444.
(22) (a) Novabiochem Catalog and Peptide Synthesis Handbook 1997/
1998, method 16. (b) MilliGen Technical Note 3.10.
(23) Thompson, L. A.; Ellman, J. A. Straightforward and General
Method for Coupling Alcohols to Solid Supports. Tetrahedron
Lett. 1994, 35, 9333-9336.
(10) (a) No¨teberg, D.; Hamelink, E.; Hulten, J.; Wahlgren, M.; Vrang,
L.; Samuelsson, B.; Hallberg, A. Design and Synthesis of

3366 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Johansson et al.
(24) Bartra, M.; Romea, P.; Urpf, F.; Vilarrasa, J. A Fast Procedure
for the Reduction of Azides and Nitro Compounds Based on the
Reducing Ability of Sn(SR)3-Species. Tetrahedron 1990, 46,
587-594.
(25) Saftig, P.; Hetman, M.; Schmahl, W.; Weber, K.; Heine, L.;
Mossmann, H.; Koster, A.; Hess, B.; Evers, M.; Figura, K. von.
Mice deficient for the lysosomal proteinase cathepsin D exhibit
progressive atrophy of the intestinal mucosa and profound
destruction of lymphoid cells. EMBO J. 1995, 14, 3599-3608.
(26) Manuscript in preparation.
(27) Enzymes: A Practical Introduction to Structure, Mechanism, and
Data Analysis, 2nd ed.; Copeland, R. A., Ed.; John Wiley &
Sons: New York, 2000.
(28) Cheng, Y.; Prusoff, W. H. Relationship between the Inhibition
Constant (Ki) and the Concentration of Inhibitor which Causes
50% Inhibition (IC50) of an Enzymatic Reaction. Biochem.
Pharmacol. 1973, 22, 3099-3108.
(29) Blackman, M. J. Methods Cell Biol. 1994, 45, 213-220.
JM031106I