Synthesis of new (-)-bestatin-based inhibitor libraries reveals a novel binding mode in the S1 pocket of the essential malaria M1 metalloaminopeptidase

Article abstract of DOI:10.1021/jm101227t

The malarial PfA-M1 metallo-aminopeptidase is considered a putative drug target. The natural product dipeptide mimetic, bestatin, is a potent inhibitor of PfA-M1. Herein we present a new, efficient, and high-yielding protocol for the synthesis of bestatin derivatives from natural and unnatural N-Boc-d-amino acids. A diverse library of bestatin derivatives was synthesized with variants at the side chain of either the α-hydroxyβ-amino acid (P1) or the adjacent naturalα-amino acid (P1′). Surprisingly, we found that extended aromatic side chains at the P1 position resulted in potent inhibition against PfA-M1. To understand these data, we determined the X-ray cocrystal structures of PfA-M1 with two derivatives having either a Tyr(OMe) 15 or Tyr(OBzl) 16 at the P1 position and observed substantial inhibitor-induced rearrangement of the primary loop within the PfA-M1 pocket that interacts with the P1 side chain. Our data provide important insights for the rational design of more potent and selective inhibitors of this enzyme that may eventually lead to new therapies for malaria.

Full text of DOI:10.1021/jm101227t

ARTICLE
pubs.acs.org/jmc
Synthesis of New (-)-Bestatin-Based Inhibitor Libraries Reveals a
Novel Binding Mode in the S1 Pocket of the Essential Malaria M1
Metalloaminopeptidase†
Geetha Velmourougane,‡ Michael B. Harbut,‡ Seema Dalal,§ Sheena McGowan,|| Christine A. Oellig,||
Nataline Meinhardt,‡ James C. Whisstock,|| Michael Klemba,§ and Doron C. Greenbaum*,‡
‡Department of Pharmacology, University of Pennsylvania, 433 South University Avenue, 304G Lynch Laboratories, Philadelphia,
Pennsylvania 19104-6018, United States
§Department of Biochemistry, Virginia Polytechnic Institute and State University, 306 Engel Hall, Blacksburg, Virginia 24061,
United States
Department of Biochemistry and Molecular Biology and Australian Research Council Centre of Excellence in Structural and Functional
Microbial Genomics, Monash University, Clayton, VIC 3800, Australia
S Supporting Information
b
ABSTRACT: The malarial PfA-M1 metallo-
aminopeptidase is considered a putative drug
target. The natural product dipeptide mimetic,
bestatin, is a potent inhibitor of PfA-M1.
Herein we present a new, efficient, and high-
yielding protocol for the synthesis of bestatin
derivatives from natural and unnatural N-Boc-
D-amino acids. A diverse library of bestatin derivatives was synthesized with variants at the side chain of either the R-hydroxy-β-amino acid
(P1) or the adjacent natural R-amino acid (P10). Surprisingly, we found that extended aromatic side chains at the P1 position resulted in
potent inhibition against PfA-M1. To understand these data, we determined the X-ray cocrystal structures of PfA-M1 with two derivatives
having either a Tyr(OMe) 15 or Tyr(OBzl) 16 at the P1 position and observed substantial inhibitor-induced rearrangement of the
primary loop within the PfA-M1 pocket that interacts with the P1 side chain. Our data provide important insights for the rational design of
more potent and selective inhibitors of this enzyme that may eventually lead to new therapies for malaria.
’ INTRODUCTION
enzyme and a potential therapeutic target.6 The P. falciparum PfA-M1
enzyme shares low percentage identity (26%) to its human ortholo-
gue, aminopeptidase N, highlighting that targeting this essential
parasite peptidase over human enzymes may be possible. It is
noteworthy that there are some conserved amino acids around the
active site, especially those that comprise and surround the HEXXH
Zn binding site motif, however, the amino acids comprising the
binding pockets show significant diversity.
MAPs catalyze cleavage on the amino terminal of peptide chains.
These enzymes are widely distributed in organisms from bacteria to
human and play essential roles in protein maturation and regulation
of the metabolism of bioactive peptides.7-9 Although the MAP
superfamily is quite large and divergent, most enzymes share a
common mechanism, exploiting the coordination of one or two Zn
atoms in the active site to activate water for nucleophilic attack of a
peptide or protein substrate. Inhibitors of this superfamily therefore
tend to incorporate a Zn-binding group into a peptide-like structure.
There have been several inhibitor scaffolds used to target the MAP
family including, most prominently, phosphinic acids,10 hydroxamic
The human parasitic pathogen Plasmodium falciparum accounts
for most of the 1-2 million malaria related deaths per year.1 This
parasite has a complex life cycle involving several stages of growth,
including a mosquito vector stage and a mammalian stage. The
human asexual erythrocytic phase (blood stage) is the cause of
malaria-associated pathology. The blood stage is a recurring lytic
cycle lasting 48 h, wherein vast amounts of host hemoglobin are
taken up by the parasite for mostly metabolic purposes.2 The roles of
many endopeptidases, such as the digestive vacuole plasmepsins and
falcipains, have been well established, however, these endopepti-
dases appear to be largely redundant.3 Interest has recently increased
in understanding the roles of aminopeptidases, several of which are
genetically essential and may contribute to several biological
processes, including the breakdown of hemoglobin and the removal
of the terminal methionine from newly synthesized proteins.
Among the 100 peptidases in the P. falciparum genome, there are
eight metallo-aminopeptidases (MAPs). Four of these are methio-
nine aminopeptidases that presumably have a housekeeping role
in the removal of the initiator methionine from newly synthesized
polypeptides.4,5 Of the remaining four peptidases, PfA-M1
(aminopeptidase N; M1 family) is thought to be an essential parasite
Received: September 21, 2010
Published: March 02, 2011
r
2011 American Chemical Society
1655
dx.doi.org/10.1021/jm101227t J. Med. Chem. 2011, 54, 1655–1666
|

Journal of Medicinal Chemistry
ARTICLE
Figure 1. Schematic of bestatin binding to the active site of PfA-M1. S1 and S10 represent canonical peptidase binding pockets. Residues that comprise
the S1 and S10 pockets are listed alongside the half moon circle. PfA-M1 side chains are represented by numbered amino acids. Solid lines = metallo-
bonds; dashed lines = hydrogen bonds; CA = R-carbon. Black spheres represent carbon atoms.
acids,11 and the bestatin family.12,13 Both phosphinic acids and
hydroxamic acids have the capacity to inhibit metallo-endopepti-
dases and peptide deformylase and therefore will be intrinsically
more difficult to design in specificity for a target MAP. Thus, we
focused on the bestatin scaffold because it is synthetically tractable,
potent, and specific for the MAP family.
or more MAP glutamate residues coordinate the free amine of
bestatin, as highlighted in a recent structure for Escherichia coli
aminopeptidase N.17 A recent cocrystal structure of bestatin
bound to malaria PfA-M1 enzyme revealed a similar set of
interactions between the enzyme and the inhibitor (Figure 1).20
Bestatin interacts with MAPs in a noncovalent, reversible manner,
and in some cases is a slow-binding inhibitor, with initial formation
of a low-affinity complex followed by a slow conformational
change leading to a high affinity complex.12,13
We previously developed a solid-phase strategy that allows for
the diversification of the P10 position of bestatin.21 Herein we seek
to expand on this previous work by the development of a facile and
general method for the preparation of bestatin analogues that are
diversified both at the leucine (P10) and at the R-hydroxy-β-amino
acid position (P1). Wedevelop a small, directedinhibitor libraryto
explore potency determinants at the S1 and the S10 pocket against
the essential malaria M1-family aminopeptidase, PfA-M1, via
kinetic and structural analysis and live parasite assays.
(-)-Bestatin is a natural product of actinomycetes that potently
inhibits multiple families of metallo-aminopeptidases (MAPs), in-
cluding the M1, M17, and M18 families.14-17 Bestatin has been
shown to modulate many biological pathways; importantly, bestatin
has been shown to inhibit growth of P. falciparum parasites in
culture.18 However, bestatin type inhibitors have not been exploited
widely for construction of inhibitor libraries and have not been
developed for use as activity-based probes. Moreover, few if any
specific inhibitors have been designed for MAPs from any organism
regardless of scaffold.19 We therefore aimed to diversify the main
binding determinants of bestatin in order to expand the repertoire of
tools with which to study MAPs. We specifically aimed to explore
structure-activity relationships with PfA-M1 as a first step toward
optimizing the potency of bestatin-like inhibitors against this enzyme.
Bestatin resembles a Phe-Leu dipeptide substrate; however, the
first residue contains a R-hydroxy-β-amino acid. Bestatin-based
inhibitors coordinate the active zinc atom of MAPs and also form
interactions using the side chains of both the R-hydroxy-β-amino
acid and the adjacent alpha amino acid which bind into the S1 and
S10 active site pockets of the target enzyme, respectively. Further-
more, the amide between the two amino acids of bestatin forms
hydrogen bonds with the enzyme backbone and in some cases one
’ RESULTS
Chemistry. Stemming from the previous work, we wanted to
design a simple solid-phase strategy for the synthesis of (-)-
bestatin and its P1/P10 derivatives for library generation
(Schemes 1,3). We decided to utilize a Boc protection strategy
at the amino group for cleavage from Wang resin, which allows
the generation of the final product in one pot.
Construction of P10 Bestatin Library. Our initial aim was to
construct a diverse set of compounds by changing the amino acid
1656
dx.doi.org/10.1021/jm101227t |J. Med. Chem. 2011, 54, 1655–1666

Journal of Medicinal Chemistry
ARTICLE
Scheme 1. Synthesis of Bestatin P10 Derivatives
at the P10 position of bestatin. Accordingly, a library of P10 bestatin
derivatives were synthesized employing solid-phase peptide synth-
esis asdepicted in Scheme 1. We chose to use Wangresin to obtain
the product with a C-terminal carboxylate. Norleucine, lysine(Z),
p-benzyloxy phenyl alanyl, 2-naphthyl alanyl, and biphenyl alanyl
amino acids were coupled to the Wang resin using HATU, DIEA,
and catalytic DMAP. Fmoc cleavage was achieved using 2%DBU
þ 2%DIEA in DMF for 20 min. Coupling of the R-hydroxy-β-
amino acid required HATU in DIEA for 1 h. Cleavage of the resin
using 95%TFA:2.5%TIS:2.5%H2O yielded the bestatin analogues
1-6 in 60-70% yields.
bis(trimethylsilyl)amide (KHMDS) as a base to give the olefin
10a-h.41 Dihydroxylation of the alkene 10a-h with osmium
tetroxide and N-methylmorpholine N-oxide gave a diastereo-
meric mixture of the diol 11a-h.42 We explored the usage of
AD-mix-R and methane sulfonamide in t-BuOH:H2O (1:1) for
dihydroxylation reaction, but it did not significantly enhance the
selectivity. The diastereomeric ratios of the diols obtained (2S,3R
and 2R,3R) when AD-mix-R was used as the dihydroxylating
agent is reported in Supporting Information Table 1. The crude
diol 11a-h was selectively oxidized at the primary hydroxyl
functionality to the R-hydroxy-β-amino acid 12:13 using TEM-
PO, NaClO2, and NaOCl in NaH2PO4.43,44 Surprisingly, the
secondary alcohol remained intact under the oxidation condi-
tions (Scheme 2). Given prior published data that the (2S,3R)
diastereomers of bestatin are much more inhibitory than the
corresponding (2R,3R) diastereomer,45,46 the active (2S,3R)
diastereomers 13a-h were purified by reverse phase HPLC.
The 1H NMR spectra of the 13a-h (2S,3R) diastereomers had
the -NH (doublet) and -Boc (singlet) protons shift at similar
ppm in all the examples under study in comparison to the phenyl
derivative which is well studied (except for the diphenyl and n-
propyl, where one diastereomer was exclusively formed). A
detailed study showing the shift of -NH and -Boc protons of
the R-hydroxy-β-amino acids (individual diastereomers) is listed
in Supporting Information Table 1. This synthesis is a straight-
forward method for the preparation of R-hydroxy-β-amino acids
from R-D-amino acids with good yields.
Construction of P1 Bestatin Library. The R-hydroxy-β-amino
acids 13a-h were converted to bestatin P1 derivatives via
coupling with preloaded Fmoc-Leu Wang resin using HATU
and DIEA. Cleavage of the Fmoc group was achieved using 20%
piperidine in DMF. Global deprotection of the Boc group and
cleavage from Wang resin was accomplished using 95%
TFA:2.5%TIS:2.5%H2O, which gave bestatin 1 and its P1
derivatives 14-20 in good yields ranging from 60% to 70%
(Scheme 3). Prior work demonstrated that the PfA-M1 MAP had
broad specificity,47 however, published structural data with
bestatin demonstrated that the S1 pocket was lined with hydro-
phobic residues packing well around the P1 Phe side chain
(Figure.1).46 Using this information, we synthesized bestatin deri-
vatives using a series of R-hydroxy-β-amino acids consisting of
aliphatic and aromatic side chains in increasing size and rigidity.
To make use of these new R-hydroxy-β-amino acids, we synthesized
a library on preloaded Wang resin wherein the second position (P10)
was fixed to a leucine, the amino acid yielding the most potent activity
against PfA-M1 at this position. Ultimately, we developed a small
library to probe the structure-activity relationships of this P1
Synthetic Strategy for R-Hydroxy-β-amino Acids for P1
Bestatin Library. After successful completion of the P10 library,
we were interested in exploring specificity determinants at the P1
amino acid side chain, which requires the synthesis of R-hydroxy-β-
amino acids. There have been several noteworthy routes reported for
the synthesis of R-hydroxy-β-amino acids, all with limitations,
including incompatibility with Boc solid-phase chemistry, which led
us to develop a more practical approach. Previous routes include: (i)
chiron approaches starting from amino acids22-25 or sugars,26-29 (ii)
asymmetric catalysis,30,31 (iii) amino hydroxylation,32,33 (iv) selective
reduction of ketones,34 and (v) chemoenzymolysis.35,36 The Passerini
approach37 (a one-pot, three-component reaction) for the synthesis
of (-)-Bestatin gave a 1.5:1 mixture of diastereomers at the hydroxy
center but is limited by difficulties in producing isocyanides in high
yields and is severely limited in side chain options. Homologation of
R-amino aldehydes via cyanohydrin is not feasible with a Boc
protection at the amine.38 Last, a majority of the methods reported
utilized expensive chiral catalysts, had lengthy preparations due to
many reaction steps, were low yielding, and were not feasible with a
Boc protection on the amino group. Herein we report a simple,
efficient, and high yielding approach for the synthesis of the R-
hydroxy-β-amino acid for bestatin and its P1 derivatives (Scheme 2).
The key intermediate R-hydroxy-β-amino acid 13a-h was
prepared in five steps from commercially available D-amino
acid 7. The transformation began with the conversion of N-Boc-
D-amino acids 7a-h into Weinreb amides 8a-h using N-methyl
morpholine, EDAC HCl, and N,O-dimethyl hydroxyl amine
3
hydrochloride in dichloromethane.39 The use of a Weinreb
amide is advantageous in that it readily gives an aldehyde upon
treatment with a reducing agent without loss of optical purity.
Reduction of the Weinreb amide 8a-h using lithium aluminum
hydride in ether at -33 °C yielded the aldehyde 9a-h.40 Wittig
methylenation of R-amino aldehydes using n-butyllithium and
methyltriphenyl phosphonium bromide in tetrahydrofuran gave
a complex mixture of products. We employed the olefination
reaction under salt-free Wittig conditions using potassium
1657
dx.doi.org/10.1021/jm101227t |J. Med. Chem. 2011, 54, 1655–1666

Journal of Medicinal Chemistry
ARTICLE
Scheme 2. Synthesis of R Hydroxy β Amino Acids
Scheme 3. Synthesis of Bestatin and Its P1 Derivatives
bestatin side chain with the S1 binding pocket of the target enzyme
PfA-M1.
inhibitors inactive, which may indicate that the lack of flexibility of
these side chains caused an unavoidable steric clash with the target
enzyme. However, more flexible large aromatic side chains, such as
Tyr(OBzl) (16), were quite potent against the enzyme, which is
unexpected given that the structure of PfA-M1 and homologous
enzymes suggested that the S1 pocket would be unlikely to
accommodate side chains much larger than phenylalanine.20 Last,
basic substituents such as lysine were also inactive (not shown),
which is surprising given that P1 lysine-containing peptide sub-
strates are processed by PfA-M1 because there is a glutamate at the
top of the S1 pocket.20 However, discrepancies between peptide
substrates and bestatin analogues are not surprising, as bestatin
contains a R-hydroxy-β-amino acid and thus presents the P1 side
chain in a different manner than do peptide substrates.17
All P1 inhibitors were then tested against live cultures of
P. falciparum. The most potent inhibitors found in the in vitro
studies also showed moderate potency against P. falciparum in
culture with IC50 values in the micromolar range (Table 2). The
most potent compound Tyr(OMe) (15) against the enzyme was
found to have an IC50 around 6 μM against parasites, which was
Biological Results. P10 and P1 Bestatin Library Screening. To
explore interactions between the side chains at the P1 and P10 sites on
the bestatin scaffold with the enzyme, a series of focused inhibitor
libraries were synthesized using natural and non-natural aliphatic and
aromatic side chains of differing length and rigidity (Schemes 1-3).
The P10 library was first tested in vitro against recombinant PfA-M1
and showed a trend that favored small hydrophobic residues at this
site, with leucine displaying the most potent activity (Table 1).
The P1 bestatin library was constructed with a P0 leucine, which
yielded the most potent activity against PfA-M1. All inhibitors were
again tested in vitro against recombinant PfA-M1. From this data, it
was observed that aliphatics, such as a leucine or norleucine, yielded
poor inhibitors against the enzyme unlike peptide substrates
(Table 2).47 Interestingly, some larger aromatic substitutions at
P1, such as Tyr(OBzl) (16) or Tyr(OMe) (15), were found to be
potent. In fact, 15 was observed to be 5-fold more potent than the
parental bestatin with a Ki of 43 nM. Larger rigid aromatic
structures, such as naphthyl or biphenyl substituents, rendered
1658
dx.doi.org/10.1021/jm101227t |J. Med. Chem. 2011, 54, 1655–1666

Journal of Medicinal Chemistry
ARTICLE
Table 1. Potency of Bestatin P10 Derivatives against
Recombinant PfA-M1a
Table 2. Potency of Bestatin P1 Derivatives against
Recombinant PfA-M1 and P. falciparuma
Ki (nM)
IC50 (μM)
Ki (nM) PfA-M1
PfA-M1
live parasites
isobutyl (bestatin)
n-butyl
1
2
3
4
5
6
190
900
p-methoxy phenyl
phenyl (bestatin)
p-benzyloxy phenyl
2-naphthyl
15
1
43
190
490
3000
5300
NI
6.4
3.2
4-benzyloxycarbonyl amino butyl
p-benzyloxyphenyl alanyl
2-naphthyl alanyl
1450
16
17
20
14
18
19
29
>15000
>15000
>15000
>50
>50
>50
>50
>50
n-propyl
biphenyl alanyl
isopropyl
a Each Ki value as determined from assays at 8 inhibitor concentrations
spanning the range 0.014-30 μM (except bestatin; see legend to
Table 2).
biphenyl
NI
gem-diphenyl
NI
a Each Ki value was determined from assays conducted at four substrate
concentrations, each with six concentrations of inhibitor. Duplicate
determinations were carried out for compounds 1, 15, and 16 and in all
cases the replicates were within 12% of the mean. NI = no inhibition
slightly less potent than bestatin, perhaps due to the lowered
permeability caused by the increased hydrophilicity with the
introduction of the methoxy group. In addition, another
Plasmodium aminopeptidase, Pf-LAP, is known to be targeted
by bestatin, which could account for the difference between IC50
values and in vitro data.
in the bestatin bound structure, with the R-hydroxy group and
neighboring carbonyl coordinating the catalytic Zn2þ. Accessible
surface area calculations revealed that inhibitor 16 also buried a
larger surface area (83.7 Å2) in comparison to 15 (65.5 Å2) and
bestatin (23.8 Å2, Figure 2B).20 These accessible surface area
maps highlight the significant movement in the S1 pocket of the
enzyme to accommodate the larger unnatural side chains.
Structural Analysis of Bestatin Derivatives 15 and 16 Bound
to PfA-M1. The X-ray crystal structure of PfA-M1 bound to
bestatin revealed that the S1 subsite in the enzyme accommodated
a P1 phenylalanine well with tight packing of enzyme side chains
around the phenylalanine side chain.20 The S1 pocket is lined
by hydrophobic residues (Val459, Tyr575, Met1034) and a single
polar residue (Glu572). Residues 570-575 (including Glu572 and
Tyr575) are located on a loop central to the active site of the
enzyme. Comparison of the unbound versus the bestatin-bound
PfA-M1 complex revealed that the side chain of only one of the
residues (Met1034) that lines the PfA-M1 S1 pocket, moved slightly
upon ligand binding. It was thus somewhat surprising that substitu-
tion of the bestatin P1 Phe position with larger groups, Tyr(OMe)
15 or Tyr(OBzl) 16, which we would predict would sterically clash
with the enzyme, resulted in molecules with good inhibitory activity.
To address this question, we determined the 1.8 Å X-ray
crystal structures of PfA-M1 bound to compounds 15 and 16
(Figure 2). Comparison of these cocrystal structures with that of
bestatin-bound PfA-M1 revealed that the S1 pocket of the
enzyme is capable of undergoing a major conformational change
in response to binding a larger P1 moiety (Figure 2A). Remark-
ably, when the PfA-M1 enzyme was inhibited by Tyr(OBzl) 16,
the CR atom of Glu572 moved by ∼5 Å, respectively. In fact, this
loop switched to an entirely different conformation with the CR
atom of the neighboring residue, Phe574, moving by ∼1.6 Å.
With the smaller 15 compound (Tyr(OMe), this loop move-
ment was less pronounced with the CR atom of Glu572 moving
only 1.2 Å to accommodate the inhibitor. Of note, however, in
the latter structure, no electron density was observed for the side
chain of Glu572, suggesting a highly mobile side chain. Two of
the surrounding residues of the loop (Met571 and Asn573)
retain the same conformation as the bestatin-bound enzyme,
however, Phe574 also shifts ∼1 Å. In contrast, the S10 pocket and
at the Zn2þ atom the interactions are unchanged from that seen
’ DISCUSSION
We anticipate that the strategy presented herein for the synthesis
of P1 derivatives of bestatin will provide a practical method for a
mixed solution and solid-phase route to synthesize novel bestatin-
based inhibitors, which will be useful for inhibition of variety of
MAPs. During the synthesis, it was surprising that we did not observe
better stereoselectivity using the Sharpless AD-mix-R reagent.
Because the (2S,3R) diastereomer was required for good inhibition
of PfA-M1, it would be useful to optimize this step in the future. The
lack of AD-mix-R stereoselectivity may be due to the steric bulkiness
of the R group and the fact that the olefin was terminal.
Screening of the inhibitor libraries revealed that the S1
pocket accommodated some large aromatic side chains well;
the (OMe)Tyr derivative (15) was the most potent inhibitor,
however, the enzyme was able to accommodate a (OBzl)Tyr side
chain (16), although the Ki decreased, possibly caused by the
energetic cost of such a large movement in the enzyme pocket.
Given this information, future efforts should be aimed at
exploiting the novel binding mode of the P1 R-hydroxy-β-amino
acid side chain of bestatin against PfA-M1. Derivatization of the
P1 side chain using a wider variety of long hydrophobic side
chains between the lengths of compounds 15 and 16 would
represent one strategy. In addition, the S10 pocket favored smaller
aliphatic side chains, and thus exploration of a series of small non-
natural amino acids at the P10 position may further increase activity.
Other possibilities to enhance the potency against the enzyme and in
live cells or animals would entail changing the peptide backbone and,
thus, reducing the peptide character of the bestatin pharmacophore.
1659
dx.doi.org/10.1021/jm101227t |J. Med. Chem. 2011, 54, 1655–1666

Journal of Medicinal Chemistry
ARTICLE
Figure 2. X-ray crystal structures of PfA-M1 bound to Tyr(OMe) (15) and Tyr(OBzl) (16). (A) Cartoon diagram of the carbon atoms of three X-ray
crystal structures of S1 pocket of PfA-M1 bound to bestatin (gray), Tyr(OMe) (15) (magenta), and Tyr(OBzl) (16) (green). The comparative movement
of the CR-atom of Glu572 is indicated by the dashed lines. (B) The electrostatic potential surface of S1 pocket of PfA-M1 with bestatin, Tyr(OMe) (15),
and Tyr(OBzl) (16) bound in active site. The zinc ion is shown as black sphere, the carbon atoms of inhibitors is in gray, and the position of Glu572 is
indicated in each panel. Surfaces were color coded according to electrostatic potential (calculated by the Poisson-Boltzmann solver within CCP4MG). Lys
and Arg residues were assigned a single positive charge, and Asp and Glu residues were assigned a single negative charge; all other residues were considered
neutral. The calculation was done assuming a uniform dielectric constant of 80 for the solvent and 2 for the protein interior. The ionic strength was set to
zero. The color of the surface represents the electrostatic potential at the protein surface, going from blue (potential of þ10 kT/e) to red (potential of -10
kT/e), where T is temperature, e is the charge of an electron, and k is the Boltzmann constant. The probe radius used was 1.4 Å.
This can be accomplished in variety of ways, however, the central
idea would entail the elimination of the central amide bond between
the R-hydroxy-β-amino acid and the leucine to create either: (i)
carbapeptides, (ii) azapeptides, or (iii) vinylogous peptides.
inhibitor bound and unbound forms.20 This information is impor-
tant for future inhibitor design efforts because it may open new
routes to building selective inhibitors that target the malaria M1
enzyme over mammalian counterparts.
Structural analyses of PfA-M1 bound to bestatin, 15 and 16
indicate that the S1 pocket loop is mobile with high B-factors for
residues 570-575. Interestingly, structural characterization of
PfA-M1 homologues, to date, has not identified this movement
in the S1 pocket and do not indicate any enhanced mobility of
this region. Taken together, our current SAR data revealed that
there is unprecedented plasticity of the S1 binding pocket in the PfA-
M1 enzyme that permits the enzyme to accommodate larger side
chains and that there is considerably more binding energy to be
gained by increasing the size of the original phenylalanyl side chain of
the parental bestatin. These data contrast with previous studies on
this enzyme family, which generally reveal little change between
It is noteworthy that there are 13 M1 family metallo-amino-
peptidase members in humans, which may complicate future
specificity studies. However, none of the enzymes share much
more than ∼26% identity with PfA-M1. Studies on various
mammalian M1 orthologues (pig, rat, and human) show sig-
nificant variation in inhibitor activity between the three closely
related enzymes, giving an indication that achieving selectivity is
plausible.48 Aminopeptidase N, the human M1 orthologue most
similar to PfA-M1, shares only 26% identity with the Plasmodium
enzyme with extensive variation at the N- and C-terminal domains,
indicating that inhibitor access to the active site may be quite altered.
Unfortunately, there is currently no structural information available
1660
dx.doi.org/10.1021/jm101227t |J. Med. Chem. 2011, 54, 1655–1666

Journal of Medicinal Chemistry
ARTICLE
for the human M1 enzyme. Ultimately designing inhibitors that are
specific for the parasite enzyme over the human M1 family members
would be ideal, and there is hope for this possibility considering the
low identity between the parasite and human enzymes.
fluorescence, indicating parasite growth, were measured on a Tristar LB
941 microplate reader (Berthold Technologies). IC50 values were
determined using GraphPad Prism.
X-ray Crystallography. rPfA-M1 enzyme was purified and crystal-
lized as previously described.20 Crystals of the PfA-M1-15 and PfA-M1-16
complex were obtained by soaking PfA-M1 crystals overnight in mother
liquor containing 1 mM ligand. Data were collected at 100 K using
synchrotron radiation at the Australian synchrotron microcrystallography
beamline 3ID1. Diffraction images were processed using MOSFLM52 and
SCALA53 from the CCP4 suite.54 Then 5% of each data set was flagged for
calculation of RFree55 with neither a σ nor a low-resolution cutoff applied to
the data. A summary of statistics is provided in Supporting Information
Table 2. Subsequent crystallographic and structural analysis was performed
using the CCP4i interface56 to the CCP4 suite,52 unless stated otherwise.
The inhibitor complex was initially solved and refined against the unbound
PfA-M1 structure (protein atoms only) as described previously45 and clearly
showed unbiased features in the active site for both structures. Super-
positions were performed using MUSTANG.57 Pymol was used to produce
structural representations (http://www.pymol.org).58 Hydrogen bonds
(excluding water-mediated bonds), were calculated using Lig_contact and
CONTACT.54 CCP4MG was used to produce electrostatic diagrams.48
Stereo diagram of both inhibitors showing electron density is represented in
Supporting Information Figure 1. The coordinates and structure factors will
be available from the Protein Data Bank (3Q43.pdb and 3Q44.pdb). Raw
data and images will be available from TARDIS.59
Chemistry. Column chromatography was performed on silica gel,
Fisher grade (170-400 mesh). TLC plates were visualized with UV, in an
iodine chamber, or with phosphomolybdic acid, ninhydrin unless otherwise
noted. Melting points were recorded on a Mel-Temp apparatus and are
uncorrected. 1H NMR and 13C NMR spectra were recorded on a Bruker
Advance DMX-360 spectrometer. The chemical shifts are reported in parts
per million (ppm, δ). The coupling constants are reported in hertz (Hz). The
purity of all final compounds was established to be >95% pure by a
combination of TLC Rf values in several solvent systems and HPLC.
Additionally, the absence of any extraneous peaks in the proton NMR
spectrum confirmed the high level of purity. The N-Boc-(2S,3R)-2-hydroxy-
3-amino-5-methylhexanoic acid was purchased from CNH technologies.
Solid-phase peptide chemistry was conducted in polypropylene
cartridges, with 2-way Nylon stopcocks (Biotage, VA). The cartridges
were connected to a 20 port vacuum manifold (Biotage, VA) that was
used to drain excess solvent and reagents from the cartridge. HRMS
were measured at the University of California Riverside Mass Spectro-
metry Service Center. All solvents were reagent grade. Unless otherwise
stated, all reagents were purchased from commercial sources and used
without additional purification. Reverse phase HPLC was conducted on
a C18 column using an Agilent 1200 HPLC.
’ CONCLUSIONS
In conclusion, we believe that this data provides robust data to
indicate that the bestatin scaffold provides an excellent pharmaco-
phore for future antimalarial inhibitor development against the PfA-
M1enzyme. Potentially, thisscaffold can also be optimized against the
other essential MAPs such as the M17 enzyme (Pf-LAP) to generate
a single scaffold that potently inhibits multiple enzyme targets, which
may reduce the ability of the parasite to generate resistance. Last,
evidence that the S1 pocket of this M1 family peptidase is quite
flexible upon inhibitor binding may be exploited to develop more
potent or selective inhibitors for other members of this family,
including other parasitic and human orthologues.
’ EXPERIMENTAL PROCEDURES
Recombinant PfA-M1. Details of the production of P. falciparum
PfA-M1 in E. coli and its purification will be described elsewhere
(D. Ragheb and M. Klemba, manuscript in preparation).
Determination of Inhibitor Ki Values. Ki values for P10
variants were determined according to the method of Dixon49 using the
fluorogenic substrate L-leucyl-7-amido-4-methylcoumarin (Leu-AMC).
Briefly, assays contained 50 mM HEPES pH 7.5, 100 mM NaCl, 0.1%
Triton X-100, 0.9 nM PfA-M1, and Leu-AMC concentrations of 12.5,
16.7, 25, and 50 μM in a volume of 200 μL. At each substrate
concentration, a range of inhibitor concentrations was assayed, typically
ranging from no inhibitor to an inhibitor concentration corresponding to
∼4 Ki. Changes in fluorescence were measured in a 96-well plate using a
Victor3 microplate fluorometer (PerkinElmer) equipped with an excita-
tion filter of 380 nm and an emission filter of 460 nm. While bestatin can
be a slow-binding inhibitor of some aminopeptidases, we found that a
steady state had been attained in the PfA-M1 assays by 40 min. Data were
plotted as 1/rate vs inhibitor concentration for each substrate concentra-
tion and a linear fit was calculated by nonlinear regression using
Kaleidagraph 4.1. A secondary plot was then made with intercepts or
slopes vs 1/[S], where S is substrate. The Ki value was determined from
the ratio of the slopes of the secondary plots (intercept:slope). Inhibitors
were assessed for uncompetitive inhibition byconstructinga/v vs i plots.50
All inhibitors tested exhibited purely competitive inhibition.
Ki values for P1 variants were determined by assaying inhibitors at
various concentrations in the range 0.014 to 30 μM with a Leu-AMC
concentration of 44 μM. Data were plotted as νi/νo vs inhibitor concentra-
tion, where νi is the inhibited rate and νo is the rate in the absence of
inhibitor and fit by nonlinear regression to the expression νi/νo = 1/(1 þ
[I]/IC50). Ki was calculated from the formula Ki IC50/(1 þS/Km)
Antimalarial Activity Assay. Plasmodium falciparum strain 3D7
was cultivated in RPMI 1640 medium (with L-glutamine) containing 20
mM HEPES, 32 mM NaHCO3, and 10% Albumax II (Invitrogen) in
erythrocytes at 4% hematocrit.51 Quantitative assessment of antimalarial
activity was determined by measurement of SYBR Green fluorescence.
Assays were performed in 96-well black, clear-bottom plates. Briefly,
75 μL of culture (in phenol red-free media RPMI 1640) was aliquoted to
each well at a starting parasitemia of 0.5%. Next, serial dilutions of
compounds in media were added to each well to bring the final volume
to 150 μL. All drugs were assayed in triplicate. Cultures were incubated
at 37 °C for 72 h. At the end of incubation 10ꢀ lysis buffer (20 mM Tris-
HCl, 5 mM EDTA, 0.16% saponin wt/vol, 1.6% Triton X vol/vol) with
10ꢀ SYBR Green I (Invitrogen; from 10000 supply) was added to each
well and incubated for 1 h at room temperature. Levels of SYBR Green
Purifications were performed at room temperature and compounds
were eluted with a concentration gradient 0-70% of acetonitrile
(0.1% Formic acid). LC/MS data were acquired using N LC/MSD SL
system (Agilent). All compounds were purified to greater than 95%
purity as assessed by LC analysis (see Supporting Information for LCMS
traces).
General Procedure for the Synthesis of Bestatin (1) and Its P10
Derivatives (2-6). Solid-phase peptide synthesis was performed on Wang
resin. The first amino acid was coupled to the Wang resin using HATU,
DIEA, and cat. DMAP for 1 h. Coupling of the R-hydroxy-β-amino acid
required HATU for 1 h. Fmoc protecting group was deprotected with 20%
piperidine/DMF (30 min). To cleave products from resin, a solution of 95%
TFA:2.5%TIS:2.5%H2O was added to the resin. After standing for 2 h, the
cleavage mixture was collected and the resin was washed with fresh cleavage
solution. The combined fractions were evaporated to dryness, and the
product was purified by reverse phase-HPLC. Fractions containing product
were pooled and lyophilized. The yields of bestatin (1) and its derivatives
(2-6) ranged from 60% to 70%, respectively, after HPLC purification.
1661
dx.doi.org/10.1021/jm101227t |J. Med. Chem. 2011, 54, 1655–1666

Journal of Medicinal Chemistry
ARTICLE
HRMS calcd for PheNleOH 2 C16H25N2O4 309.1809 (Mþ þ H),
found 309.1801.
CHCl3). 1H NMR (360 MHz, CDCl3) δ 1.39 (s, 9H), 2.92-2.94 (m, 1H),
3.07-3.13 (m, 1H), 3.19 (s, 3H), 3.69 (s, 3H), 4.99 (br s, 1H), 5.19 (br s,
1H), 7.23-7.58 (m, 9H). 13C NMR (90 MHz, CDCl3) δ 28.3, 28.5, 32.3,
32.3, 38.7, 38.9, 51.7, 61.8, 79.8, 118.9, 127.2, 127.3, 127.4, 127.6,
128.9,130.1, 135.9, 139.9, 141.2, 145.6, 155.4, 172.5. HRMS calcd for
C22H29N2O4 (Mþ þ H) = 385.2127, found = 385.2116.
HRMS calcd for PheLys(Z)OH 3 C24H32N3O6 458.2286 (Mþ þ H),
found 458.2278.
HRMS calcd for PheTyr(Bzl)OH 4 C26H29N2O5 449.2071 (Mþ
þ
H), found 449.2068.
HRMS calcd for PheNaphOH 5 C23H25N2O4 393.1809 (Mþ þ H),
found 393.1816.
(R)-N-(tert-Butoxycarbonyl)-2-amino-3,3-diphenyl-N-methoxy-N-met-
hylpropionamide (8g). Prepared according to the general procedure and
was purified by flash chromatography (n-hexane/EtOAc = 4/1) to give 8g
(88%) as a colorless solid; mpt 83 °C. 1H NMR (360 MHz, CDCl3) δ 1.31
(s, 9H), 2.93 (s, 3H), 3.61 (s, 3H), 4.33 (d, 1H, J = 10.8 Hz, -NH), 5.01
(d, 1H, J = 10.1 Hz, -CH(Ph)2), 5.68 (t, 1H, J = 10.1 Hz, NHCHCO),
7.15-7.36 (m, 10H). 13C NMR (90 MHz, CDCl3) δ 28.4, 28.6, 31.9, 52.0,
54.7, 61.9, 79.8, 79.9, 126.9, 127.0, 128.4, 128.6, 128.8, 129.0, 129.4, 140.4,
140.6, 155.2, 172.6. HRMS calcd for C22H29N2O4 (Mþ þ H) = 385.2127,
found = 385.2119.
HRMS calcd for PheBipOH 6 C25H27N2O4 419.1965 (Mþ þ H),
found 419.1959.
Synthesis of r-Hydroxy-β-aminoacids. General Procedure
for the Synthesis of N-Methoxy-N-methyl Acetamide (Weinreb Amides).
In an oven-dried, 50 mL, two-neck, round-bottom flask equipped with a
magnetic stirring bar and argon balloon was placed N-Boc-D-amino acid
(1.997 g, 6.33 mmol) in dry CH2Cl2 (15 mL) and the solution was cooled
to -15 °C. N,O-Dimethylhydroxylamine hydrochloride (0.636 g, 6.52 mmol)
was added, followed by N-methylmorpholine (0.72 mL, 6.55 mmol). After
5 min, 1-(3-methylaminopropyl-3-ethylcarbodiimide hydrochloride (1.238
g, 6.46 mmol) was added in five portions over 30 min. The reaction was
warmed to room temperature, and after 5 h, H2O(2mL) wasaddedandthe
solution was extracted with CH2Cl2 (3 ꢀ 5 mL). The combined organic
phases were washed with brine (5 mL), dried (Na2SO4), and concentrated.
The residue was purified by flash chromatography.
(R)-N-(tert-Butoxycarbonyl)-2-amino-3-phenyl-N-methoxy-methyl-
propionamide (8b). Prepared according to the general procedure and was
purified by flash chromatography (n-hexane/EtOAc = 5/1) to give 8b
(96%) as an oily liquid; [R]D25 = -24.26 (c = 0.68, CHCl3). 1HNMR(360
MHz, CDCl3) δ 1.39 (s, 9H), 2.87-2.90 (m, 1H), 3.02-3.08 (m, 1H),
3.16 (s, 3H), 3.66 (s, 3H), 4.94 (br s, 1H), 5.15 (br s, 1H), 7.16-7.30 (m,
5H). 13C NMR (90 MHz, CDCl3) δ 28.5, 32.2, 32.3, 39.1, 51.8, 61.7, 79.8,
127.0, 128.6, 129.7, 136.9, 155.4.
(R)-N-(tert-Butoxycarbonyl)-2-amino-3-(p-methyloxyphenyl)-N-meth-
oxy-N-methylpropionamide (8c). Prepared according to the general pro-
cedure and was purified by flash chromatography (n-hexane/EtOAc =
5/1) to give 8c (97%) as an oily liquid; [R]D25 = -22.12 (c = 1.08, CHCl3).
1H NMR (360 MHz, CDCl3) δ 1.39 (s, 9H), 2.82-2.86 (m, 1H), 2.97-3.02
(m, 1H), 3.16 (s, 3H), 3.66 (s, 3H), 3.77 (s, 3H), 4.90 (br s, 1H), 5.14 (br s,
1H), 6.81 (d, J = 8.3 Hz, 2H), 7.08 (d, J = 8.3 Hz, 2H). 13C NMR (90 MHz,
CDCl3) δ 28.5, 32.3, 38.2, 51.8, 55.4, 61.7, 79.7, 114.0, 128.8, 130.6, 155.4,
158.7. HRMS calcd for C17H27N2O5 (Mþ þ H) = 339.1914, found =
339.1912.
(R)-N-(tert-Butoxycarbonyl)-2-amino-3-propyl-N-methoxy-N-methyl-
propionamide (8h). Prepared according to the general procedure and was
purified by flash chromatography (n-hexane/EtOAc = 4/1) to give 8h
(90%) as an oily liquid. 1H NMR (360 MHz, CDCl3) δ 0.89 (t, 3H, J = 6.8
Hz), 1.26-1.55 (m, 14H), 1.66-1.72 (m, 1H), 3.21 (s, 3H), 3.77 (s, 3H),
1.09 (brs, 1H), 5.15 (d, 1H, J = 7.6 Hz).
General Procedure for the Wittig Reaction. In an oven-dried, 50 mL,
single-necked, round-bottom flask equipped with a magnetic stirring bar,
rubber septum, and argon balloon was placed Weinreb amides (1.860 g,
5.19 mmol) in anhydrous Et2O (20 mL). The solution was cooled to -
33 °C and LiAlH4 (1 M in THF, 6.2 mL, 6.23 mmol) was added dropwise,
and the reaction mixture was warmed to 0 °C. After 45 min, the solution was
cooled to -33 °C and 10% aq KHSO4 (10 mL) was added to quench the
reaction. After warming to room temperature 1 N HCl (10 mL) was added,
and the organic phases were separated, the aqueous phase was extracted
with EtOAc (2 ꢀ 10 mL), and the combined organic phases were dried
(MgSO4) and concentrated to yield the crude aldehyde. In a separate 50
mL, single-necked, round-bottom flask was placed methyltriphenylpho-
sphonium bromide (3.65 g, 10.24 mmol) in THF (20 mL), and KHMDS
(0.5 M in toluene, 19.5 mL, 9.76 mmol) was added. The resulting yellow
suspension was stirred at room temperature for 2 h, then cooled to -78 °C
and the crude aldehyde (1.75 g, 5.85 mmol) in THF (5 mL) was added
dropwise. It was warmed to room temperature over 2 h and quenched with
MeOH (5 mL). The resulting mixture was poured into a mixture of
saturated potassium sodium tartarate and water (1:1, 15 mL), extracted with
Et2O (2 ꢀ 50 mL), dried (MgSO4), and concentrated. The residue was
purified by flash chromatography to afford the respective alkenes.
(R)-N-(tert-Butoxycarbonyl)-3-amino-4-phenyl-1-butene
(R)-N-(tert-Butoxycarbonyl)-2-amino-3-(p-benzyloxyphenyl)-N-meth-
oxy-N-methylpropionamide (8d). Prepared according to the general
procedure and was purified by flash chromatography (n-hexane/EtOAc =
5/1) to give 8d (85%) as a colorless solid; mp 102 °C; [R]D25 = -14.54
(c = 0.55, CHCl3). 1H NMR (360 MHz, CDCl3) δ 1.39 (s, 9H), 2.80-2.85
(m, 1H), 2.96-3.01 (m, 1H), 3.15 (s, 3H), 3.64 (s, 3H), 4.89 (br s, 1H), 5.02
(s, 2H), 5.13 (s, 1H), 6.88 (d, 2H, J=9.0Hz), 7.07(d, 2H,J= 9.0 Hz), 7.25-
7.42 (m, 5H). 13CNMR(90MHz,CDCl3) δ28.5, 32.3, 38.2, 38.2, 51.8, 61.7,
70.2, 79.8, 115.0, 127.6, 128.1, 128.7, 129.2, 130.7, 137.4, 158.0, 207.1. HRMS
calcd for C23H30N2O5 (Mþ þ H) = 415.2233, found = 415.2229.
(R)-N-(tert-Butoxycarbonyl)-2-amino-3-(2-naphthyl)-N-methoxy-N-
methylpropionamide (8e). Prepared according to the general procedure
and was purified by flash chromatography (n-hexane/EtOAc = 5/1) to give
(10b). Prepared according to the general procedure and was purified by
flash chromatography (n-hexane/EtOAc = 20/1) to give 10b (71%) as a
colorless solid; mp 62 °C; [R]D25 = -37.19 (c = 0.28, CHCl3). 1H NMR
(360 MHz, CDCl3) δ 1.39 (s, 9H), 2.83 (d, J = 6.5 Hz, 1H), 4.40 (br s,
1H), 5.05-5.11 (m, 1H), 5.74-5.83 (m, 1H), 7.16-7.30 (m, 5H).
(R)-N-(tert-Butoxycarbonyl)-3-amino-4-(p-methyloxyphenyl)-1-butene
(10c). Prepared according to the general procedure and was purified
by flash chromatography (n-hexane/EtOAc = 20/1) to give 10c (67%)
25
as a colorless solid; mp 58 °C ; [R]D = -36.33. 1H NMR
(360 MHz, CDCl3) δ 1.41 (s, 9H), 2.77 (d, J = 6.5 Hz, 2H), 3.78
(s, 3H), 4.36 (br s, 1H), 5.06-5.12 (m, 2H), 5.74-5.83 (m, 1H), 6.82 (d,
J = 8.6 Hz, 2H), 7.09 (d, J = 8.6 Hz, 2H). 13C NMR (90 MHz, CDCl3) δ
28.6, 40.8, 40.8, 53.8, 54.0, 55.5, 79.6, 114.0, 114.9, 129.6, 130.7, 138.4,
155.5, 158.5. HRMS calcd for C16H24NO3 (Mþ þ H) = 278.1751, found =
278.1749.
25
8e (93%) as a colorless solid; mp 103 °C; [R]D = -20.19 (c = 1.02,
CHCl3). 1H NMR (360 MHz, CDCl3) δ 1.35 (s, 9H), 3.01-3.25 (m, 5H),
3.65 (s, 3H), 5.04 (br s, 1H), 5.20 (br s, 1H), 7.31 (d, J = 10.0 Hz, 1H),
7.39-7.46 (m, 2H), 7.62 (s, 1H), 7.75-7.80 (m, 3H). 13CNMR(90MHz,
CDCl3) δ 28.2, 28.5, 32.3, 39.2, 51.7, 61.8, 79.8, 125.7, 126.2, 127.8, 127.8,
128.1, 128.3, 132.6, 133.7, 134.3, 172.6, 207.1.
(R)-N-(tert-Butoxycarbonyl)-3-amino-4-(p-benzyloxyphenyl)-1-butene
(10d). Prepared according to the general procedure and was purified by
flash chromatography (n-hexane/EtOAc = 20/1) to give 10d (71%) as a
colorless solid; mp 87 °C; [R]D25 = -29.62 (c = 0.53, CHCl3). 1H NMR
(360 MHz, CDCl3) δ 1.40 (s, 9H), 2.77 (d, J = 6.7 Hz, 2H), 4.40 (br s, 1H),
(R)-N-(tert-Butoxycarbonyl)-2-amino-3-(p-phenylphenyl)-N-methoxy-
N-methylpropionamide (8f). Prepared according to the general procedure
and was purified by flash chromatography (n-hexane/EtOAc = 5/1) to give
25
8f (87%) as a colorless solid; mp 106 °C; [R]D = -11.37 (c = 0.73,
1662
dx.doi.org/10.1021/jm101227t |J. Med. Chem. 2011, 54, 1655–1666

Journal of Medicinal Chemistry
ARTICLE
5.04 (s, 2H), 5.06-5.12 (m, 2H), 5.74-5.83 (m, 1H), 6.90 (d, J = 11.2 Hz,
2H), 7.08 (d, J = 11.2 Hz, 2H), 7.30-7.44 (m, 5H). 13C NMR (90 MHz,
CDCl3) δ 28.5, 40.7, 53.8, 70.2, 79.4, 114.8, 114.9, 127.6, 128.0, 128.7, 130.0,
130.7, 137.3, 138.4, 155.4, 157.7. HRMS calcd for C22H27NO3 (Mþ þ Na) =
376.1889, found 376.1879.
(R)-N-(tert-Butoxycarbonyl)-3-amino-4-(2-naphtyl)-1-butene (10e).
Prepared according to the general procedure and was purified by flash
chromatography (n-hexane/EtOAc = 20/1) to give 10e (80%) as a
colorless solid; mpt 104 °C; [R]D25 = -42.46 (c = 0.32). 1H NMR (360
MHz, CDCl3) δ 1.38 (s, 9H), 2.97-3.01 (m, 2H), 4.50 (br s, 1H), 5.07-
5.14 (m, 2H), 5.79-5.87 (m, 1H), 7.33 (d, J = 9.7 Hz, 1H), 7.40-7.47
(m, 2H), 7.63 (s, 1H), 7.76-7.82 (m, 3H). 13CNMR(90MHz, CDCl3) δ
28.5, 41.9, 53.5, 115.1, 125.7, 126.2, 127.8, 127.8, 128.1, 128.2, 128.3, 132.5,
133.7, 135.2, 138.3, 155.4, tert-butyl carbon not observed. HRMS calcd
for C19H23NO2 (Mþ þ Na) = 320.1624, found 320.1620.
to 70%. HPLC purification of the mixture provided the individual
diastereomers.
(2R,3R)-N-(tert-Butoxycarbonyl)-3-amino-2-hydroxy-4-phenyl Buta-
noic Acid (12b). Mp 148 °C. 1H NMR (360 MHz, DMSO-d6) δ 1.27 (s,
9H), 2.66-2.68 (m, 2H), 3.92-4.00 (m, 2H), 6.66 (d, J = 9.0 Hz, 1H,
NH), 7.16-7.26 (m, 5H). 13C NMR (90 MHz, DMSO-d6) δ 28.1, 34.9,
54.5, 72.6, 77.5, 125.8, 127.9, 129.0, 139.2, 154.9, 173.9.
(2S,3R)-N-(tert-Butoxycarbonyl)-3-amino-2-hydroxy-4-phenyl Buta-
1
noic Acid (13b). Mp 126 °C. H NMR (360 MHz, DMSO-d6) δ 1.30
(s, 9H), 2.66-2.71 (m, 1H), 2.78-2.83 (m, 1H), 3.96-4.03
(m, 1H), 5.14 (br s, 1H), 6.37 (d, J = 9.4 Hz, 1H, NH), 7.17-7.30 (m,
5H), 12.5 (s, 1H). 13C NMR (90 MHz, DMSO-d6) δ 27.7, 28.1, 37.5, 54.7,
70.4, 77.8, 126.0, 128.1, 129.1, 138.6, 154.8, 174.0.
(2R,3R)-N-(tert-Butoxycarbonyl)-3-amino-2-hydroxy-4-(p-methyloxy-
phenyl)-butanoic Acid (12c). 1H NMR (360 MHz, DMSO-d6) δ 1.28
(s, 9H), 2.60-2.62 (m, 2H), 3.70 (s, 3H), 3.36-3.88 (m, 1H), 3.99-4.0
(m, 1H), 6.53 (d, J = 8.6 Hz, 1H, NH), 6.81 (d, J = 8.6 Hz, 2H), 7.08
(d, J= 8.6 Hz, 2H). 13C NMR (90 MHz, DMSO-d6) δ28.1, 33.9, 54.7, 54.9,
72.5, 77.5, 107.8, 113.4, 129.9, 130.9, 154.9, 157.5, 174.0.
(2S,3R)-(tert-Butoxycarbonyl)-3-amino-2-hydroxy-4-(p-methyloxy-
phenyl)-butanoic Acid (13c). Mp 55 °C. 1HNMR(360MHz, DMSO-d6)
δ 1.33 (s, 9H), 2.632.65 (m, 1H), 2.71-2.73 (m, 1H), 3.72 (s, 3H),
3.84-3.94 (m, 2H), 6.32 (d, J = 9.36 Hz, 1H, NH), 6.84 (d, J =
8.6 Hz, 2H), 7.12 (d, J = 8.6 Hz, 2H). 13C NMR (90 MHz, DMSO-
d6) δ 27.8, 28.2, 36.6, 54.9, 55.0, 70.3, 77.8, 113.7, 130.1, 130.5,
154.9, 157.7, 174.1. HRMS calcd for C16H24NO6 (Mþ þ H)
326.1598, found 326.1599.
(2R,3R)-N-(tert-Butoxycarbonyl)-3-amino-2-hydroxy-4-(p-benzyloxy
phenyl)-butanoic Acid (12d). Mp 129 °C. 1H NMR (360 MHz, DMSO-
d6) δ1.27 (s, 9H), 2.60-2.61 (m, 2H), 3.85-3.86 (m, 1H), 3.96-3.99 (m,
1H), 5.04 (s, 2H), 6.61 (d, J = 8.6 Hz, 1H, NH), 6.88 (d, J = 8.3 Hz, 2H),
7.08 (d, J = 8.3 Hz, 2H), 7.31-7.43 (m, 5H). 13C NMR (90 MHz, DMSO-
d6) δ 28.1, 33.9, 54.7, 69.1, 72.5, 77.5, 114.3, 127.5, 127.7, 128.3, 130.0,
131.2, 137.3, 155.0, 156.6, 174.0.
(R)-N-(tert-Butoxycarbonyl)-3-amino-4-(p-phenylphenyl)-1-butene
10f). Prepared according to the general procedure and was purified by
flash chromatography (n-hexane/EtOAc = 20/1) to give 10f (67%) as a
colorless solid. 1H NMR (360 MHz, CDCl3) δ 1.40 (s, 9H), 2.88 (d, J =
5.8 Hz, 2H), 4.47 (br s, 2H), 5.09-5.16 (m, 2H), 5.79-5.88 (m, 1H),
7.24-7.59 (m, 9H). 13C NMR (90 MHz, CDCl3) δ 28.6, 41.4, 115.0,
127.2, 127.3, 127.4, 129.0, 130.2, 136.8, 138.3, 139.6, 141.1, 155.4.
(R)-N-(tert-Butoxycarbonyl)-3-amino-5,5-diphenyl-1-pentene (10g).
Prepared according to the general procedure and was purified by flash
chromatography (n-hexane/EtOAc = 20/1) to give 10g (78%) as a
25
1
colorless solid; mp 98 °C; [R]D = -56.89 (c = 0.58, CHCl3). H
NMR (360 MHz, CDCl3) δ 1.36 (s, 9H), 3.97 (d, J = 9.4 Hz, 2H), 4.39
(br s, 1H), 4.99 (br s, 1H), 5.03-5.16 (m, 2H), 5.71-5.80 (m, 1H), 7.15-
7.30 (m, 10H). 13C NMR (90 MHz, CDCl3) δ 28.5, 55.4, 57.0, 115.5,
126.8, 127.0, 128.7, 128.7, 128.8, 128.9, 137.7, 141.8, 155.5, tert-butyl carbon
not observed. HRMS calcd for C21H25NO2 (Mþ þ Na) = 346.1783, found
346.1784.
(R)-N-(tert-Butoxycarbonyl)-3-amino-4-propyl-1-butene (10h).
Prepared according to the general procedure and was purified by flash
chromatography (n-hexane/EtOAc = 20/1) to give 10h (65%) as an
(2S,3R)-N-(tert-Butoxycarbonyl)-3-amino-2-hydroxy-4-(p-benzyl-
25
1
oxyphenyl)-butanoic Acid (13d). Mp 52 °C; [R]D = þ56.84
oily liquid. H NMR (360 MHz, CDCl3) δ 0.89 (t, 3H, J = 6.8 Hz),
1
(c = 0.19, MeOH). H NMR (360 MHz, DMSO-d6) δ 1.31 (s, 9H),
1.27-1.33 (m, 4H), 1.42-1.51 (s, 11H), 4.07 (brs, 1H), 4.53 (brs,
1H), 5.05-5.16 (m, 2H), 5.70-5.77 (m, 1H).
2.62-2.66 (m, 1H), 2.72-2.74 (m, 1H), 3.86-3.96 (m, 2H), 5.05 (s, 2H),
6.33 (d, J = 9.4 Hz, 1H, NH), 6.93 (d, J = 8.6 Hz, 2H), 7.13 (d, J = 8.6 Hz,
2H), 7.31-7.44 (m, 5H). 13C NMR (90 MHz, DMSO-d6) δ 28.1, 36.6,
54.9, 69.2, 70.3, 77.8, 114.6, 127.6, 127.7, 128.4, 130.2, 130.7, 137.2, 154.9,
156.9, 174.1. HRMS calcd for C22H27NO6 (Mþ - H) = 400.1760,
found =400.1755.
(2R,3R)-N-(tert-Butoxycarbonyl)-3-amino-2-hydroxy-4-(2-naphtyl)-
butanoic Acid (12e). Yield 77%; mp 154-155 °C. 1H NMR (360 MHz,
DMSO-d6) δ 1.20 (s, 9H), 2.84-2.86 (m, 2H), 4.01-4.04 (m, 2H), 6.72
(d, J = 8.6 Hz, 1H, NH), 7.34-7.47 (m, 3H), 7.66 (s, 1H), 7.79-7.86
(m, 3H).
(2S,3R)-N-(tert-Butoxycarbonyl)-3-amino-2-hydroxy-4-(2-naphtyl)-
butanoic Acid (13e). Mp 113 °C; [R]D25 = þ55.18 (c = 0.54, MeOH).
1H NMR (360 MHz, DMSO-d6) δ 1.26 (s, 9H), 2.88-2.90 (m, 1H),
2.97-2.99 (m, 1H), 3.93 (s, 1H), 4.11-4.13 (m, 1H), 6.42 (d, J = 9.7 Hz,
1H, NH), 7.39-7.48 (m, 3H), 7.71 (s, 1H), 7.82-7.87 (m, 3H). 13CNMR
(90 MHz, DMSO-d6) δ 28.1, 37.7, 54.7, 70.6, 77.8, 125.3, 125.9, 127.3,
127.4, 127.4, 127.6, 127.8, 131.8, 133.1, 136.2, 154.9, 174.0. HRMS calcd for
C19H23NO5 (Mþ - H) = 344.1498, found = 344.1497.
(2R,3R)-N-(tert-Butoxycabonyl)-3-amino-2-hydroxy-4-(p-phenylphe-
nyl)-butanoic Acid (12f). 1HNMR(360MHz, DMSO-d6) δ1.27(s, 9H),
2.73 (br s, 2H), 3.95-4.03 (m, 2H), 6.71 (d, J = 8.8 Hz, 1H, NH), 7.26-
7.63 (m, 9H). 13C NMR (90 MHz, DMSO-d6) δ 27.7, 28.1, 34.5, 54.6, 72.6,
77.5, 126.2, 126.4, 127.1, 128.8, 129.6, 137.7, 138.5, 140.1, 155.0, 174.0.
(2S,3R)-N-(tert-Butoxycarbonyl)-3-amino-2-hydroxy-4-(p-phenylphe-
nyl)-butanoic Acid (13f). Mp 162-163 °C. 1H NMR (360 MHz,
DMSO-d6) δ ; 1.31 (s, 9H), 2.73-2.76 (m, 1H), 2.83-2.89
General Method for the Synthesis of R-Hydroxy-β-amino Acid. In
an oven-dried, 50 mL, single-necked, round-bottom flask equipped with
a magnetic stirring bar, rubber septum, and argon balloon was placed the
alkene (1.20 g, 4.33 mmol) in t-BuOH:H2O (12 mL, 1:1) and
methanesulfonamide (0.412 g, 4.33 mmol) was added. The reaction
mixture was cooled to 0 °C and AD-mix-R (6.54 g) was added. The
resulting mixture was stirred at 0 °C for 36 h and at room temperature
for 8 h and then quenched with solid Na2SO3 (7.0 g) and extracted with
ethyl acetate. The organic extracts were washed with NaOH (1M, 10
mL), water and brine, dried (Mg2SO4), and concentrated. The crude
diol was used as such for the next step.
The residue was dissolved in phosphate buffer (NaH2PO4, pH 6.7, 6 mL)
and MeCN (6 mL). To the solution was added 2,2,6,6-tetramethylpiperidin-
1-oxyl (TEMPO, 0.169 g, 1.07 mmol) and sodium chlorite (0.697 g, 7.71
mmol). The reaction mixture was warmed to 35 °C, and 4% aqueous sodium
hypochlorite (NaOCl) solution (0.008 g, 0.12 mmol) was added. The
mixture was stirred at this temperature for 6.5 h, and TEMPO (0.053 g, 0.34
mmol) along with 4% aqueous NaOCl solution (0.001 g, 0.02 mmol) were
added. After stirring at 35 °C for 24 h, H2O (5 mL) was added, and thepH of
the mixture was adjusted to 8.0 with 2.0 M aqueous NaOH solution. The
reaction flask was cooled to 20 °C and 6% aq sodium sulfite was added until
the solution had a pH of 9. After warming to rt for 30 min, Et2O (5 mL) was
added and the organic phase was separated. To the aqueous layer 1N HCl was
added until the pH of the solution was 3, and extracted with ethyl acetate,
washed with brine dried and concentrated to yield the R-hydroxy-β-amino
acids. The combined yields of the R-hydroxy-β-amino acids ranged from 65%
1663
dx.doi.org/10.1021/jm101227t |J. Med. Chem. 2011, 54, 1655–1666

Journal of Medicinal Chemistry
ARTICLE
(m, 1H), 3.92-4.04 (m, 2H), 6.39 (d, J = 9.4 Hz, 1H, NH), 7.29-7.64
(m, 9H). 13C NMR (90 MHz, DMSO-d6) δ 27.7, 28.1, 37.1, 54.7, 70.5,
77.8, 126.5, 127.2, 128.9, 129.8, 129.9, 137.9, 138.0, 140.0, 154.9, 174.0.
HRMS calcd for C21H25NO5 (Mþ - H) = 370.1654, found = 370.1650.
N-(tert-Butoxycarbonyl)-3-amino-4,4-diphenyl-2-hydroxypenta-
noic Acid (12g). 1H NMR (360 MHz, DMSO-d6) δ 1.18 (s, 9H), 3.68
(s, 1H), 4.21 (d, J= 11.8 Hz, 1H), 4.79 (t, J =11.6 Hz, 1H), 6.24 (d, J=10.04
Hz, 1H, NH), 7.08-7.37 (m, 10H). 13C NMR (90 MHz, DMSO-d6) δ27.7,
27.9, 53.1, 55.1, 69.6, 77.6, 125.9, 126.5, 127.9, 128.0, 128.4, 128.7, 142.2,
142.5, 154.8, 174.0. HRMS calcd for C21H25NO5 (Mþ - H) = 370.1654,
found = 370.1655.
N-(tert-Butoxycarbonyl)-3-amino-2-hydroxy-4-propylbutanoic
Acid (12h). 1H NMR (360 MHz, DMSO-d6) δ 0.83 (t, 3H, J = 6.8 Hz),
1.15-1.40 (m, 15H), 3.67-3.69 (m, 1H), 3.92 (d, 1H, J = 4.3 Hz), 6.44 (d,
1H, J = 9 Hz). 13C NMR (90 MHz, DMSO-d6) δ 13.8, 21.9, 27.7, 28.2, 28.3,
52.6, 72.6, 77.5, 120.7, 155.3, 174.1. ESIMS calcd for C12H23NO5 (Mþ þ H)
= 261.1, found = 284.1 (Mþ þ 23).
5T32AI007532. M.K. is supported by NIH grant AI077638. J.C.
W. is an Australian Research Council Federation Fellow and a
National Health and Medical Research Council (NHMRC)
Principal Research Fellow. We thank the NHMRC and the
ARC for funding support. We thank the Australian Synchrotron
for beamtime and Tom Caradoc-Davis in particular for technical
assistance.
’ ABBREVIATIONS USED
Pf-LAP, Plasmodium falciparum-leucine amino peptidase; Tyr-
(OMe), p-methoxy phenyl; Tyr(OBzl), p-benzyloxy phenyl; Boc,
tert-butyloxy carbonyl; DIEA, N,N-diisopropylethylamine; DMAP,
4-dimethylaminopyridine; HATU, (2-(7-aza-1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate); DBU, 1,8-diaza-
bicyclo[5.4.0]undec-7-ene; Fmoc, fluorenylmethyloxycarbonyl;
TFA, trifluoroacetic acid; TIS, triisopropyl silane; EDAC HCl, N-(3-
3
dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride;
General Procedure for the Synthesis of Bestatin (1) and its P1
Derivatives (14-20). Standard solid-phase peptide synthesis was per-
formed on LeuWang resin. Coupling of the R-hydroxy-β-amino acid required
HATU for 1 h. Fmoc protecting groups were removed with 20% piperidine/
DMF (30 min). To cleave products from resin, a solution of 95%TFA:2.5%
TIS:2.5%H2O was added to the resin. After standing for 2 h, the cleavage
mixture was collected and the resin was washed with fresh cleavage solution.
The combined fractions were evaporated to dryness, and the product was
purified by reverse phase-HPLC. Fractions containing product were pooled
and lyophilized. The yields of bestatin (1) and its derivatives 14-20 ranged
from 60% to 70%, respectively.
TEMPO, 2,2,6,6-tetramethylpiperidine-1-oxyl
’ REFERENCES
(1) Snow, R. W.; Guerra, C. A.; Noor, A. M.; Myint, H. Y.; Hay, S. I.
The global distribution of clinical episodes of Plasmodium falciparum
malaria. Nature 2005, 434, 214–217.
(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) Liu, J.; Istvan, E. S.; Gluzman, I. Y.; Gross, J.; Goldberg, D. E.
Plasmodium falciparum ensures its amino acid supply with multiple
acquisition pathways and redundant proteolytic enzyme systems. Proc.
Natl. Acad. Sci. U.S.A. 2006, 103, 8840–8845.
(4) Chen, X.; Chong, C. R.; Shi, L.; Yoshimoto, T.; Sullivan, D. J., Jr.;
Liu, J. O. Inhibitors of Plasmodium falciparum methionine aminopepti-
dase 1b possess antimalarial activity. Proc. Natl. Acad. Sci. U.S.A. 2006,
103, 14548–14553.
(5) Chen, X.; Xie, S.; Bhat, S.; Kumar, N.; Shapiro, T. A.; Liu, J. O.
Fumagillin and fumarranol interact with P. falciparum methionine
aminopeptidase 2 and inhibit malaria parasite growth in vitro and in
vivo. Chem. Biol. 2009, 16, 193–202.
(6) Dalal, S.; Klemba, M. Roles for two aminopeptidases in vacuolar
hemoglobin catabolism in Plasmodium falciparum. J. Biol. Chem. 2007,
282, 35978–35987.
HRMS calcd for LeuLeuOH 14 C13H27N2O4 (Mþ þ H) 275.1965,
found 275.1973.
HRMS calcd for Tyr(OMe)LeuOH 15 C17H27N2O5 (Mþ þ H)
339.1914, found 339.1913.
HRMS calcd for Tyr(OBzl)LeuOH 16 C23H31N2O5 (Mþ þ H)
415.2227, found 415.2221.
ESIMS calcd for NaphLeuOH 17 C20H27N2O4 (Mþ þ H) 359.1,
found 359.1, 330.2 (Mþ - 29).
ESIMS calcd for BipLeuOH 18 C22H30N2O4 (Mþ þ H) 385.2,
found 385.1, 355.1 (Mþ - 29).
ESIMS calcd for DipLeuOH 19 C22H30N2O4 (Mþ þ H) 385.2,
found 355.1 (Mþ - 29).
ESIMS calcd for NleLeuOH 20 C13H26N2O4 (Mþ þ H) 275.1965,
found 275.1964.
(7) Luan, Y.; Xu, W. The structure and main functions of amino-
peptidase N. Curr. Med. Chem. 2007, 14, 639–647.
(8) Sato, Y. Role of aminopeptidase in angiogenesis. Biol. Pharm.
Bull. 2004, 27, 772–776.
(9) Foulon, T.; Cadel, S.; Cohen, P. Aminopeptidase B (EC
3.4.11.6). Int. J. Biochem. Cell Biol. 1999, 31, 747–750.
’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra and
S
b
LCMS profiles of compounds. This material is available free of
(10) Giannousis, P. P.; Bartlett, P. A. Phosphorus amino acid analogues
as inhibitors of leucine aminopeptidase. J. Med. Chem. 1987, 30, 1603–1609.
(11) Chan, W. W.; Dennis, P.; Demmer, W.; Brand, K. Inhibition of
leucine aminopeptidase by amino acid hydroxamates. J. Biol. Chem.
1982, 257, 7955–7957.
charge via the Internet at http://pubs.acs.org.
Accession Codes
†PDB ID Codes: The coordinates and structure factors are
available from the Protein Data Bank (3Q43 and 3Q44).
(12) Wilkes, S. H.; Prescott, J. M. The slow, tight binding of bestatin and
amastatin to aminopeptidases. J. Biol. Chem. 1985, 260, 13154–13162.
(13) Rich, D. H.; Moon, B. J.; Harbeson, S. Inhibition of amino-
peptidases by amastatin and bestatin derivatives. Effect of inhibitor
structure on slow-binding processes. J. Med. Chem. 1984, 27, 417–422.
(14) Suda, H.; Aoyagi, T.; Takeuchi, T.; Umezawa, H. Inhibition of
aminopeptidase B and leucine aminopeptidase by bestatin and its
stereoisomer. Arch. Biochem. Biophys. 1976, 177, 196–200.
(15) Burley, S. K.; David, P. R.; Lipscomb, W. N. Leucine amino-
peptidase: bestatin inhibition and a model for enzyme-catalyzed peptide
hydrolysis. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 6916–6920.
(16) Tsuge, H.; Ago, H.; Aoki, M.; Furuno, M.; Noma, M.; Miyano,
M.; Minami, M.; Izumi, T.; Shimizu, T. Crystallization and preliminary
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (þ)1-215-746-2992, Fax: (þ)1-215-746-6697, E-mail:
dorong@mail.med.upenn.edu.
’ ACKNOWLEDGMENT
D.C.G. acknowledges support from McCabe Pilot Award,
Penn Genome Frontiers Institute, 1R56AI081770-01A2, and the
Ritter Foundation. M.B.H. is supported by NIH training grant
1664
dx.doi.org/10.1021/jm101227t |J. Med. Chem. 2011, 54, 1655–1666

Journal of Medicinal Chemistry
ARTICLE
X-ray crystallographic studies of recombinant human leukotriene A4
hydrolase complexed with bestatin. J. Mol. Biol. 1994, 238, 854–856.
(17) Addlagatta, A.; Gay, L.; Matthews, B. W. Structure of amino-
peptidase N from Escherichia coli suggests a compartmentalized, gated
active site. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 13339–13344.
(18) Nankya-Kitaka, M. F.; Curley, G. P.; Gavigan, C. S.; Bell, A.;
Dalton, J. P. Plasmodium chabaudi chabaudi and P. falciparum: inhibition
of aminopeptidase and parasite growth by bestatin and nitrobestatin.
Parasitol. Res. 1998, 84, 552–558.
(19) Chauvel, E. N.; Llorens-Cortes, C.; Coric, P.; Wilk, S.; Roques,
B. P.; Fournie-Zaluski, M. C. Differential inhibition of aminopeptidase A
and aminopeptidase N by new beta-amino thiols. J. Med. Chem. 1994, 37,
2950–2957.
(20) McGowan, S.; Porter, C. J.; Lowther, J.; Stack, C. M.; Golding,
S. J.; Skinner-Adams, T. S.; Trenholme, K. R.; Teuscher, F.; Donnelly,
S. M.; Grembecka, J.; Mucha, A.; Kafarski, P.; Degori, R.; Buckle, A. M.;
Gardiner, D. L.; Whisstock, J. C.; Dalton, J. P. Structural basis for the
inhibition of the essential Plasmodium falciparum M1 neutral amino-
peptidase. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 2537–2542.
(21) Harbut, M. B.;Velmourougane, G.;Reiss, G.;Chandramohanadas,
R.; Greenbaum, D. C. Development of bestatin-based activity-based
probes for metallo-aminopeptidases. Bioorg. Med. Chem. Lett. 2008, 18,
5932–5936.
(37) Semple, J. E.; Owens, T. D.; Nguyen, K.; Levy, O. E. New
Synthetic Technology for Efficient Construction of alpha-Hydroxy-beta-
amino Amides via the Passerini Reaction. Org. Lett. 2000, 2, 2769–
2772.
(38) Herranz, R.; Castro-Pichel, J.; Vinuesa, S.; Garcia-Lopez, M. T.
An improved one-pot method for the stereoselective synthesis of the
(2S,3R)-3-amino-2-hydroxy acids: key intermediates for bestatin and
amastatin. J. Org. Chem. 1990, 55, 2232–2234.
(39) So, R. C.; Ndonye, R.; Izmirian, D. P.; Richardson, S. K.;
Guerrera, R. L.; Howell, A. R. Straightforward Synthesis of Sphinganines
via a Serine-Derived Weinreb Amide. J. Org. Chem. 2004, 69, 3233–
3235.
(40) de Koning, M. C.; Petersen, L.; Weterings, J. J.; Overhand, M.;
van der Marel, G. A.; Filippov, D. V. Synthesis of thiol-modified peptide
nucleic acids designed for post-assembly conjugation reactions. Tetra-
hedron 2006, 62, 3248–3258.
(41) McKillop, A. T.; Watson, R. J.; Lewis, N. An improved
procedure for the preparation of the garner aldehyde and its use for
the synthesis of N-protected 1-halo-2-(R)-amino-3-butenes. Synthesis
1994, 31–33.
(42) Cong, X.; Liao, Q.-J.; Yao, Z.-J. RCM Approaches toward the
Diastereoselective Synthesis of Vicinal trans-Diaminocyclitols from
l-Serine. J. Org. Chem. 2004, 69, 5314–5321.
(22) Suda, H.; Takita, T.; Aoyagi, T.; Umezawa, H. The chemical
synthesis of bestatin. J. Antibiot. 1976, 29, 600–601.
(23) Lee, B. W.; Lee, J. H.; Jang, K. C.; Kang, J. E.; Kim, J. H.; Park,
K.-M.; Park, K. H. Diastereoselective synthesis of syn-aminoalcohols via
contributing CH-[pi] interaction: simple synthesis of (-)-bestatin.
Tetrahedron Lett. 2003, 44, 5905–5907.
(43) Wovkulich, P. M.; Shankaran, K.; Kiegiel, J.; Uskokovic, M. R.
Total synthesis of 1233A. J. Org. Chem. 1993, 58, 832–839.
(44) Zhao, M.; Li, J.; Mano, E.; Song, Z.; Tschaen, D. M.; Grabowski,
E. J. J.; Reider, P. J. Oxidation of Primary Alcohols to Carboxylic Acids
with Sodium Chlorite Catalyzed by TEMPO and Bleach. J. Org. Chem.
1999, 64, 2564–2566.
(24) Kobayashi, S.; Isobe, T.; Ohno, M. A stereocontrolled synthesis
of (-)-bestatin from an acyclic allylamine by iodocyclocarbamation.
Tetrahedron Lett. 1984, 25, 5079–5082.
(25) Govindaswamy, M.; Motomu, K.; Groger, H.; Shibasaki, M.
Anti- and syn-selective cyanosilylation reactions promoted by a sugar-based
bifunctional catalyst: stereoselective syntheses of essential building blocks for
HIV protease inhibitors and bestatin. Synlett 2001, 5, 617–620.
(26) Koseki, K.; Matsushita, H. Synthesis of Bestatin, a potent
inhibitor of leukotriene A4 hydrolase, by an N3 nucleophile. Biosci.,
Biotechnol., Biochem. 1996, 60, 534–536.
(45) Nishizawa, R.; Saino, T.; Takita, T.; Suda, H.; Aoyagi, T.
Synthesis and structure-activity relationships of bestatin analogues,
inhibitors of aminopeptidase B. J. Med. Chem. 1977, 20, 510–515.
(46) Stockel-Maschek, A.; Stiebitz, B.; Koelsch, R.; Neubert, K.
Novel 3-amino-2-hydroxy acids containing protease inhibitors. Part 1:
Synthesis and kinetic characterization as aminopeptidase P inhibitors.
Bioorg. Med. Chem. 2005, 13, 4806–4820.
(47) Skinner-Adams, T. S.; Stack, C. M.; Trenholme, K. R.; Brown,
C. L.; Grembecka, J.; Lowther, J.; Mucha, A.; Drag, M.; Kafarski, P.;
McGowan, S.; Whisstock, J. C.; Gardiner, D. L.; Dalton, J. P. Plasmodium
falciparum neutral aminopeptidases: new targets for antimalarials. Trends
Biochem. Sci. 2010, 35, 53–61.
(27) Levene, P. A. S., E. T. Acetone derivatives of D-ribose. J. Biol.
Chem. 1933, 102, 187–201.
(28) Bergmeier, S. C.; Stanchina, D. M. Acylnitrene Route to Vicinal
Amino Alcohols. Application to the Synthesis of (-)-Bestatin and
Analogues. J. Org. Chem. 1999, 64, 2852–2859.
(29) Lee, J. H.; Jang, K. C.; Jeong, I.-Y.; Yang, M. S.; Lee, S. G.; Park,
K. H. Chirospecific synthesis of the (2S,3R)- and (2S,3S)-3-amino-2-
hydroxy-4-phenylbutanoic acids from sugar: Application to (-)-Bestatin.
Synthesis 2003, 6, 829–836.
(30) Gogoi, N.; Boruwa, J.; Barua, N. C. A total synthesis of (-)-
bestatin using Shibasaki’s asymmetric Henry reaction. Tetrahedron Lett.
2005, 46, 7581–7582.
(31) George, S.; Suryavanshi, G. S.; Sudalai, A. A short enantiose-
lective synthesis of (-)-bestatin via l-proline-catalyzed [alpha]-amina-
tion of an aldehyde. Tetrahedron Lett. 2008, 49, 6791–6793.
(32) Rubin, A. E.; Sharpless, K. B. A Highly Efficient Aminohydrox-
ylation Process. Angew. Chem., Int. Ed. Engl. 1997, 36, 2637–2640.
(33) Donohoe, T. J.; Johnson, P. D.; Pye, R. J. The tethered aminohy-
droxylation (TA) reaction. Org. Biomol. Chem. 2003, 1, 2025–2028.
(34) Fraser, D. S.; Park, S. B.; Chong, J. M. Diastereoselective
reduction of alpha-aminoketones: synthesis of anti- and syn-beta-ami-
noalcohols. Can. J. Chem. 2004, 82, 87–101.
(48) Potterton, L.; McNicholas, S.; Krissinel, E.; Gruber, J.; Cowtan,
K.; Emsley, P.; Murshudov, G. N.; Cohen, S.; Perrakis, A.; Noble, M.
Developments in the CCP4 molecular-graphics project. Acta Crystal-
logr., Sect. D: Biol. Crystallogr. 2004, 60, 2288–2294.
(49) Dixon, M. The determination of enzyme inhibitor constants.
Biochem. J. 1953, 55, 170–171.
(50) Cornish-Bowden, A. A simple graphical method for determin-
ing the inhibition constants of mixed, uncompetitive and noncompeti-
tive inhibitors. Biochem. J. 1974, 137, 143–144.
(51) Trager, W.; Jensen, J. B. Human malaria parasites in continuous
culture. Science 1976, 193, 673–675.
(52) Leslie, A. G. W. Recent changes to the MOSFLM package for
processing film and image plate data. Joint CCP4 þ ESF-EAMCB
Newsletter on Protein Crystallography 1992, 26, 27-33.
(53) Evans, P. Scaling and assessment of data quality. Acta Crystal-
logr., Sect. D: Biol. Crystallogr. 2006, 62, 72–82.
(54) The CCP4 suite: programs for protein crystallography. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 1994, 50, 760-763.
(55) Brunger, A. T. Assessment of phase accuracy by cross valida-
tion: the free R value. Methods and applications. Acta Crystallogr., Sect.
D: Biol. Crystallogr. 1993, 49, 24–36.
(35) Anand, N.; Kapoor, M.; Koul, S.; Taneja, S. C.; Sharma, R. L.;
Qazi, G. N. Chemoenzymatic approach to optically active phenylglyci-
dates: resolution of bromo- and iodohydrins. Tetrahedron: Asymmetry
2004, 15, 3131–3138.
(56) Potterton, E.; Briggs, P.; Turkenburg, M.; Dodson, E. A
graphical user interface to the CCP4 program suite. Acta Crystallogr.,
Sect. D: Biol. Crystallogr. 2003, 59, 1131–1137.
(57) Konagurthu, A. S.; Whisstock, J. C.; Stuckey, P. J.; Lesk, A. M.
MUSTANG: a multiple structural alignment algorithm. Proteins 2006,
64, 559–574.
(36) Feske, B. D.; Stewart, J. D. Chemoenzymatic formal total
synthesis of (-)-bestatin. Tetrahedron: Asymmetry 2005, 16, 3124–
3127.
1665
dx.doi.org/10.1021/jm101227t |J. Med. Chem. 2011, 54, 1655–1666

Journal of Medicinal Chemistry
ARTICLE
(58) DeLano, W. The PyMOL Molecular Graphics System; Scientific
D DeLano Scientific: San Carlos, CA USA, 2002.
(59) Androulakis, S.; Schmidberger, J.; Bate, M. A.; DeGori, R.;
Beitz, A.; Keong, C.; Cameron, B.; McGowan, S.; Porter, C. J.; Harrison,
A.; Hunter, J.; Martin, J. L.; Kobe, B.; Dobson, R. C.; Parker, M. W.;
Whisstock, J. C.; Gray, J.; Treloar, A.; Groenewegen, D.; Dickson, N.;
Buckle, A. M. Federated repositories of X-ray diffraction images. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 2008, 64, 810–814.
1666
dx.doi.org/10.1021/jm101227t |J. Med. Chem. 2011, 54, 1655–1666