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1275
structure 1F29, PDB code) in structure in the active site.20 This
O
O
O
i
ii
N2
computational information, coupled with the report by Ellman
and others21 of compound 2 as a peptidomimetic inhibitor of cruz-
ain, we proceeded with docking studies of cruzain inhibitor 2 in FP-
2. The studies revealed that inhibitors of cruzain would be a good
starting structural motif for the design of novel inhibitors of falci-
pain cysteine proteases. The overlap in structural features of our
designed motif 3 and the cruzain inhibitor 2 are shown in blue in
Figure 1.
BocHN
BocHN
BocHN
Cl
OH
Ph
Ph
Ph
4
5
6
Scheme 1. Synthesis of
morpholine, isobutyl chloroformate, 1 h, reflux; THF, 0 °C, CH2N2, 2 h, 72%; (ii) HCl–
AcOH (1:1), ether, 0 °C, 1 h, 89%.
a
-chloroketone 6. Reagents and conditions: (i) 4-Methyl-
Of particular note, one of the phenylalanine residues in 2 has
been replaced with a homophenylalanine in 3 and one of the carb-
oxybenzyl-protected (Cbz-protected) phenylalanine amino acids
in 2 has been replaced with a relatively constrained Cbz-protected
proline derivative in 3. The use of unnatural amino acids was delib-
erately made for two reasons: to by-pass peptidase metabolism,
thereby increasing the compound half-life; and to decrease analog
flexibility to improve binding thermodynamics. For the later, utiliza-
tion of cyclic derivatives such as b-amino acids was appropriate. To
probe the available 3D-chemical space for the substrate binding site
of FP-2 and to explore the SAR, we designed and synthesized twenty-
two compounds (21–42) from readily available building blocks.
The syntheses of the twenty-two pseudodipeptidic cysteine
protease inhibitors are dependent upon the construction of a hom-
studies against the W2-strain (chloroquine resistant) of P. falcipa-
rum were also carried out.8,19,22–25 The biological activities of the
derivatives are summarized in Table 1. While several peptidomi-
metics showed moderate activity in one of the three biological
tests, compound 39 was clearly a superior candidate in both iso-
forms of FP-2, FP-3, and W2 inhibition of 80 nM, 60 nM, and
7.70 lM respectively.
In an attempt to understand the SAR of the peptidomimetics,
we first considered the binding affinity in the active site of FP-2
and FP-3. From the experimental SAR, it was evident that hydroxyl
proline in the S2 pocket, an
a-hydroxyketone electrophile in the
S1–S0 pocket, and homophenylalanine in the S1 pocket were suit-
2
able substituents for high binding affinity peptidomimetics. Inter-
estingly, efforts to understand the SAR of this series by means of
steric and electrostatic interactions derived from the docking stud-
ies in FP-2 and FP-3 were not sufficient to explain the variation in
biological data. Van der Waals energy, electrostatics, hydrogen
bonds, or Docking Score from Glide SP26,27 docking calculations
did not provide a statistical correlation with experimental binding
affinity. Furthermore, implicit solvent binding energy estimations
from MM–GBSA (as implemented in the program Prime)28,29 did
not show a significant correlation with experimental binding
affinity.
In light of the above findings, we anticipated the involvement of
water molecules in the binding of these inhibitors, since explicit
water solvation is neglected from all of the above analyses. There-
fore, we generated thermodynamic profiles of water molecules
present in the ligand binding domain (LBD) of FP-2 and FP-3 using
WaterMap (Schrodinger, LLC) in an attempt to understand the ob-
served SAR among the designed pseudopeptidomimetics. Water-
Map computes the location and energetics of water molecules
around a protein using explicit solvent molecular dynamics (MD),
solvent clustering, and statistical thermodynamics.30,31 WaterMap
was chosen to study the protein solvation effects because it has
been effectively applied to a broad range of pharmaceutically rele-
vant targets including PDZ domains,32 kinases,33 G-protein coupled
receptors (GPRCs),34 protein–protein interaction interfaces,35 and
serine proteases.36 Recently, a WaterMap study was reported by
our group to understand binding modes of small molecule inhibi-
tors of falcipain (FP-2 & FP-3) identified by virtual screening.37
ophenylalanine-based scaffold representing the C-terminus: an a-
substituted ketone intended to interact with the catalytic cysteine
thiol moiety; and the N-terminal residue. Peptide-like coupling of
the
N-Cbz proline gives such a pseudodipeptide (3).
Specifically, synthesis of the -chloroketone 6 was accom-
a-chloroketone corresponding to homophenylalanine, 6, with
a
plished by in situ activation of the amino acid 4 as its mixed anhy-
dride followed by quench with diazomethane to yield the
diazoketone 5, best immediately converted to the more stable
a-
chloroketone 6 with HCl in AcOH (Scheme 1). We chose eleven dif-
ferent synthetic amino acids for coupling to 6, N-terminal residues
which occupy the opposing S2/S3 site of falcipain. Hence, the Boc-
group of 6 was removed with 2 M HCl in ether and the resulting
amine-HCl salt 7 was then coupled with the eleven Cbz-protected
unnatural amino acids furnishing the target compounds 9–20
(Scheme 2).
The N-terminal amino acids were all cyclic except for the use of
a homophenylalanine (e.g. 36). Most of these amino acids were
b-aminoacids, normally cis, and include b,b (e.g. 26) or
a,a (27)
1-aminocyclohexyl carboxylic acids. While the amino acids used
for peptide coupling are denoted as R in Scheme 2 for the sake of
simplicity, the exact structural details of the final compounds are
shown in Figure 2. The reactive
a-chloroketone of the derivatives
was easily displaced by O, N, or S nucleophiles resulting in
twenty-two target derivatives, 21–42 (Scheme 3).
The synthesized peptidomimetics were tested against FP-2 and
FP-3 isoforms in vitro. Additionally, in vitro growth inhibition
P3 site P2 site P1 site
P1' site
O
Ph
O
O
H
N
O
R2
O
S
Ph
H
N
Ph
O
N
H
3
S
R3
N
H
R1'
O
Ph
O
R1
1
2
P2 site P1 site
H
P1' site
R1
O
N
N
O
O
O
P3 site
3
Ph
Ph
Figure 1. Structural similarity of vinyl sulfone ligand 1 from the cruzain crystal structure and cruzain inhibitor 2 with our designed inhibitor 3.