On-bead screening of a combinatorial fumaric acid derived peptide library yields antiplasmodial cysteine protease inhibitors with unusual peptide sequences

Article abstract of DOI:10.1021/jm900629w

A new class of cysteine protease inhibitors based on fumaric acid derived oligopeptides was successfully identified from a high-throughput screening of a solid-phase bound combinatorial library. As target enzymes falcipain and rhodesain were used, which play important roles in the life cycles of the parasites which cause malaria (Plasmodium falciparum) and African sleeping sickness (Trypanosoma brucei rhodesiense). The best inhibitors with unusual amino acid sequences not reported before for this type of enzyme were also fully analyzed in detail in solution. K_i values in the lower micromolar and even nanomolar region were found. Some inhibitors are even active against plasmodia and show good selectivity relative to other enzymes. Also the mechanism of action was studied and could be shown to be irreversible inhibition.

Full text of DOI:10.1021/jm900629w

5662 J. Med. Chem. 2009, 52, 5662–5672
DOI: 10.1021/jm900629w
On-Bead Screening of a Combinatorial Fumaric Acid Derived Peptide Library Yields Antiplasmodial
Cysteine Protease Inhibitors with Unusual Peptide Sequences
Uwe Machon,†,# Christian Buchold,‡ Martin Stempka,‡ Tanja Schirmeister,*,‡ Christoph Gelhaus,§ Matthias Leippe,§ Jiri Gut,
Philip J. Rosenthal, Caroline Kisker,^ Matthias Leyh,^ and Carsten Schmuck*,†,#
†Institute of Organic Chemistry, University of Duisburg-Essen, Universitatstrasse 7, 45141 Essen, Germany, ‡Institute of Pharmacy and Food
Chemistry, University of Wurzburg, Am Hubland, 97074 Wurzburg, Germany, §Institute of Zoology, University of Kiel, Olshausenstrasse 40,
24098 Kiel, Germany, Department of Medicine, San Francisco General Hospital, University of California, San Francisco, Box 0811, San
Francisco, California 94143, and ^Rudolf-Virchow-Zentrum DFG-Forschungszentrum fur Experimentelle Biomedizin, Versbacher Strasse 9,
97078 Wurzburg, Germany. # Former address: Institute of Organic Chemistry, University of Wurzburg, Am Hubland, 97074 Wurzburg, Germany.
Received May 13, 2009
A new class of cysteine protease inhibitors based on fumaric acid derived oligopeptides was successfully
identified from a high-throughput screening of a solid-phase bound combinatorial library. As target
enzymes falcipain and rhodesain were used, which play important roles in the life cycles of the parasites
which cause malaria (Plasmodium falciparum) and African sleeping sickness (Trypanosoma brucei
rhodesiense). The best inhibitors with unusual amino acid sequences not reported before for this type of
enzyme were also fully analyzed in detail in solution. Ki values in the lower micromolar and even
nanomolar region were found. Some inhibitors are even active against plasmodia and show good
selectivity relative to other enzymes. Also the mechanism of action was studied and could be shown to be
irreversible inhibition.
Introduction
invasion, and hemoglobin degradation. Treatment with cys-
teine protease inhibitors blocks hemoglobin hydrolysis and
development of the parasite.12 While falcipain-1 is suggested
to play a principal role in nonerythrocytic stages,13 falcipain-2
and falcipain-3 are likely the major hemoglobinases in
the food vacuole of erythrocytic parasites.14 Therefore, the
inhibition of cysteine proteases presents a promising strategy
for combating these infections. However, no drugs that target
these proteases are yet on the market.
Proteases specifically recognize their protein or oligopeptide
substrate, and knowledge about substrate specificity aids the
development of inhibitors. This information can be obtained
by using combinatorial libraries of labeled peptides.15,16 On the
other hand, combinatorial libraries of small molecules prepared
with mechanism-based scaffolds have resulted in the identifica-
tion of potent inhibitors of various proteases.17,18 Such inhibi-
tors, if cell-permeable, can serve as lead compounds for the
development of new drugs or as biochemical tools to study the
function of the respective proteases in vitro and in vivo.
In this paper we describe the synthesis of a combinatorial
heptapeptide library containing fumaric acid as an electro-
philic building block which can covalently block the active site
Cys residue of falcipain-2 and rhodesain via a Michael-type
reaction. Compounds from this library also showed promis-
ing antiplasmodial activity in vitro.
The burden of tropical diseases of humans caused by
protozoan parasites is large, both in terms of mortality and
morbidity and because these diseases impede economic
growth and prosperity.1 Diseases such as malaria (caused by
various species of Plasmodium) and African trypanosomiasis
(sleeping sickness caused by Trypanosoma brucei gambiense
and Trypanosoma brucei rhodesiense) are among the most
severe.2,3 Late-stage trypanosomiasis is characterized by somno-
lence and coma, leading invariably to death, if untreated.
Chemotherapy depends principally on drugs developed dec-
ades ago that lack adequate efficacy and cause serious side
effects. Further, the emergence ofdrug-resistant Trypanosoma
strains has been reported.2 Similarly in malaria,4 increasing
resistance5 of malaria parasites to antimalarial drugs, the lack
of highly effective vaccines, and inadequate control of mos-
quito vectors demand new approaches to drug development.
In both diseases one promising strategy to develop new
drugs has been to target parasite cysteine proteases.3,6 These
enzymes, termed rhodesain7 (RD) in T. b. rhodesiense, bruci-
pain in T. brucei brucei (infective to animals), and falcipains8
(falcipain-1, falcipain-2, falcipain-20, and falcipain-3)9,10 in
Plasmodium falciparum, belong to the cathepsin L subfamily
of the papain family (clan CA, family C1; CAC1) of cysteine
proteases.11 Cysteine protease inhibitors have been shown to
kill African trypanosomes in vitro and in animal models of
the disease.3 Proteases of malaria parasites play pivotal roles
in the processes of host erythrocyte rupture, erythrocyte
Several efficient irreversible inhibitors of proteases based
on activated olefins are known. For example, the fumaric acid
derivative of the well-known epoxysuccinyl peptide (S,S)-
epoxysuccinyl-(S)-leucylamido(4-guanidino)butane (E-64), the
papain inhibitor DC-11,19 has been shown to irreversibly
block CAC1 cysteine proteases, and recently related peptidyl
ene diones20 and azapeptide Michael acceptors21 have been
identified as potent inactivators of cysteine proteases. Other
*Corresponding authors. T.S.: phone, þ49 931 8885440; fax,
þ49 931 8885494; e-mail, schirmei@pzlc.uni-wuerzburg.de. C.S.: phone,
þ49 201 1833097; fax, þ49 201 1834259; e-mail, carsten.schmuck@
uni-due.de.
r
pubs.acs.org/jmc
Published on Web 08/31/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5663
Table 1. Composition of the Heptapeptide Fumaric Acid Diamide
Librarya
and mix method25 on amino functionalized PEGA resin.a
The use of PEGA resin is essential as initial experiments
showed. The screening is performed in aqueous solutions so
that the applied resin must have good swelling properties in
polar solvents. On-bead enzyme assays described in the
literature (vide infra) used libraries based on TentaGel26 or
PEGA resin.27,28 Both resins contain polyethylene glycol
(PEG) chains and therefore swell efficiently in water so that
large biomolecules like enzymes can reach the resin-bound
compounds. However, TentaGel, a polystyrene resin that is
grafted with PEG chains, shows a weak but nevertheless
existing interaction with proteins which can interfere with
the screening results.29 Thus, we decided to use the amino
PEGA resin that is completely based on PEG chains and
therefore is virtually inert toward proteins. We used the
IRORI technique with radio frequency tagged micro chips
to be able to determine the amino acid sequence during and
after the synthesis.30 Each of the 150 members was synthe-
sized on 30 mg of resin in a MikroKan that was scanned and
redistributed to individual reactors after each coupling step
to obtain the combinatorial library spatially separated. The
coupling steps were monitored by the Kaiser test31 for free
amino functions and the malachite green test32 for carboxy-
lates. For all coupling reactions HCTU was used as coupling
agent, NMM as base, and DMF as solvent. Every coupling
step was performed twice overnight. The first four amino
acids were introduced by the standard Fmoc strategy, and
the protecting group was removed with a solution of 20%
piperidine in DMF. Then the monoprotected fumaric acid 2
(Scheme 2) was introduced and deprotected with 50% TFA
in dichloromethane. The other peptide arm was synthe-
sized via an N- to C-coupling strategy using tert-butyl ester
protected amino acids with free amino functions.33 The ester
group was cleaved with 50% TFA in dichloromethane after
each coupling step. Finally, the whole library was washed
thoroughly with DMF, methanol, and dichloromethane.
The monoprotected fumaric acid building block 2 was
synthesized as shown in Scheme 2. Fumaric acid was con-
verted to the monochloride with 1 equiv of oxalyl chloride in
dichloromethane. This acid chloride was reacted with potas-
sium tert-butylate to obtain the tert-butyl ester 2.
P4
P3
P2
P1
P10
P20
P30
Val
Val
Ala
Leu
Leu
Ile
Gln
Asn
Gly
Ala
Gly
Phe
Phe
Chg
Phg
C1-NHAc
Ala
Phe
a Due to the insertion of the fumaric acid moiety the definition of the
amino acid residues as P1, P2, P10, P20, etc. does not necessarily implicate
binding of these residues into the respective substrate binding pockets
(S1, S2, S10, S20, etc.) of the enzyme.
fumaric acid derivatives have also been tested against these
proteases.22,23 In order to further explore the suitability of
fumaric acid derivatives, we established a synthetic strategy
for a fumaric acid diamide library and developed on-bead
screening assays for falcipain-2 and rhodesain. This allowed
fast high-throughput screening and selective identification of
new inhibitors with amino acids unusual for cathepsin L-like
cysteine proteases. Several inhibitors identified by on-bead
screening of the library were synthesized in larger scale, and
inhibition constants against the target enzymes as well as
in vitro antiparasitic activities were determined.
Results
Library Design and Synthesis. The library was designed
according to Table 1. The selection of amino acids mainly
accounted for the known preference of CAC1 proteases for
hydrophobic amino acids in the P2 position (Leu, Ile, Phe,
Chg, Phg). The unnatural amino acids cyclohexylglycine
(Chg) and phenylglycine (Phg) were chosen in order to
enhance proteolytic stability. For P3 different nonpolar
amino acids were used (Val, Ala, Leu) whereas P4 as well
as the primed-site amino acids were held constant or only
marginally varied in P10 (Gly, Ala). The designation of
residues as P1, P10, etc. is simply used here as a convenient
numbering scheme but does not imply binding of these
residues into the respective substrate binding pockets (S1,
S2, S10, S20, etc.) of the enzymes. Due to the central fumarate
core the residues are shifted with respect to a normal peptidic
substrate and might very well occupy different binding
pockets.
CAC1 cysteine proteases strongly prefer Arg in the P1
position. A library which includes Arg as amino acid would
most likely lead to a hit list dominated by this amino acid. In
order to avoid this and to find new amino acids possibly
fitting into the S1 binding site, we did not use Arg, but
instead either the aromatic amino acid Phe, Ala, or amino
acids with amide side chains (Gln, Asn, C1-NHAc) were
chosen. This library design with amino acids such as Gln or
Asn in the P1-position offers the advantage that the inhibi-
tors can also be tested against viral cysteine proteases.24
However, as published recently, falcipains accommodate
Gln in the P1-position as well.15 In total, the library consisted
of 150 compounds (a complete list of all library members can
be found in the Supporting Information).
All of the amino acids used for the synthesis of the library
were commercially available except the unnatural amino
acid Fmoc-C1-NHAc 7. Its synthesis is shown in Scheme 3
starting from Cbz-protected Asn 3. The first step is a
literature known Hofmann degradation.34 Then the free
amine was acetylated with 1 equiv of acetic anhydride. In
the last steps the amino function was deprotected with H2
and Pd/C, and the Fmoc group was introduced.
On-Bead Enzyme Assays. There are several on-bead en-
zyme assays described in the literature for different enzymes.
Assays are described for the trypanothione reductase,35 the
aldose reductase,36 the human serine racemase,37 the protein
disulfide isomerase,38 and Brk protein tyrosine kinase,39 but
the best investigated enzymes are proteases. On-bead assays
have been described for all four classes of proteases: metallo,
aspartic, serine, and cysteine proteases.40 In these assays in
a Abbreviations: AMC, aminomethylcoumarin; HCTU, 2-(6-chloro-
1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium hexafluorophos-
phate; IRORI, company which developed the radiofrequency tagging
technology used here; NMM, N-methylmorpholine; MBHA resin,
4-methylbenzhydrylamine hydrochloride salt resin; PEGA resin, beaded
polyethylene glycol polyacrylamide copolymer; PEG, polyethylene
glycol; TFMSA, trifluoromethanesulfonic acid.
Synthesis of the Library. The solid-phase synthesis of the
inhibitor library (Scheme 1) was performed by using the split

5664 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18
Machon et al.
Scheme 1. Synthesis of the Fumaric Acid Heptapetide Library
Scheme 2. Synthesis of Fumaric Acid tert-Butyl Ester 2
We chose a different approach here. Our screening assay is
based on a one-bead-one-compound library of potential
inhibitors. As the IRORI-radiofrequency tagging methodo-
logy provides the individual library members spatially sepa-
rated, they can be separately incubated with both substrate
and enzyme in solution. Furthermore, using a microtiter
plate reader the assay can provide quantitative inhibition
data for each library member. Individual library members
can then be compared, and an initial structure-activity
relationship can be derived. After incubation of the library
with the target enzymes (falcipain-2 or rhodesain) for 12 h at
9 ꢀC the substrate (Cbz-Phe-Arg-AMC, 40 μM) was added,
and the residual enzyme activity was monitored by fluore-
scence spectroscopy. The enzyme hydrolyzes the substrate,
thus liberating the fluorescent aminomethylcoumarin (AMC).
The more efficient the inhibition, the lower the fluorescence
increase. With this method we obtained quantitative on-bead
inhibition data for all 150 inhibitors in the library for both
falcipain-2 and rhodesain. The inhibition for both proteases
ranged between 7% and 77% under these experimental
conditions. Hence, our library spanned the whole range from
poor to good inhibitors. Since the Km values of the substrate
used (Cbz-Phe-Arg-AMC) are quite different for the target
enzymes (falcipain-2, 21.5 μM; rhodesain, 827 nM; res-
pectively), the percentages of inhibition for the two enzymes
found in the on-bead screenings cannot be compared di-
rectly. The substrate is a much better competitor in the case
of rhodesain due to its higher affinity for this enzyme, so that,
e.g., 25% inhibition of rhodesain means much better inhibi-
tion than 25% inhibition of falcipain-2. However, for both
enzymes the results were similar concerning the preferences
for the various amino acids as expected based on the
homology of both proteins. The active sites are largely
general a combinatorial library is incubated with an enzyme
of interest, and the hits are detected by colorimetric or
fluorogenic tools, then sorted, and finally analyzed by Ed-
man sequencing, MALDI-TOF mass spectrometry, or MAS
NMR spectroscopy. There are both protease substrate
specificity assays and inhibitor screening assays based on
fluorescence resonance energy transfer (FRET). Substrate
specificity assays are based on libraries of FRET-substrates
in the form of one-bead-one-compound libraries. These
assays can provide information about the preferences for
the hydrolysis of peptide sequences by a given enzyme.
During incubation of the library with a protease, some beads
show fluorescence due to a fast cleavage of the attached
substrates by the enzyme.
In contrast, inhibitor screening assays so far have been based
on one-bead-two-compound libraries: Each bead has a good
FRET-substrate and one member of the library of potential
inhibitors attached. After incubation with a protease, nonfluor-
escent beads indicate inhibitors.41 This methodology requires
the synthesis of both the inhibitor and the FRET-substrate on
the bead, which increases the synthetic effort and also limits the
library screening to one type of substrate/enzyme combination.
Furthermore, all on-bead assays reported so far, whether
substrate specificity assays or inhibitor screenings, provide only
qualitative yes/no information. One can only decide whether
there is a hit or not due to the occurrence of fluorescence or
color of individual beads.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5665
Scheme 3. Synthesis of the Fmoc-Protected Amino Acid Fmoc-C1-NHAc
Table 2. Classification of the Inhibitors in Groups A-E by Their On-Bead Inhibitory Potential and Number of Library Members Included in Each
Subset
A
B
C
D
E
inhibition in on-bead
falcipain-2
rhodesain
I g 60%
60% > I g 50%
50% > I g 40%
40% > I g 30%
30% > I
19
37
45
34
42
32
29
24
15
23
Figure 1. Relative frequencies of amino acids at P3 for falcipain-2 (left) and rhodesain (right). Subsets were defined as follows according to the
inhibition I observed in the on-bead screening: subset A, I g 60%; subset B, 60% > I g 50%; subset C, 50% > I g 40%; subset D, 40% > I g
30%; subset E, 30% > I.
Figure 2. Relative frequencies of amino acids at P2 for falcipain-2 (left) and rhodesain (right). Subsets were defined as follows according to
the inhibition I observed in the on-bead screening: subset A, I g 60%; subset B, 60% > I g 50%; subset C, 50% > I g 40%; subset D, 40% >
I g 30%; subset E, 30% > I.
identical.42 However, there are some differences which might
be interesting as starting points to develop more specific
inhibitors for just one of the two enzymes (vide infra).
By comparison of the quantitative inhibition data, we were
furthermore able to derive some structure-activity relation-
ships. For this, we subdivided the library into five groups,
A-E, according to the inhibitory potential of the com-
pounds (Table 2). Then the relative frequencies of the various
amino acids in each of the four variable positions for each
subset were analyzed.
The relative frequencies of each amino acid in position P1
to P3 are illustrated in Figures 1-3. At P3 for both enzymes
Leu is the most efficient amino acid. However, this prefer-
ence is much more pronounced for rhodesain than for
falcipain-2. The former does not tolerate any other amino
acid in P3. The inhibition significantly drops for both valine
and alanine. About 80% of the most potent inhibitors
(subset A) have Leu in P3 and only about 15% Val and even
less for Ala. In contrast, Ala in P3 leads to mostly inactive
inhibitors for rhodesain (>95% occurrence in subset E). For

5666 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18
Machon et al.
Figure 3. Relative frequencies of amino acids at P1 for falcipain-2 (left) and rhodesain (right). Subsets were defined as follows according to the
inhibition I observed in the on-bead screening: subset A, I g 60%; subset B, 60% > I g 50%; subset C, 50% > I g 40%; subset D, 40% > I g
30%; subset E, 30% > I.
Scheme 4. Solid-Phase Synthesis of the 14 Selected Library
Members
falciapin-2 the choice of amino acid in P3 is much less
decisive. The three amino acids are more equally distributed
over all five subsets. For example, in the most potent
inhibitors (subset A), Ala is nearly as often found as Leu
(both ca. 40%) and even Val is found in 20% of the potent
inhibitors. In the moderate inhibitors (subset C) again all
three amino acids are found with similar frequency. The only
significant selectivity is that Leu is not found in the worst
inhibitors (<5% occurrence). Therefore, the amino acid in
P3 might provide a starting point for the development of
inhibitors which can efficiently differentiate between falci-
pain-2 and rhodesain despite their overall similarity.
In contrast to this, at P2 we did not observe any clear
preferences for any amino acid in both enzymes (Figure 2).
This means that all five amino acids which were included in
the library at this position (Leu, Phg, Chg, Phe, Ile) are
equally well accepted by both enzymes. This position is
important for neither the potency of the inhibitors nor the
enzyme selectivity at least with this subset of amino acids
used here. This seems at the first glance to contradict results
from other substrate selectivity screenings which indicate a
strong preference for Leu at this position.43 However, the
two unnatural amino acids Phg and Chg we used here have
not been tested in those earlier reports which concentrated
development of enzyme-selective inhibitors. At P10 for both
on standard proteinogenic amino acids. Also, it is known
from other enzymes (e.g., SARS-coronavirus main protease)
that Leu can be substituted by Phg and Chg while maintain-
ing activity.44 Furthermore, lacking any structural data on
these new inhibitors, we do not know their exact positioning
in the active site of the enzymes. The fumaric acid moiety in
the central part of our inhibitors might affect the binding
mode of the peptide in the enzyme, causing a slightly
different selectivity pattern for the individual binding sites
as compared to a natural peptide substrate.
enzymes Gly is slightly preferred compared to Ala; however,
the difference is not significant (data not shown).
To conclude, our combinatorial quantitative on-bead
screening not only revealed new and potent inhibitors for
two medically important cysteine proteases, falcipain-2 and
rhodesain, but also provided insight into some interesting
structure-activity relationships which might prove useful
for the further development of a next generation of enzyme-
selective inhibitors.
The situation for P1 is similar to that of P3 but with a
reversed behavior of both enzymes (Figure 3). Rhodesain does
not show any clear preference for any of the five amino acids
used at P1. In contrast to this, falcipain-2 now significantly
prefers Asn in P1 whereas the “reversed amide” in C1-NHAc
leads to a nearly complete loss of inhibition. About 47% of the
most potent inhibitors (subset A) contain Asn in P1, followed
by Phe (ca. 26%) but no C1-NHAc. In contrast, nearly 60% of
the worst inhibitors had C1-NHAc at P1. This is remarkable in
two aspects. The chemical difference between Asn and C1-
NHAc is rather small, as both contain an amide group but in a
reversed order. Second, this preference for Asn and the
complete intolerance for C1-NHAc by falcipain-2 but not
rhodesain again might be useful as a starting point for the
Synthesis of Selected Library-Derived Inhibitors. However,
in order to validate these on-bead screening results, 14 inhibi-
tors from the library were selected for further evaluation
in solution. The selected inhibitors fulfilled the following
requirements: (1) They were selected from all five subsets
A-E, so that both highly potent and inactive inhibitors
could be tested. (2) The best amino acids for each of the four
variable positions were included. The synthesis of these
inhibitors (Scheme 4) followed the synthesis of the library
as described above, but this time an amino-functionalized
MBHA resin was used. After the coupling of the last amino
acid the peptidic compounds were cleaved from the resin
under acidic conditions with 10% TFMSA in TFA and
isolated by precipitation from diluted hydrochloric acid.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5667
Table 3. Inhibition of Falcipain-2 (FP-2) and Antiplasmodial Activities of 14 Selected Library Members
on-bead inhibition
(%) of FP-2
inhibition (%) of
FP-2 at 100 μM
Ki (μM),
FP-2
IC50 (μM),
P. falciparum
no.
structurea
8
H2N-ValrLeurLeurAsnrFum-Gly-Gly-Phe-OH
H2N-ValrAlarLeurAsnrFum-Gly-Gly-Phe-OH
H2N-ValrLeurPherAsnrFum-Gly-Gly-Phe-OH
H2N-ValrLeurPhgrAsnrFum-Gly-Gly-Phe-OH
H2N-ValrLeurLeurPherFum-Gly-Gly-Phe-OH
H2N-ValrValrLeurGlnrFum-Ala-Gly-Phe-OH
H2N-ValrAlarChgrAsnrFum-Gly-Gly-Phe-OH
H2N-ValrValrChgrAsnrFum-Gly-Gly-Phe-OH
H2N-ValrValrChgrGlnrFum-Gly-Gly-Phe-OH
H2N-ValrAlarChgrC1-NHAcrFum-Gly-Gly-Phe-OH
H2N-ValrLeurLeurAlarFum-Gly-Gly-Phe-OH
H2N-ValrLeurLeurAlarFum-Ala-Gly-Phe-OH
H2N-ValrLeurLeurC1-NHAcrFum-Gly-Gly-Phe-OH
H2N-ValrAlarLeurC1-NHAcrFum-Gly-Gly-Phe-OH
70
41
70
51
69
48
73
61
51
23
55
58
54
32
92
33
49
76
100
45
79
90
83
83
73
86
79
18
10.1
67.5
nd
74 ( 9b
nd
nd
9
10
11
12
13
14
15
16
17
18
19
20
21
21.3
0.8
nib
nib
nd
nd
12.2
5.2
75 ( 9b
32 ( 7b
27 ( 10b
22 ( 4b
nic
1.5
7.0
nd
9.5d
11.8d
nd
nic
nic
nic
Reference Compounds
E-64
CQ
nd
nd
nd
nd
0.29e
nd
5.3b,e
0.24c,e
a Fum, fumaric acid (OdC;HCdCH;CdO); Chg, cyclohexylglycine; Phg, phenylglycine; C1-NHAc, acetylaminoalanine; r, peptide sequence
reversed from the C- to N-terminus; nd, not determined. All values are mean values of at least two independent assays; mean standard deviations are less
than 5-10% unless otherwise indicated. b Strain FCBR. c Strain W2. d Cbz-Leu-Arg-AMC ([S] = 25 μM, Km = 8.4 μM) used as substrate. e Taken from
ref 45; E-64, (S,S)-epoxy-succinyl-(S)-leucylamido(4-guanidino)butane; CQ, chloroquine.
The compounds are initially isolated as the free carboxylic
acids, some of which have a limited solubility in aqueous
solutions. To obtain better soluble compounds, the triethyl-
ammonium salts of the inhibitors were therefore prepared.
Biological Activities. These 14 inhibitors were tested
against falcipain-2 (FP-2) and rhodesain (RD) in standard
fluorescence assays as published previously.45,46 The hydro-
lyses of Cbz-Phe-Arg-AMC or Cbz-Leu-Arg-AMC in the
absence or presence of the respective inhibitor were mea-
sured by following the fluorescence increase due to release of
AMC. As a positive control the well-known cysteine pro-
tease inhibitor E-6447,48 and as negative control the solvent
DMSO were used. In addition, inhibition was tested against
mammalian analogues of these two cysteine proteases,
namely, cathepsins L and B. Since falcipain-2 and rhodesain
belong to the cathepsin L-like cysteine proteases, this study
should also clarify the subtype selectivity of the inhibitors.
With the most potent inhibitors dialysis assays were per-
formed in order to verify the irreversible inhibition mech-
anism. Unfortunately, the limited long-time stability of
falcipain-2 and rhodesain prevented any dialysis studies with
these two enzymes. We therefore tested inhibitor 15, which
revealed to be also quite active against cathepsin B, in a
dialysis experiment. These studies proved the expected irre-
versible inhibition mechanism, since after 3 h of continuous
dialysis against assay buffer the enzyme activity did not
recover (inhibitor concentration 200 μM, 74% and 73%
inhibition before and after dialysis, respectively).
controls, and the solvent DMSO was used as a negative
control. Antitrypanosomal activities were determined
against T. b. brucei as published earlier.46 The well-known
drugs melarsoprol, eflornithine, suramine, nifurtimox, and
pentamidine were used as positive controls. All inhibition
data are summarized in Tables 3, 4, and 5.
Summary of the Results. Comparing the results obtained in
the on-bead screening with the standard solution assays
at 100 μM inhibitor concentration, a good correlation is
observed. In general, for both enzymes, rhodesain and
falcipain-2, >50% inhibition in the on-bead screening re-
sults in >50% inhibition at 100 μM in solution. Even more
importantly, the relative trends in the inhibition data are
similar for both solid-phase-bound inhibitors and free in-
hibitors in solution. Therefore, we successfully could identify
new and potent inhibitors using our combinatorial on-bead
screening assay.
The most potent inhibitors are peptides 12, 15, and 16 with
Ki values in the lower micromolar or even nanomolar region
(Ki(12) = 0.8/0.2 μM, Ki(15) = 5.2/0.08 μM, and Ki(16) =
1.5/0.08, for falcipain-2/rhodesain, respectively). Again,
small changes in the structure can lead to significant differ-
ences in inhibition potency as was also observed in the on-
bead assay. For example, replacing Phe in P1 position in
12 against an amino acid with an amide side chain (Asn (8)
or C1-NHAc (20)) leads to an at least 10-fold decrease
in inhibition potency for both falcipain-2 (Ki = 10.1 and
11.8 μM, respectively) and rhodesain (Ki=2.7 and 1.8 μM,
respectively). In agreement with the results from the on-bead
screening no significant differences can be found for Gly or
Ala in P10 (compare inhibitors 18 and 19) or the exchange of
an amino acid in P2 (compare inhibitors 10 and 11 with
either Phe or Phg in P2 or inhibitors 9 and 14 with either Ala
or Chg in P2).
First, initial screenings with 100 μM inhibitor concentra-
tion were performed. For inhibitors which displayed con-
siderable activity in this screening, dissociation constants
Ki were determined in order to specify the affinities to the
target enzymes.49 Inhibitors displaying Ki values in the lower
micromolar range were further tested for antiplasmodial
and antitrypanosomal activities. Antiplasmodial activities
were determined with the P. falciparum strain FCBR using
a recently published assay50 or by flow cytometry accord-
ing to a previously published method using P. falciparum
strain W2.10 Chloroquine51 and E-6452 were used as positive
Inhibitor 12 was also explicitly tested with or without
added detergent to exclude that nonspecific aggregate for-
mation53 is the reason for the observed inhibition results
(which would have been very unlikely, in light of the positive
on-bead screening assay in which aggregation cannot occur,

5668 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18
Machon et al.
Table 4. Inhibition of Rhodesain (RD) and Cathepsin B (CB) and Antitrypanosomal Activities of 14 Selected Library Members
on-bead
inhibition (%)
of RD
inhibition (%)
of RD at
inhibition (%)
of CB at
Ki (μM), IC50 (μM),
Ki (μM),
CB
no.
structurea
100 μM
RD
T. b. b.
100 μM
8
9
H2N-ValrLeurLeurAsnrFum-Gly-Gly-Phe-OH
H2N-ValrAlarLeurAsnrFum-Gly-Gly-Phe-OH
71
24
61
50
69
64
26
46
53
25
65
67
60
32
63
8
2.7
nd
nd
nd
nd
nd
nd
nd
nd
ni
21
17
24
13
73
23
78
100
81
83
38
45
ni
nd
nd
10 H2N-ValrLeurPherAsnrFum-Gly-Gly-Phe-OH
11 H2N-ValrLeurPhgrAsnrFum-Gly-Gly-Phe-OH
12 H2N-ValrLeurLeurPherFum-Gly-Gly-Phe-OH
13 H2N-ValrValrLeurGlnrFum-Ala-Gly-Phe-OH
14 H2N-ValrAlarChgrAsnrFum-Gly-Gly-Phe-OH
15 H2N-ValrValrChgrAsnrFum-Gly-Gly-Phe-OH
16 H2N-ValrValrChgrGlnrFum-Gly-Gly-Phe-OH
17 H2N-ValrAlarChgrC1-NHAcrFum-Gly-Gly-Phe-OH
18 H2N-ValrLeurLeurAlarFum-Gly-Gly-Phe-OH
19 H2N-ValrLeurLeurAlarFum-Ala-Gly-Phe-OH
20 H2N-ValrLeurLeurC1-NHAcrFum-Gly-Gly-Phe-OH
21 H2N-ValrAlarLeurC1-NHAcrFum-Gly-Gly-Phe-OH
6
nd
nd
nd
nd
26
68
37
98
96
99
95
99
96
87
30
0.2
nd
22.9
nd
0.4
0.08
0.08
0.3
1.0
1.3
1.8
nd
22.2
1.4
nd
ni
ni
12.4
nd
nd
nd
nd
nd
nd
nd
ni
nd
Reference Compounds
MS
EF
SA
NX
PN
nd
nd
94
31
nd
nd
nd
nd
nd
0.0026b
30.7b
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
32
nd
nd/28c
0.31b
3.4b
0.0029b
nd/nd
a Fum, fumaric acid (OdC;HCdCH;CdO); Chg, cyclohexylglycine; Phg, phenylglycine; C1-NHAc, acetylaminoalanine; r, peptide sequence
reversed from the C- to N-terminus; nd, not determined. All values are mean values of at least two independent assays; mean standard deviations are less
than 5-10% unless otherwise indicated. b Taken from ref 46 c [I] = 200 μM. MS, melarsoprol; EF, eflornithine; SA, suramine; NX, nifurtimox; PN,
pentamidine.
Table 5. Inhibition of Cathepsin B (CB) and Cathepsin L (CL) of Selected Library Members
structurea
no.
Ki (μM), CB
Ki (μM), CL
12
14
15
17
18
19
20
21
H2N-ValrLeurLeurPherFum-Gly-Gly-Phe-OH
H2N-ValrAlarChgrAsnrFum-Gly-Gly-Phe-OH
H2N-ValrValrChgrAsnrFum-Gly-Gly-Phe-OH
H2N-ValrAlarChgrC1-NHAcrFum-Gly-Gly-Phe-OH
H2N-ValrLeurLeurAlarFum-Gly-Gly-Phe-OH
H2N-ValrLeurLeurAlarFum-Ala-Gly-Phe-OH
H2N-ValrLeurLeurC1-NHAcrFum-Gly-Gly-Phe-OH
H2N-ValrAlarLeurC1-NHAcrFum-Gly-Gly-Phe-OH
22.9
22.2
1.4
12.4
nd
34.8
nd
37.5
nd
30.7
33.6
40.0
20.8
nd
nd
nd
a Fum, fumaric acid (OdC;HCdCH;CdO); Chg, cyclohexylglycine; Phg, phenylglycine; C1-NHAc, acetylaminoalanine; r, peptide sequence
reversed from the C- to N-terminus; nd, not determined. All values are mean values of at least two independent assays; mean standard deviations are less
than 5-10% unless otherwise indicated.
but nevertheless we wanted to address this question expli-
citly). The Ki values were identical, proving that any non-
specific enzyme inhibition in solution did not occur.
14 or 17 show intermediate activity against falcipain-2 and
cathepsins.
With the exception of 12 which is not active against
Plasmodium, the other most potent falcipain-2 inhibitors
such as 15-17 also display moderate antiplasmodial activity
(IC50 ca. 20-30 μM). In contrast, no antitrypanosomal
activity can be observed, even with the most potent rhode-
sain inhibitors 15, 16, and 17. Most likely, activity was
limited either by the bioavailability of these overall rather
nonpolar peptides or by problems with long-time hydrolytic
stability within the assay conditions.
Some of the most potent inhibitors, namely, 12 and 15,
also show good selectivity when measured against cathepsin
B (CB) or cathepsin L (CL). With the exception of inhibi-
tor 15 which displays good inhibition of cathepsin B (Ki =
1.4 μM) all other inhibitors exhibit Ki values in the upper
micromolar range (12-40 μM) against the mammalian
enzymes. Especially the selectivity indices for rhodesain are
quite high (CB, 18, CL, 470 (15); CB, 115, CL, 174 (12); CB,
56 (14); CB, 41 (17); CL, 31 (18); respectively). For falcipain
the selectivity was somewhat lower; e.g., inhibitors such as
Summary
A solid-phase-bound combinatorial library of 150 fumaric
acid derived peptides was synthesized and tested as cysteine
protease inhibitors against falcipain and rhodesain, essential
proteases of the two parasites P. falciparum and T. b. rhode-
siense responsible for severe tropical infections (malaria and
sleeping sickness, respectively). The inhibitors were synthe-
sized in macroscopic amounts using a combinatorial split-mix
approach and the IRORI-radio frequency tagging techno-
logy. The inhibitors were then screened directly on-bead in a
newly developed quantitative high through-put fluorescence
assay with the two target enzymes. Efficient inhibition of
both enzymes was observed. Based on the quantitative inhibi-
tion data for all library members thus obtained, a first
structure-activity relationship for this new class of protease
inhibitors was derived. Unusual combinations of amino
acids, not reported before for these enzymes, were found to
be most active. Selected members of the library were then

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5669
resynthesized in larger quantities and studied in detail to
obtain the Ki values. The most potent inhibitors (peptides
12, 15, and 16) have Ki values in the lower micromolar or even
nanomolar region (Ki(12)=0.8/0.2 μM, Ki(15)=5.2/0.08 μM,
and Ki(16)=1.5/0.08 for falcipain-2/rhodesain, respectively)
and are also selective relative to other enzymes such as
cathepsin B or cathepsin L. The best inhibitors are also active
against Plasmodium whereas no activity against Trypanosoma
was found. Further studies revealed that the mechanism of
action is irreversible inhibition of the enzymes.
Nr-Fmoc-β-acetylamino-L-alanine (7). A mixture of 5 (8.01 g,
28.6 mmol) and Pd/C (801 mg) in methanol (150 mL) was
hydrogenated at 35 ꢀC for 7 h. The mixture was filtered through
Celite to remove Pd/C. The Celite was washed with a mixture of
5% hydrochloric acid and methanol (1/1, 100 mL), and the
solvent of the combined filtrates was evaporated to give the
deprotected amino acid (6.34 g) as a white solid. This crude
product was used in the next step without further purification.
A solution of Fmoc-Cl (8.14 g, 31.5 mmol) in dioxane was
added dropwise at 0 ꢀC to a solution of the crude amino acid
6 (6.34 g) in an aqueous solution of sodium carbonate (100 mL,
10%). The reaction mixture was stirred at room temperature for
20 h and then acidified with concentrated hydrochloric acid
(pH 2). The mixture was extracted with dichloromethane (3 ꢀ
100 mL). The solvent of the combined organic layers was evapo-
rated to obtain a yellow oil. The crude product was purified by
flash column chromatography on silica gel (dichloromethane/
methanol=9/1, 0.1% acetic acid) to give 7 (3.45 g, 33%) as a
Experimental Section
General Remarks. Reaction solvents were dried and distilled
before use. All other reagents were used as obtained from
Aldrich, Fluka, IRIS, or GL Biochem. NR-Cbz-β-amino-L-
alanine (4) was synthesized according to literature.34 All experi-
ments were run in oven-dried glassware. 1H and 13C NMR
spectra were recorded with a Bruker Avance 400 spectrometer.
The chemical shifts are reported relative to the deuteriated
solvents. Peak assignments are based on either DEPT and/or
comparison with literature data. The ESI and HR mass spectra
were recorded with a Finnigan MAT 900 S spectrometer.
Melting points are not corrected. Purity of compounds was
determined using analytical HPLC with a Dionex HPLC appa-
ratus consisting of a P680 HPLC pump, an ASI-100 automated
sample injector, and a UVD 340U UV detector with a Supelcosil
LC-18 (25 cm ꢀ 4.6 mm, 5 μm) column. Commercially available
HPLC grade solvents were used as eluents. All compounds used
for solution phase assays had a purity of >95%.
1
white solid: mp 237 ꢀC (dec). H NMR (400 MHz, DMSO-d6,
27 ꢀC): δ = 1.80 (s, 3H, CH3), 3.27-3.34 (m, 1H, CH2),
3.44-3.48 (m, 1H, CH2), 4.04-4.09 (m, 1H, CH), 4.23-4.30
(m, 3H, Fmoc-CH2, Fmoc-CH), 7.33 (t, JHH = 7.3 Hz, 2H,
3
Fmoc-aryl CH), 7.42 (m, 3H, Fmoc-aryl CH, Fmoc-NH), 7.72
(d, 3JHH=7.4 Hz, 2H, Fmoc-aryl CH), 7.90 (m, 3H, Fmoc-aryl
CH, CH2NH) ppm. 13C NMR (100 MHz, DMSO-d6, 27 ꢀC):
δ=22.5 (CH3), 39.8 (CH2NH2), 46.6 (Fmoc-CH), 54.1 (CH),
65.7 (Fmoc-CH2), 120.1, 125.2, 127.1, 127.6 (Fmoc-aryl CH),
140.7, 143.8 (Fmoc-aryl Cq), 155.9, 169.7, 172.1 (CdO) ppm.
FT-IR (KBr pellet): ν~=3405 (m), 2931 (w), 1700 (s), 1660 (s),
1545 (s), 1265 (m), 1035 (m) cm-1. HR-MS (positive ESI): calcd
for C20H20N2O5 [M þ Naþ] 391.12644; found 391.12575.
Solid-Phase Synthesis of the Peptidic Inhibitors 8-21. The
peptidic inhibitors were synthesized on MBHA resin according
to a standard protocol. MBHA resin (300 mg, 1.3 mmol/g,
390 μmol) was swollen in DMF for 1 h, and the first four amino
acids were coupled to the resin by the use of Fmoc-protected
amino acids (2.5 equiv), HCTU (2.5 equiv), and N-methylmor-
pholine (NMM, 300 μL) in DMF (10 mL) for 20 h. Each
coupling step was repeated twice, and the resin was washed
with DMF (3 ꢀ 10 mL). The Fmoc group was then cleaved
with piperidine in DMF (20%, 10 mL), and the resin was washed
with DMF (6 ꢀ 10 mL). The fumarate 2 was introduced under
the same coupling conditions using HCTU, NMM, and DMF.
The tert-butyl ester was removed by the following conditions:
the resin was washed with dichloromethane (3 ꢀ 10 mL), treated
for 5 min with a solution of TFA in dichloromethane (25%,
10 mL), and treated for 30 min with a solution of TFA in
dichloromethane (50%, 10 mL). The resin was then washed with
dichloromethane (3 ꢀ 10 mL) and with DMF (3 ꢀ 10 mL).
The next three amino acids were coupled using the tert-butyl
ester protected amino acid (2.5 equiv), HCTU (2.5 equiv),
NMM (300 μL), and DMF (10 mL) for 20 h. Each coupling
step was repeated twice, the tert-butyl ester was removed as
described above, and the resin was washed with dichloro-
methane (3 ꢀ 10 mL), methanol (3 ꢀ 10 mL), diethyl ether
(2 ꢀ 10 mL), and dichloromethane (3 ꢀ 10 mL). Cleavage from
the resin was performed by exposure to TFA/TFMSA (9/1)
for 3 h. The resin was washed with TFA (3 ꢀ 10 mL), and the
solvent of the combined filtrates was then evaporated to obtain a
yellow oil. The residue was treated with hydrochloric acid
(0.1 M, 20 mL), and the white precipitated solid was collected.
In order to obtain a better solubility water and triethylamine
(20%) were added, and the suspension was lyophilized. The
ratio of contained triethylamine was different for each com-
pound and shown in parentheses after the molecular formula.
The amounts of triethylamine were determined by the integrals
in the 1H NMR spectra of the inhibitors. In some cases we
observed the formation of triethylammonium chloride that
could not be removed. The additional salt did not have any
influence on the inhibition of the cysteine proteases.
Mono-tert-butyl Fumarate (2). A solution of oxalyl chloride
(24.0 g, 189 mmol) in dichloromethane (20 mL) was added
dropwise to a solution of fumaric acid (1, 20.0 g, 172 mmol) in
dichloromethane (100 mL) and DMF (5 mL). The mixture was
stirred for 1 h at room temperature, and the solvent was
removed by distillation under reduced pressure. The remaining
residue was dissolved in tert-butyl alcohol (75 mL). The solution
was cooled to 0 ꢀC in an ice bath, and potassium tert-butylate
(21.2 g, 189 mmol) was added. The mixture was stirred for 2 h at
room temperature, and the solvent was removed under reduced
pressure. The crude product was purified by flash column
chromatography on silica gel (hexane/ethyl acetate, 0.1% acetic
acid) to give 2 (3.40 g, 12%) as a white solid: mp 66 ꢀC. 1H NMR
(400 MHz, DMSO-d6, 27 ꢀC): δ=1.46 (s, 9H, CH3), 6.59 (dd,
3JHH=15.6 Hz, 4JHH=19.8 Hz, 2H, 2 ꢀ olefin CH), 13.08 (br s,
1H, COOH) ppm. 13C NMR (100 MHz, DMSO-d6, 27 ꢀC): δ=
27.6 (CH3), 81.4 (CqMe3), 133.8, 134.3 (olefin CH), 163.7, 165.8
(CdO) ppm. FT-IR (KBr pellet): ν~=2985 (m), 1714 (s), 1472
(w), 1423 (w), 1370 (m), 1305 (m), 1272 (s), 1160 (s) cm-1
.
HR-MS (positive ESI): calcd for C8H11O4 [M - Hþ] 171.06628;
found 171.06623.
Nr-Cbz-β-acetylamino-L-alanine (5). Acetic anhydride (3.60 mL,
37.4 mmol) was added dropwise to a suspension of NR-Cbz-
β-amino-L-alanine (4, 8.10 g, 34.0 mmol) in dichloromethane
(200 mL). Then triethylamine (10.5 mL, 74.8 mmol) was added,
and the reaction mixture was stirred for 5 h at room temperature
until the suspension turned into a clear solution. The solvent was
removed under reduced pressure. The obtained oil was sus-
pended in water and lyophilized to give 5 (8.01 g, 84%) as a white
solid: mp 43 ꢀC (dec). 1H NMR (400 MHz, DMSO-d6, 27 ꢀC):
δ=1.77 (s, 3H, CH3), 3.23-3.37 (m, 2H, CH2), 3.84-3.89 (m,
3
1H, CH), 5.01 (s, 2 H, Cbz-CH2), 6.90 (d, 1H, JHH=7.4 Hz,
NHCbz), 7.29-7.38 (m, 5H, aryl CH), 7.79 (br s, 1H, NH) ppm.
13C NMR (100 MHz, ]DMSO-d6, 27 ꢀC): δ=22.5 (CH3), 39.8
(CH2NH), 54.0 (CH), 65.5 (Cbz-CH2), 127.7, 127.8, 128.3
(aryl CH), 136.9 (Cq), 156.0, 169.9, 172.0 (CdO) ppm. FT-IR
(KBr pellet): ν~=3350 (m), 3060 (w), 2950 (w), 1720 (s), 1540 (s),
1225 (m), 1065 (m) cm-1. HR-MS (negative ESI): calcd for
C13H15N2O5 [M - Hþ] 279.09865; found 279.09864.

5670 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18
Machon et al.
Detailed spectroscopic data of these inhibitors can be found
in the Supporting Information.
NH2-ValrLeurLeurAsnrFum-Gly-Gly-Phe-OH (8, 8/9
NEt3). C38H57N9O11 (8/9 C6H15N): 905.86 g/mol. Yield 265 mg
(293 μmol, 75%). Mp 228 ꢀC (dec). MS (negative MALDI): calcd
for C38H56N9O11- [M - Hþ] 814.411; found 814.629.
6.5, 2 mM DTT, 5 mM EDTA, 200 mM NaCl. Substrates and
inhibitor stock solutions were prepared in DMSO and diluted
with the respective assay buffer (final DMSO concentration:
10%). Total volume: 250 μL (215 μL of assay buffer, 10 μL of
enzyme solution, 20 μL of DMSO or inhibitor solution, 5 μL of
substrate solution). Enzyme stock solutions were diluted with
assay buffer to yield a fluorescence increase of dF/min ≈ 20.
A Varian Cary eclipse fluorescence spectrophotometer (Varian,
NH2-ValrAlarLeurAsnrFum-Gly-Gly-Phe-OH (9, NEt3,
1/2 HNEt3Cl). C35H51N9O11 (C6H15N, 1/2 C6H15ClN): 1034.91 g/
mol. Yield 266 mg (257 μmol, 66%). Mp 193 ꢀC (dec). MS (negative
Darmstadt, Germany) with 96-well plates was used; λex
380 nm and λem=460 nm.
=
-
MALDI): calcd for C35H50N9O11 [M - Hþ] 772.364; found
Screening. The hydrolysis of the substrate Cbz-Phe-Arg-
AMC (40 μM) or Cbz-Leu-Arg-AMC (in the case of 12 and
13, 25 μM) by the respective enzyme was monitored in the
absence or presence of 100 μM inhibitor (final concentration).
The slope of the progress curve in the absence of inhibitor was
set to 100% (=vc). Percentage of inhibition was calculated using
the equation % inhibition=100(1 - vi/vc) with vi as the inhibited
rate of the reaction.
Determination of Ki Values. The hydrolyses of the substrates
were monitored over a period of 5-10 min in the presence of
0-80 μM inhibitor (final inhibitor concentrations: 0.1, 1, 2, 4, 5,
6, 8, 10, 20, 40, 50, 60, 80 μM). The Ki values were calculated
using the Dixon equation vo/vi=1 þ ([I]/Kiapp) and correction to
zero substrate concentration with Ki=Kiapp/(1 þ [S]/Km]) with
[S] = 40 μM and Km = 21.5 μM for falcipain-2, 827 nM for
rhodesain, 150 μM for cathepsin B, and 6.25 and 6.5 μM for
cathepsin L. The GraFit6 software was used to calculate the
Kiapp values.
772.362.
NH2-ValrLeurPherAsnrFum-Gly-Gly-Phe-OH (10, 7/9
NEt3). C41H55N9O11 (7/9 C6H15N): 928.63 g/mol. Yield 293 mg
(316 μmol, 81%). Mp 165 ꢀC (dec). MS (negative MALDI):
calcd for C41H54N9O11- [M - Hþ] 848.395; found 848.733.
NH2-ValrLeurPhgrAsnrFum-Gly-Gly-Phe-OH (11, NEt3).
C40H54N9O11 (C6H15N): 937.09 g/mol. Yield 314 mg (335 μmol,
86%). Mp 205 ꢀC (dec). MS (negative MALDI): calcd for
C40H53N9O11- (M - Hþ) 834.379; found 834.382.
NH2-ValrLeurLeurPherFum-Gly-Gly-Phe-OH (12, 8/9
NEt3). C43H60N8O10 (8/9 C6H15N): 938.93 g/mol. Yield 308 mg
(328 μmol, 84%). Mp 160 ꢀC (dec). MS (negative MALDI):
calcd for C43H59N8O10- [M - Hþ] 847.436; found 847.796.
NH2-ValrValrLeurGlnrFum-Ala-Gly-Phe-OH (13, 2/3
NEt3). C39H60N9O11 (2/3 C6H15N): 897.40 g/mol. Yield 311
mg (347 μmol, 89%). Mp 240 ꢀC (dec). MS (negative MALDI):
calcd for C39H59N9O11- [M - Hþ] 828.426; found 828.751.
NH2-ValrAlarChgrAsnrFum-Gly-Gly-Phe-OH (14, NEt3,
1/3 HNEt3Cl). C43H68N10O11 (1/3 C6H15N): 946.94 g/mol. Yield
262 mg (277 μmol, 71%). Mp 245 ꢀC (dec). MS (negative MAL-
DI): calcd for C43H67N10O11- (M - Hþ) 798.379; found 798.733.
NH2-ValrValrChgrAsnrFum-Gly-Gly-Phe-OH (15, NEt3).
C39H57N9O11 (C6H15N): 929.11 g/mol. Yield 250 mg (269 μmol,
69%). Mp 232 ꢀC (dec). MS (negative MALDI): calcd for
C39H56N9O11- [M - Hþ] 826.411; found 826.661.
Evaluations of Cultured Malaria Parasites. These were done as
described previously.50
Antitrypanosomal Activities. These were done as described
previously.55
On-Bead Screening. The respective enzyme was incubated
with samples of the resin-bound inhibitors. The resin was added
in isopyknotic suspensions in DMSO (20 μL, 20 mg/mL). The
hydrolysis of the substrate Cbz-Phe-Arg-AMC (40 μM) by the
proteases was monitored in the presence of 0.64 mM inhibitor
(final concentration, determined by the theoretical loading of
the resin, 0.40 mmol/g). The obtained kinetic data were com-
pared with a sample of amino-functionalized PEGA resin
(without inhibitor). The slope of the progress curve of the PEGA
resin was set to 100% so that the inhibition could be determined
using the equation: % inhibition=100(1 - vi/vc).
NH2-ValrValrChgrGlnrFum-Gly-Gly-Phe-OH (16, NEt3,
2/3 HNEt3Cl). C40H59N9O11 (C6H15N, 2/3 C6H16ClN): 1034.91 g/
mol. Yield 319 mg (308 μmol, 79%). Mp 250 ꢀC (dec). MS
(negative MALDI): calcd for C40H58N9O11- [M - Hþ] 840.426;
found 840.661.
NH2-ValrAlarChgrC1(Ac)rFum-Gly-Gly-Phe-OH (17,
NEt3, 4/9 HNEt3Cl). C38H55N9O11 (C6H15N, 4/9 C6H15ClN):
976.27 g/mol. Yield 263 mg (269 mmol, 69%). Mp 225 ꢀC (dec).
-
MS (negative MALDI): calcd for C38H54N9O11 [M - Hþ]
812.395; found 812.660.
Acknowledgment. We thank the DFG (Deutsche For-
schungsgemeinschaft) for ongoing financial support of our
work (SFB 630, TP A3 and TP A4). We also thank the Z1
NH2-ValrLeurLeurAlarFum-Gly-Gly-Phe-OH (18, 2/3-
NEt3). C37H56N8O10 (2/3 C6H15N): 840.35 g/mol. Yield 161 mg
(191 μmol, 49%). Mp 244 ꢀC (dec). MS (negative MALDI):
calcd for C37H55N8O10- [M - Hþ] 771.400; found 771.432.
NH2-ValrLeurLeurAlarFum-Ala-Gly-Phe-OH (19, 7/9
NEt3). C38H58N8O10 (7/9 C6H15N): 540.65 g/mol. Yield 287
mg (332 μmol, 85%). Mp 228 ꢀC (dec). MS (negative MALDI):
calcd for C38H57N8O10- [M - Hþ] 785.420; found 785.421.
NH2-ValrLeurLeurC1(Ac)rFum-Gly-Gly-Phe-OH (20,
8/9 NEt3). C39H59N9O11 (8/9 C6H15N): 919.89 g/mol. Yield
190 mg (207 μmol, 53%). Mp 231 ꢀC (dec). MS (negative
€
project of SFB 630 (PD Dr. A. Stich, Dr. T. Olschlager, and
their staff) for the tests against Trypanosoma.
Supporting Information Available: On-bead inhibition data of
the complete combinatorial library for falcipain-2 (FP-2) and
rhodesain (Rd) and spectroscopic data of peptidic inhibitors
8-21. This material is available free of charge via the Internet at
http://pubs.acs.org.
-
MALDI): calcd for C39H58N9O11 [M - Hþ] 828.430; found
828.408.
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NH2-ValrAlarLeurC1(Ac)rFum-Gly-Gly-Phe-OH (21,
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