Michael acceptor based antiplasmodial and antitrypanosomal cysteine protease inhibitors with unusual amino acids

Article abstract of DOI:10.1021/jm900946n

New peptidic Michael acceptor based cysteine protease inhibitors displaying antiparasitic activity were identified by testing a broad series of 45 compounds in total, containing Asn, Gln, or Phe. As target enzymes, falcipain-2 and -3 from P. falciparum and rhodesain from T. b. rhodesiense were used. In the case of the Asn/Gln containing compounds, the trityl-protected, diastereomeric ?'-configured vinylogous dipeptide esters 16 (Boc-(S)-Phg-(R/S)-vGln(Trt) -OEt) were discovered as most active inhibitors concerning both protease inhibition and antiparasitic acitivity, with inhibition constants in the submicromolar range. The compounds were shown to display time-dependent and competitive inhibition. In the case of the Phe containing compounds, the maleic acid derivatives 42 and 43 (BnOPhe←Mal-Phe-OBn, BnO-Phe-Mal←Phe-Ala- OBn, Mal = maleic acid) displayed good inhibition of rhodesain as well as good antitrypanosomal activity, while the fumaric acid derived E-analogue 14 (BnO-Phe-Fum-Phe-OBn) only displayed inhibition of the target enzymes but no antiparasitic activity. Inhibition by these Phe derivatives was shown to be time-independent and competitive.

Full text of DOI:10.1021/jm900946n

J. Med. Chem. 2010, 53, 1951–1963 1951
DOI: 10.1021/jm900946n
Michael Acceptor Based Antiplasmodial and Antitrypanosomal Cysteine Protease Inhibitors with
Unusual Amino Acids
†
†
†
†
†,
Alexander Breuning, Bjorn Degel, Franziska Schulz, Christian Buchold, Martin Stempka, Uwe Machon,‡
Saskia Heppner,§ Christoph Gelhaus,§ Matthias Leippe,§ Matthias Leyh, Caroline Kisker, Jennifer Rath,¥ August Stich,¥
Jiri Gut,^ Philip J. Rosenthal,^ Carsten Schmuck,*,‡ and Tanja Schirmeister*,†
€
€
†Institute of Pharmacy and Food Chemistry, University of Wu€rzburg, Am Hubland, 97074 Wu€rzburg, Germany, ‡Institute of Organic
§
Chemistry, University of Duisburg-Essen, Universitatstrasse 7, 45141 Essen, Germany, Institute of Zoology, University of Kiel,
€
Olshausenstrasse 40, 24098 Kiel, Germany, Rudolf-Virchow-Zentrum, DFG-Forschungszentrum fu€r Experimentelle Biomedizin,
University of Wu€rzburg, Josef-Schneider-Strasse 2, 97080 Wu€rzburg, Germany, ^Department of Medicine, San Francisco General Hospital,
University of California, San Francisco, Box 0811, San Francisco, California 94143, and ¥Department of Tropical Medicine,
Medical Mission Institute, Salvatorstrasse 7, 97074 Wu€rzburg, Germany
Received June 29, 2009
New peptidic Michael acceptor based cysteine protease inhibitors displaying antiparasitic activity were
identified by testing a broad series of 45 compounds in total, containing Asn, Gln, or Phe. As target
enzymes, falcipain-2 and -3 from P. falciparum and rhodesain from T. b. rhodesiense were used. In the
case of the Asn/Gln containing compounds, the trityl-protected, diastereomeric E-configured viny-
logous dipeptide esters 16 (Boc-(S)-Phg-(R/S)-vGln(Trt)-OEt) were discovered as most active inhibitors
concerning both protease inhibition and antiparasitic acitivity, with inhibition constants in the
submicromolar range. The compounds were shown to display time-dependent and competitive
inhibition. In the case of the Phe containing compounds, the maleic acid derivatives 42 and 43 (BnO-
PherMal-Phe-OBn, BnO-PherMal-Phe-Ala-OBn, Mal = maleic acid) displayed good inhibition of
rhodesain as well as good antitrypanosomal activity, while the fumaric acid derived E-analogue 14
(BnO-PherFum-Phe-OBn) only displayed inhibition of the target enzymes but no antiparasitic
activity. Inhibition by these Phe derivatives was shown to be time-independent and competitive.
Introduction
Trypanosoma strains has been reported.2 Similarly in malaria,
increasing resistance of malaria parasites to antimalarial
drugs, the lack of highly effective vaccines, and inadequate
control of mosquito vectors demand new approaches to drug
development.4-7
The burden of tropical diseases of humans caused by proto-
zoan parasites is large, 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 T. b. rhodesiense)
are among the most severe.2,3 Late-stage trypanosomiasis is
characterized by somnolence and coma, leading invariably to
death if untreated. Chemotherapy depends principally on drugs
developed decades ago that lack adequate efficacy and cause
serious side effects. Further, the emergence of drug-resistant
In both diseases one promising strategy to develop new drugs
has been to target parasite cysteine proteases.3,8 These enzymes,
termed rhodesain9 (RDa) in T. b. rhodesiense, brucipain in T. b.
brucei (infective to animals), and falcipains10-13 (falcipain-2,
falcipain-20, and falcipain-3) in Plasmodium falciparum, belong
to the cathepsin L subfamily of the papain family (clan CA,
family C1; CAC1)14 of cysteine proteases. Cysteine protease
inhibitors have been shown to kill African trypanosomes in
vitro and in animal models of the disease,3 although it is not yet
clear whether rhodesain is the only target of the inhibitors.15
Proteases of malaria parasites play pivotal roles in the processes
of host erythrocyte rupture, erythrocyte invasion, and hemo-
globin degradation. Treatment with cysteine protease inhibi-
tors blocks hemoglobin hydrolysis and development of the
parasite.6,16-19 Among the falcipains, falcipain-2 and falcipain-3
are likely the major hemoglobinases in the food vacuole of
erythrocytic parasites.8,12,20 Therefore, the inhibition of cysteine
proteases presents a promising strategy for combating these
infections.
*To whom correspondence should be addressed. For C.S.: phone,
þ49 201 1833097; fax, þ49 201 1834259; e-mail, carsten.schmuck@
uni-due.de. For T.S.: phone, þ49 931 8885440; fax, þ49 931 8885494;
e-mail, schirmei@pzlc.uni-wuerzburg.de.
a Abbreviations: ACN, acetonitrile; C1-NHAc, acetylaminoalanine;
cc, column chromatography; Chg, cyclohexylglycine; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; DCC, dicyclohexylcarbodiimide; DCM,
dichloromethane; DIBAL-H, diisobutylaluminum hydride; DIPEA, di-
isopropylethylamine; DMAP, 4-dimethylaminopyridine; DMF, dimethyl-
formamide; DPPA, diphenylphosphorylazide; DTT, dithiothreitole; FP-2,
falcipain-2; FP-3, falcipain-3; Fum, fumaric acid; HCTU, 2-(6-chloro1H-
benzotriazole-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate;
IBCF, isobutyl chloroformate; Mal, maleic acid; NBS, N-bromosuccini-
mide; NMM, N-methylmorpholine; Phg, phenylglycine; PyBOP, benzo-
triazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate; RD,
rhodesain;rt,roomtemperature;TEA, triethylamine;TFA, trifluoroacetic
acid; TLC, thin layer chromatography; Trt, trityl; vGln, vinyl-Gln. Amino
acids are (S)-configured unless otherwise indicated.
In a recent paper we described the synthesis of a combina-
torial heptapeptide library (Figure 1) based on fumaric acid as
an electrophilic building block.21 The selection of amino acids
r
2010 American Chemical Society
Published on Web 02/04/2010
pubs.acs.org/jmc

1952 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Breuning et al.
for the library considered the known preference of CAC1
proteases for hydrophobic amino acids in the P222 position
(Leu, Ile, Phe, cyclohexylglycine Chg, phenylglycine Phg).
For P3, different unpolar amino acids were used (Val, Ala,
Leu), whereas P4 amino acids were held constant and those at
P10 only marginally varied (Gly, Ala). For the P1-position
either nonpolar amino acids (Phe, Ala) or amino acids
with amide side chains (Gln, Asn, C1-NHAc) were chosen.
Via a new high-throughput on-bead assay, inhibitors with
amino acids unusual for cathepsin-L like cysteine proteases
were identified,21 namely, Gln and Asn at P1 and Phg and
Chg at P2.
We now present the synthesis and testing of a large series of
related peptidic inhibitors containing these amino acids or
respective peptides but with various modified Michael sys-
tems, e.g., trans- and cis-configured vinylogous amino acid or
peptide esters, crotonic acid derivatives, and acrylic acid
derivatives. Notably, these studies revealed trityl-protected
Asn- and Gln-derivatives as highly potent inhibitors.
The manuscript is organized as follows: first, we describe
thesyntheses of the potential inhibitors. Second, the biological
activities against the target enzymes (falcipain-2, falcipain-3,
rhodesain) and the parasites (P. falciparum, T. b. brucei) and
cytotoxicity data are presented. A chapter about the clarifica-
tion of enzyme inhibition mechanisms and the discussion of
the structure-activity relationships (SAR) complete the
manuscript.
Results
Since the recently described studies21 yielded inhibitor
H2N-ValrLeurLeurPherFum-Gly-Gly-Phe-OH (1) con-
taining Phe in the P1-position and inhibitors H2N-Valr
ValrChgrAsnrFum-Gly-Gly-Phe-OH (2) and H2N-Valr
ValrChgrGlnrFum-Gly-Gly-Phe-OH (3) containing
Asn or Gln in the P1-position as the most potent inhibitors
(Figure 1) (Ki values in μM for falcipain-2/rhodesain: (1)
0.8/0.2; (2) 5.2/0.08; (3) 7.0/0.3), we now prepared com-
pounds with the same amino acids but with a modified
Michael system. The various Michael acceptors, which are
bound to Gln, Asn, and Phe, or di-, tri- and tetrapeptide
sequences of the nonprimed site of the fumaric acid library
(Phg/Chg/Leu-Asn/Gln; Ala-Leu-Gln; Ala-Val-Chg-Gln; Val-
Val-Leu-Gln) were synthesized as depicted in Schemes 1-10.
Since the syntheses of the respective peptides yielded several
trityl-protected peptides (Trt = trityl) as intermediates,
these were also coupled to the various Michael acceptors,
and the resulting inhibitors were included for testing. In
addition, for selected compounds, the analogues with
Z-configured activated double bonds were synthesized
and tested (Schemes 11 - 13). Inhibitor structures and
results of biological tests are summarized in Table 1.
Syntheses. Compounds 4 and 5 were synthesized by
Steglich esterification of fumaric acid monoethyl ester with
Boc-asparaginol or Boc-glutaminol (Scheme 1). Deprotec-
tion with TFA and DPPA-mediated peptide coupling with
Boc-Phg yielded inhibitors 6 and 7.
The fumaric acid amides 8-12 (Scheme 2) were synthe-
sized starting from fumaric acid monoethyl ester and Asn-O-
t-But (f8), Trt-protected Asn-OMe (f9), Gln-O-t-But
(f10), Trt-protected Gln-OMe (f11), or Phe-OBn (f12)
by various standard peptide coupling methods. For the
synthesis of the free acid 13 fumaric acid mono-tert-butyl
ester21 was used. Final cleavage of the tert-butyl ester with
TFA in dichloromethane yielded compound 13.
Figure 1. Composition of the heptapeptide fumaric acid diamide
library: structures of inhibitors 1-3.21. (a) Because of the insertion
of the fumaric acid moiety into the peptide, the definition of the
amino acid residues as P1, P2, P10, P20, etc. does not implicate
binding of these residues into the respective substrate binding
pockets (S1, S2, S10, S20, etc.) of the enzyme. Depending on the
carbon atom attacked by the nucleophilic cysteine residue of
the enzyme, the binding mode may be shifted to either the P- or
P0-direction.21 (b) C1-NHAc, acetylaminoalanine.
Scheme 1. Syntheses of Fumaric Acid Diester Based Inhibitors 4-7

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1953
Scheme 2. Syntheses of the Fumaric Acid Amides 8-13
Scheme 3. Synthesis of the Fumaric Acid Diamide 14
Scheme 4. Synthesis of the Vinylogous Amino Acid (15) and Dipeptide Esters (16)
Scheme 5. Synthesis of Aminocrotonic Acid Esters 17-20
The fumaric acid diamide 14 was synthesized by reac-
tion of fumaric acid dichloride with 2 equiv of Phe-OBn
(Scheme 3).
The vinylogous amino acid ethyl esters 15 and 16 were
obtained by Masamune reaction or Horner-Wadsworth-
Emmons olefination (Scheme 4) of the aminoaldehyde

1954 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Breuning et al.
Scheme 6. Synthesis of Hydroxy- or Aminoacrylic Acid Esters 21-23
Scheme 7. Synthesis of Hydroxycrotonic Acid Esters 24-30
Scheme 9. Synthesis of Crotonic Acid Amide 32
Scheme 10. Synthesis of the Phe Ester 33
16b in a ratio of 8:2, providing us with a very small amount of
the minor isomer 16b after chromatographic separation.
Thus, this compound was at first only tested against the
parasites. Since these assays showed 16b to display very good
antiparasitic activity, weresynthesized this diastereomerusing
(R)-Gln(Trt) instead of (S)-Gln(Trt).
Scheme 8. Synthesis of the Hydroxycrotonic Acid Amide 31
The aminocrotonic acid esters 17-19 (Scheme 5) were
synthesized by N-alkylation of the respective amino acid
esters Asn-O-t-But (f17), Gln-O-t-But (f18), or Phe-OBn
(f19) with ethyl 4-bromocrotonate. Deprotection of the
tert-butyl ester 18 with TFA/anisole and PyBOP-mediated
peptide coupling with the tripeptide Chg-Val-Ala-OBn
yielded compound 20.
The Michael-type addition (Scheme 6) of either Boc-
glutaminol (f21) or Asn- or Gln-O-t-But (f22, 23) to ethyl
propiolate yielded the hydroxyacrylic acid ester (21) and the
aminoacrylic acid esters 22 and 23. While the addition of the
amino alcohol exclusively led to the E-isomer, the addition of
the amino acids yielded the E- and Z-isomers in a ratio
of about 1:2. Since no inhibition could be observed with the
E/Z-mixture in preliminary studies, we did not separate the
diastereomers.
The hydroxycrotonic acid esters 24 and 25 (Scheme 7) were
obtained by Steglich esterification of 4-hydroxycrotonic acid
with the respective Boc-protected amino acids. Compounds
26-30 (Scheme 7) were obtained by deprotection of 24 and
25, respectively, and PyBOP mediated peptide coupling with
amino acids or peptides.
Compound 31 (Scheme 8) was synthesized on solid phase
by using amino functionalized Sieber amide resin23 and
Fmoc-protected amino acids. The amino acids were acti-
vated with PyBOP in DMF and NMM as a base. An excess
of NMM and 2.5 equiv of the amino acids and PyBOP were
used in each coupling step. The 4-hydroxycrotonic acid was
synthesized starting from crotonic acid based on published
Boc-Gln(Trt)-H (f15) or the peptidylaldehydeBoc-Phg-Gln-
(Trt)-H (f16) at room temperature. At this temperature only
the respective E-isomers were obtained. In the case of the
dipeptide 16 the DIBAL-H reduction of the methyl ester led to
partial epimerization at the R-carbon atom of Gln(Trt). Thus,
the subsequent olefination yielded two diasteromers 16a and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1955
Scheme 11. Synthesis of Maleic Acid Esters and Amides 34-36, 38, and 40
Scheme 12. Synthesis of Maleic Acids 37, 39, 41 and Diamides
42 and 43
Scheme 13. Syntheis of the Vinylogous Z-Configured Amino
Acid Derivatives 44 and 45
procedures24-27 and coupled after activation with PyBOP in
DMF and NMM. Only 1.25 equiv of the reagents were used
this time to prevent the reaction of the unprotected alcohol
with the active ester. The crotonic acid amide 31 was
obtained after cleavage from the resin with 1% TFA and
purification by MPLC (RP-18 column, water and methanol).
Amide coupling (Scheme 9) of crotonic acid with Phe-OBn
using isobutyl chloroformate as coupling reagent yielded the
amide 32.
(E)-5-Hydroxypent-2-enoic acid ethyl ester, which was
synthesized by Wittig reaction of 3-hydroxypropanal (obtained
from reaction of acrolein with dilute sulfuric acid) with carbo-
ethoxymethyltriphenylphosphonium bromide, was reacted with
the acid chloride of Cbz-protected Phe to yield the ester 33
(Scheme 10).
The maleic acid diester 34 and the amides 35, 36, 38, and 40
(Scheme 11) were obtained either by Steglich esterification of
maleic acid monoethyl ester with Boc-phenylalaninol (f34)
or by various peptide coupling procedures with amino acid
or peptide esters.
Acylation of amino acid or peptide esters with maleic
acid anhydride yielded the maleic acids 37, 39, and 41. The
acid 37 was coupled via the PyBOP method to Phe-OBn or
Phe-Ala-OBn in order to obtain the diamides 42 and 43
(Scheme 12).
Biological Activities. The inhibitors were tested against
falcipain-2 (FP-2), falcipain-3 (FP-3), and rhodesain (RD) in
standard fluorescence assays as published previously.28,29 The
hydrolyses of Cbz-Phe-Arg-AMC (for RD) or Cbz-Leu-Arg-
AMC (for FP-2 and FP-3) in the absence or presence of the
respective inhibitor were measured by following the fluores-
cence increase due to release of AMC. The well-known cysteine
protease inhibitor E-64,30-32 as a positive control, and the
solvent DMSO, as negative control, were used. Studies to clarify
the inhibition mechanisms (time-dependent vs time-indepen-
dent inhibition, competitive vs noncompetitive inhibition,
effects of the low-molecular weight sulfur nucleophile DTT)
as well as studies to exclude nonspecific inhibition by aggrega-
tion33,34 were performed with the most active compounds.
Inhibitors were further tested for antiplasmodial and antitry-
panosomal activities. Antiplasmodial activities were determined
by flow cytometry according to a previously published method
using P. falciparum strain W212 or with the P. falciparum strain
FCBR using a previously published assay.35 Chloroquine36 and
E-6437 were used as positive controls, and the solvent DMSO
was used as a negative control. Antitrypanosomal activities were
determined against T. brucei brucei as published earlier.29 The
well-known drugs melarsoprol, eflornithine, suramine, nifurti-
mox, and pentamidine were used as positive controls. All
inhibition data are summarized in Table 1. For several com-
pounds assays on mammalian macrophages (J774.1) to evaluate
nonspecific toxicity were performed as previously described
(Table 2).29
Finally, the vinylogous amino acid ester 44 was obtained
with high Z-selectivity (Z/E = 13/1) by Horner-Wadsworth-
Emmons olefination (Scheme 13) of Boc-phenylalaninal at low
temperature. The olefination of Boc-glycinal yielded the inter-
mediate Z-configured vinylogous amino acid ester (R = H, Z/
E = 2.5/1), which (after column chromatography to separate
the Z- from the E-isomer) was then deprotected with TFA and
coupled to Boc-Phe using PyBOP as coupling reagent to give
inhibitor 45.
Studies on Inhibition Mechanisms. With some of the most
active compounds (namely, inhibitors 14, 15, 16a, 16b, 20,

1956 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Breuning et al.
Table 1. Inhibition of Falcipains (FP) and Rhodesain (RD) and Antiparasitic Activity of Michael Acceptors Containing Phe, Gln, or Asnd

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1957
Table 1. Continued

1958 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Table 1. Continued
Breuning et al.
a Strain FCBR. b Taken from ref 29. c Strain FCBR: 5.3 μM.28 d Chg, cyclohexyl glycine; Phg, phenylglycine; C1-NHAc, acetylaminoalanine; n.d., not
determined; n.i., no inhibition at 100 μM; E-64, (S,S)-epoxysuccinyl-(S)-leucylamido(4-guanidino)butane. All values are mean values of at least two independent
assays, with mean standard deviations less than 15% for antiprotease activity and less than 25% for antiparasitic activity unless otherwise explicitly indicated.
Table 2. Cytotoxicity Data for the Most Active Compoundsa
and 42), studies to elucidate the inhibition mechanisms were
performed.
compd
RD Ki (μM)
T. b. b. IC50 (μM)
J774.1 IC50 (μM)
Inhibitor 14 was subjected to dilution assays.38 The residual
activity of rhodesain was measured after 0, 5, 20, 35, and 50 min
incubation times tinc of enzyme and inhibitor prior to substrate
addition ([S] = 10 μM). This was done for 15 different inhibitor
concentrations of [I] = 0-100 μM. The inhibitor displayed
time-independent inhibition. Additionally, these experiments
were performed in the presence of 5 mM DTT. No differences
in Ki values were found. Both experiments showed that inhibitor
14 is a reversible inhibitor that does not undergo nucleophilic
Michael addition with either the enzyme or the low-molecular
weight thiol DTT. The same experiments were performed
with the Z-configured analogue 42. These assays also showed
inhibitor 42 to be a time-independent inhibitor.
12
15
16b
19
32
33
36
38
40
42
43
45
7.6
4.3
0.25
2.8
3.5
>100
60
n.d.
n.i.
3.5
25.0
2.8
36.1
45.2
>100
3.3
n.i.
36% (100 μM)
15% (100 μM)
43% (100 μM)
29% (100 μM)
0.15
19.0
8
2.7
3.0
29
14.9
2.9
31
32
1.4
47% (100 μM)
2.2
29.0
>100
a RD, rhodesain; T. b. b., Trypanosoma brucei brucei, J774.1, macro-
phage cell line; n.d., not determined; n.i., no inhibition at 100 μM.
In contrast, inhibitor 15 showed time-dependent inhibition
of rhodesain in the dilution assays.38 The second-order rate
Also, for the related inhibitors 16a and 16b time-dependent
inhibition of rhodesain was observed. The k2nd values were
determined to be 12 250 M-1 s-1 (16a) and 26483 M-1 s-1
(16b; ki = 0.19 ( 0.019 min-1; Ki = 0.14 ( 0.05 μM). A
similar second-order rate constant k2nd was found for inhi-
bition of falcipain-2 by 16b: 15 263 M-1 s-1 (ki = 0.44 (
0.053 min-1; Ki = 0.49 ( 0.015 μM).
In order to prove that inhibition is competitive with
respect to the substrate, Kiapp values for inhibition of rho-
desain by 16a were determined from the slopes of the first
minutes of the progress curves obtained without preincuba-
tion of enzyme and inhibitor for various substrate concen-
trations ([S] = 5, 10, 20, 50, and 100 μM). The apparent
dissociations constants Kiapp increased with increasing sub-
strate concentrations, proving that inhibition is competitive.
constant of inhibition k2nd was calculated to be 11 100 M-1 s-1
.
Again, inhibition was determined in the absence and presence
of 5 mM DTT in the assay buffer. The Ki values,39,40 which were
determined using the slopes of the first minutes of progress
curves obtained without preincubation of enzyme and inhibi-
tor, were determined to 4.3 μM (without DTT, Table 1) and
3.6 μM (with DTT), showing that the compound does not react
with low molecular weight thiols but only with the Cys residue
of the enzyme. In order to exclude nonspecific inhibition by
aggregation,33 which could be due to the trityl moiety, the
inhibition of falcipain-2 was also determined in the presence
of various concentrations of the nonionic detergent Brij 35
(0.005%, 0.01%, 0.025%). No differences were found, excluding
a nonspecific inhibition mechanism.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1959
A plot41-43 of Kiapp vs [S] yielded a Ki value of 0.67 μM. In
order to again exclude nonspecific inhibition by aggrega-
tion,33 the Ki values for inhibition of rhodesain were deter-
mined in the absence and presence of nonionic detergent,
both with the normally used enzyme concentration and with
a 2-fold higher enzyme concentration, and additionally, the
percentage of inhibition at 10 μM inhibitor concentration
was determined after 0, 0.5, 2, and 5 min of spinning a
solution containing enzyme and inhibitor in a microfuge.
The experiments showed that the inhibition was independent
of the absence or presence of nonionic detergent and inde-
pendent of the enzyme concentration (Ki values: low enzyme
concentration þ detergent, 0.30 μM; low enzyme concentra-
tion without detergent, 0.64 μM; high enzyme concentration þ
detergent, 0.40 μM; high enzyme concentration without
detergent, 0.64 μM) and also does not decrease after spinning
the solution in a microfuge. Together with the observed
competitive inhibition mechanisms, these results further prove
that inhibition was not due to aggregation.
Inhibition of falcipain-2 by compound 20 was determined
with either the substrate Cbz-Phe-Arg-AMC or Cbz-Leu-
Arg-AMC. The Kiapp values were corrected to zero substrate
concentration, yielding Ki values of 5.4 μM (Cbz-Phe-Arg-
AMC) and 6.7 μM (Cbz-Leu-Arg-AMC), proving that
inhibition is competitive with respect to the substrate.
Structure-Activity Relationships (SARs). The previous
high-throughput on-bead screening21 yielded fumaric acid
peptides with Gln or Asn in P1-position and with Phg or Chg
in P2-position or with Phe in P1-position as most potent
inhibitors. Now we included compounds with these amino
acids or respective peptides but modified acceptor-substi-
tuted alkene moieties. Thus, the following discussion of the
SARs will consider the amino acids of the P1-position (Asn,
Gln/Phe) and the Michael system.
Inhibitors with Asn or Gln. The fumaric acid diesters 6 and
7, which contain the dipeptide moieties Asn-Phg and Gln-
Phg instead of only Asn or Gln alone (compounds 4 and 5),
were more active against the enzymes and the parasites than
4 and 5. Also, the fumaric acid diamides 8 and 10 containing
only Asn or Gln were not active or only weakly active.
Interestingly, inhibition potency against both enzymes and
parasites was enhanced if the amino acid residues contained
a trityl group, especially in the case of Gln (compounds 9 and
11). This is also true for the vinylogous amino acid esters and
dipeptides 15 and 16, which were among the most active
inhibitors against enzymes and parasites. Again, the addi-
tional Phg moiety in inhibitors 16 led to improvement.
Notably, the minor diastereomer 16b with (R)-Gln instead
of (S)-Gln, which first could only be isolated in low yields but
was resynthesized using (R)-Gln, displayed very high activity
against the parasites and target proteases. Since a trityl group
is a large lipophilic moiety that may lead to aggregation of
the compounds, we examined the inhibition mechanisms of
inhibitors 15 and 16a/b (see above). All assays showed that
inhibition was not due to aggregation but rather was time-
dependent and competitive, indicating that the inactivation
results from a Michael-type reaction between the Cys residue
of the enzyme and the inhibitor. In a comparison of inhibi-
tors 11 and 15, the reversed peptide sequence (C-N in 15 vs
N-C in 11) and/or the shorter distance between the activated
double bond and the Trt-proteced Gln residue seem to be
superior for the inhibition of the proteases. Like inhibi-
tors 4, 5, 8, and 10, also the amino crotonic acid esters 17
(Asn) and 18 (Gln), the hydroxy or aminoacrylic acid
esters 21 (Gln), 22 (Asn), and 23 (Gln), and the hydro-
xycrotonic acid esters 24 (Asn) and 25 (Gln) were inactive
or only weakly active. Prolongation of the peptide to Gln-
Chg (29), Gln-Leu-Ala (30), or Gln-Chg-Val-Ala (20) again
improved inhibition potency, at least against falcipains and
plasmodia. This is in line with the good potency of the library
derived heptapeptide inhibitors 2 and 3 (Figure 1, Ki values in
M for falcipain-2: (2) 5.2; (3) 7.0), which are even more
active. Z-Configuration of the activated double bond
(compound 35 vs 11) seems to be unfavorable. The inactivity
of the Trt-containing Z-configured compound 35 compared
to its E-analogue 11 additionally emphasizes that inhibition
by 11 is not due to aggregation, since this should be inde-
pendent from the configuration of the double bond.
Inhibitors with Phe. In the case of the Asn and Gln
containing compounds, enzyme inhibition and antiparasitic
activity correlated quite well; i.e., compounds not active
against the enzymes did not inhibit parasite growth, and
the most potent enzyme inhibitors (11, 15, 16, Ki values
of 0.45-3.3 μM for FP-2, 1.4-14.7 μM for FP-3, and
0.14-4.3 μM for RD) were also quite active against both
plasmodia and trypanosomes (IC50 of 0.09-4.7 for plasmodia,
2.8-31 μM for trypanosomes). In contrast, some of the Phe
containing compounds displayed strong enzyme inhibition
while being inactive against parasites or were active against
parasites while being inactive or only weakly active against the
target enzymes.
Results showing inhibition of parasite growth without inhibi-
tion of proteases especially concern rhodesain/trypanosoma;
examples are compounds 19, 32, 33, 36, 38, 40, and 45. These
results raise a question regarding the target of the antitry-
panosomal activity of these compounds.
On the other hand, highly active enzyme inhibitors did not
display antiparasitic activity. The striking example for rho-
desain/trypanosomes is compound 14, which was equally
potent to its Z-isomer 42 (Ki = 0.34 μM/0.15 μM) but in
contrast to the latter did not display antitrypanosomal
activity up to 100 μM. Compound 14 also exhibits good
inhibition of falcipains (Ki = 0.98, 2.8 μM) but not against
plasmodia. As described above, 14 and 42 are reversible
inhibitors. Interestingly, replacement of the ester moiety in
inhibitor 12 by Phe benzyl ester (14) totally abolishes anti-
trypanosomal activity but on the other hand improves
enzyme inhibition. In the case of the corresponding
Z-isomers (36 vs 42), however, both, enzyme inhibition
and parasite inhibition were improved. Polar groups like a
free acid (inhibitors 13, 37, 41) or the hydroxymethyl group
in 31 decreased antiprotease and antiparasitic activity.
Among the various Michael systems (compare E-config-
ured inhibitors 12, 19, 32, 33 or Z-configured inhibitors 34,
36, 44, 45), the fumaric acid ester 12 and the corresponding
maleic acid ester 36 were best concerning antitrypanosomal
activity.
In summary, the results show that there are some quite
good starting points for falcipain (12, 14, 43) and rhodesain
(12, 14, 42, 43) inhibitors on the one hand and for antitry-
panosomal (12, 32, 38, 42, 43) and antiplasmodial (43)
activity on the other hand, with inhibitor 43 displaying the
best overall activity within the Phe containing compound
series.
Toxicity Studies. The most active antitrypanosomal com-
pounds, namely, 12, 15, 16b, 42, 43 as well as those that were
active against trypanosomes but not against rhodesain (19,
32, 33, 36, 38, 40, 45), were subjected to cytotoxicity assays

1960 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Breuning et al.
with the macrophage cell line J774.1. Results are shown in
Table 2, which additionally contains the data for inhibition
of rhodesain and trypanosomes.
Experimental Section
Biological Activities. General. Falcipains and rhodesain were
recombinantly expressed as described previously (FP-2 in ref 31,
FP-3 in ref 44, and RD in ref 45). Substrates (Cbz-Phe-Arg-
AMC for rhodesain, Cbz-Leu-Arg-AMC for falcipains) were
purchased from Bachem. Assay buffer was 50 mM acetate,
pH 5.5, 5 mM EDTA, 200 mM NaCl. Enzyme buffer was
50 mM acetate, pH 5.5, 5 mM EDTA, 200 mM NaCl, 5 mM
DTT. Substrates and inhibitor stock solutions were prepared in
DMSO and diluted with assay buffer (final DMSO concentra-
tion of 10%). A Varian Cary Eclipse fluorescence spectrophot-
ometer (Varian, Darmstadt, Germany) with 96-well plates was
used: λex = 380 nm and λem = 460 nm.
These studies show that in the case of the Phe containing
compounds displaying antitrypanosomal activity but
lacking good rhodesain inhibition, nonspecific cytotoxi-
city may indeed be one reason for the antiparasitic activity
(compounds 19, 32, 36, 38, 40). Selectivity indices
(IC50(J774.1)/IC50(T.b.b.)) of about 10 are found for inhi-
bitors 12, 42, and 43. In contrast, the Gln(Trt) containing
inhibitors are only weakly toxic (16b: 60 μM) or do not
show nonspecific toxicity below 100 μM (15), yielding
selectivity indices of 17 and >36.
Determination of Ki Values. The hydrolyses of the substrates
were monitored over 5-10 min in the presence of inhibitor. The
Ki values were calculated using the Dixon equation,41 vo/vi =
1 þ ([I]/Kiapp), and correction to zero substrate concentration41
was done with Ki = Kiapp/(1 þ [S]/Km]) with [S] = 25 μM and
Summary and Conclusion
Starting from the results of a combinatorial fumaric acid
based peptide library that yielded peptides with Phe, Asn, or
Gln in P1-position to display high inhibitory potency against
falcipain-2 andrhodesain, wesynthesizedand tested aseries of
analoguous peptides containing various Michael acceptors.
Both, E- and Z-configured compounds were included and
tested against the target enzymes (falcipain-2, falcipain-3,
rhodesain), the parasites (Plasmodium falciparum, Trypano-
soma brucei brucei), and macrophages. In the case of
E-configured compounds, those with a Gln(Trt) residue,
namely, 11, 15, 16a, and 16b, displayed the highest protease
inhibiting and antiparasitic properties with the diastereomeric
vinylogous dipeptide esters 16a and 16b (dipeptide sequence
(S/R)-Gln(Trt)-Phg) as the most active inhibitors in the whole
series (16a(b) Ki (FP-2/FP-3/RD) = 0.45(0.49)/1.4(nd.)/
0.47(0.14) μM; 16a(b) IC50 (P. falciparum/T. brucei) = 3.9
(0.9)/31(3.5) μM). Inhibitionofthe target enzymes was shown
to be time-dependent (15/16a/16b: k2nd = 11100/12250/15263
(FP-2), 16b: 26483 (RD) M-1 s-1) and competitive with respect
to the substrate. Unspecific inhibition by aggregation was
excluded by various experiments (addition of detergent,
increased enzyme concentration, spinning in a microfuge).
The analoguous Z-configured compound was inactive.
The most active inhibitors, especially against rhodesain and
Trypanosoma, among the Z-configured compounds are the
Phe-containing maleic acid derivatives 42 and 43 (Ki (FP-2/
RD) = 10.2/3.8 μM for 42 and 3.8/1.4 μM for 43; IC50
(P. falciparum/T. brucei) = 100/2.9 μM for 42 and 8.0/2.2 μM
for 43). However, in contrast to the Gln derivatives described
above, which were shown to be not toxic, unspecific cytotoxi-
city may also be a reason for the antitrypanosomal activity
(IC50 (macrophages) ≈ 30 μM). Interestingly, the E-config-
ured analogue 14 displayed even a bit higher protease inhibi-
tion (Ki (FP-2/FP-3/RD) = 0.98/2.8/0.34) but no antiparasitic
activity. Both the E-configured (14) and the Z-configured
derivatives (42) showed time-independent, reversible, and
competitive inhibition. In summary, we have discovered new
peptidic Michael-type cysteine protease inhibitors with pro-
mising properties displaying different inhibition mechanisms
depending on the configuration of the double bond and the
peptide sequence: on the one hand, the E-configured vinylo-
gous Gln(Trt) esters as time-dependent inhibitors, and on the
other hand, Z- or E-configured maleic and fumaric acid
derivaites containing two Phe residues as reversible inhibitors.
The Gln(Trt) derivatives especially are promising starting
points for new inhibitors, since they display high antiparasitic
activity while being nontoxic against macrophages (selectivity
index of >36).
K
m = 8.4 μM for falcipain-2, with Km = 72 μM for falcipain-3,
and with [S] = 10 μM and Km = 827 nM for rhodesain. GraFit
software was used to calculate the Kiapp values.
Determination of k2nd Values. The residual activities of rho-
desain were measured after 0, 5, 20, 35, and 50 min of incubation
time tinc of inhibitor (15, 16a) and enzyme prior to addition of
substrate ([S] = 10 μM). The residual enzyme activities vi for
various inhibitor concentrations ranging between 0 and 100 μM
were fitted to the incubation times tinc using equation vi = vo
exp(-kobs inc
t
) þ constant. The thus obtained pseudo-first-order
rate constants kobs were plotted against the inhibitor concentra-
tions. The graphs were observed to be linear; thus, the second-
order rate constants of inhibition k2nd were calculated by the
slopes of the curves, with correction to zero substrate concen-
tration: k2nd = (1 þ [S]/Km)(kobs/[I]). In the case of inhibitor 16b,
continuous assays with rhodesain and falcipain-2 were per-
formed, and kobs values were obtained by fitting the progress
curves to the exponential equation y = lim(1 - exp(-kobst)) þ
offset. The thus obtained pseudo-first-order rate constants kobs
were plotted against the inhibitor concentrations. The graphs
showed a hyperbolic shape, and the second-order rate constants
of inhibition were thus calculated using equations kobs = ki[I]/
(Kiapp þ [I]) (yielding the first-order rate constant ki and the
apparent dissociation constant Kiapp) and k2nd = (1 þ [S]/Km)
(ki/Kiapp). GraFit software was used to calculate the inhibition
constants.
Evaluations of cultured malaria parasites were as described
previously.12,28,35
Antitrypanosomal activities and studies on macrophages
were as described previously.29
Syntheses of Inhibitors. General. Reaction solvents were
dried and distilled before use. All other reagents were used as
obtained from Aldrich, Fluka, IRIS, or GL Biochem. All
experiments were run in oven-dried glassware under N2 atmo-
sphere. NMR spectra were recorded on an Avance 400 MHz
spectrometer from Bruker Biospin GmbH, Germany (solvent
CDCl3; 1H NMR, 400.13 MHz; 13C NMR, 100.61 MHz). Peak
assignments are based on DEPT. ESI mass spectra were
recorded on an Agilent 1100 LC/MSD trap. Characterization
of the purity of the compounds by LC analyses was done with
the following parameters: HPLC system 1100 from Agilent and
a Jupiter 4 μm Proteo 90A RP C-18 column (4.6 mm ꢀ 150 mm),
gradient from 40% acetonitrile with 0.1% formic acid (5 min),
40-95% acetonitrile with 0.1% formic acid (25 min) and 95%
acetonitrile with 0.1% formic acid for 15 min, flow rate of
600 μL/min, and UV detection at 220 and 254 nm. The purities
of the compounds are generally >98% unless otherwise indi-
cated. Melting points (not corrected) were determined in open
€
capillary on a type 510 apparatus from Buchi, Switzerland.
IR spectra were recorded on a FT-IR spectrometer, type Phar-
malyzIR, from Bio-Rad. The R values were detemined on a 241

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1961
polarimeter from PerkinElmer. Column chromatography (cc)
was performed with silica gel 60 from Merck (0.063-0.2 mm or
70-230 mesh). For TLC (thin layer chromatography) alumina
sheets from Merck coated with silica gel 60 F254 were used. All
amino acids used are L-configured.
Synthetic Methods. Method A (Steglich Esterification). An
amount of 1.00 equiv of carboxylic acid was suspended in
CH2Cl2 abs (∼5 mL/mmol) and cooled to 0 ꢀC. Then 1.10 equiv
of alcohol, 0.08-0.10 equiv of DMAP, and 1.10 equiv of DCC
were added. The reaction mixture was stirred at rt for 24 h, and
the precipitated dicyclohexylurea was filtered off. The organic
phase was washed with saturated solutions of NaHCO3, NH4Cl,
and NaCl and dried with sodium sulfate, the solvent was
removed in vacuo, and the residue was purified by cc.
Method G (Reaction of Maleic Anhydride with Amino Acids or
Peptides). To a mixture of amino acid, di- or tripeptide (1 equiv),
and NEt3 (2.1 equiv) in methylene chloride, maleic anhydride
(1 equiv) was added at 0 ꢀC. The solution was stirred at 0 ꢀC for
2 h and at rt for a further 24 h. The solution was acidified to pH 2
with 2 M HCl and extracted with methylene chloride. The
combined organic phases were washed with saturated NaCl
solution and dried over Na2SO4, and the solvent was evaporated
to yield the Z-olefin. The crude product was purified by cc.
Syntheses and Analytical Data of the Selected Inhibitors 11, 14,
15, 16a, 16b, 42, 43. (S,E )-Methyl 2-(4-Ethoxy-4-oxobut-2-
enamido)-5-oxo-5-(tritylamino)pentanoate (11). Method B with
75.0 mg (522 μmol) of monoethyl fumarate, 200 mg (497 μmol)
of H-Gln(Trt)-OMe, 118 μL (151 mg, 547 μmol) of DPPA, and
73 μL (53 mg, 522 μmol) of TEA in 5 mL of DMFabs was used.
Purification was done by cc (silica gel 60, cyclohexane/ethyl acetate
1/1. Yield: 56 mg (106 μmol, 21%), colorless solid. Mp: 138-
141 ꢀC. [R]2D1 -13.46 (c 0.56, MeOH). ESI-MS (m/z): calcd for
C31H32N2O6, 528.23 [M]; found, 529.0 [M þ 1], 551.5 [M þ
Naþ]. 1H NMR (CDCl3): δ (ppm) = 7.16-7.35 (m, 16 H), 6.88
(s, 1 H), 6.73 (d, 1 H, J = 15.7 Hz), 6.68 (d, 1 H, J = 15.7 Hz),
4.49-4.54 (m, 1 H), 4.25 (q, 2 H, J = 7.3 Hz), 3.70 (s, 3 H),
2.36-2.49 (m, 2 H), 2.14-2.20 (m, 1 H), 1.99-2.06 (m, 1 H),
1.30 (t, 3 H, J = 7.3 Hz). IR (neat): ν~ = 3302, 2962, 1723, 1659,
Method B (Boc Deprotection and Peptide Coupling with
DPPA). An amount of 1.00 mmol of Boc-protected compound
was dissolved in 6 mL of dichloromethane and cooled to 0 ꢀC.
An amount of 6 mL of TFA was added, and the mixture was
stirred at 0 ꢀC. After completion of the deprotection (TLC
control) the solvent was removed in vacuo, and the remaining
residue was dried and directly submitted to peptide coupling.
Amounts of 1.00 equiv of acid and 1.05 equiv of amine dissolved
in DMF were cooled to 0 ꢀC. An amount of 1.10 equiv of DPPA
was added, and the reaction mixture was stirred for 30 min.
Then an amount of 2.05 equiv of TEA was added, and the
mixture was stirred for 18-36 h at 0 ꢀC and for 2-5 days at rt
(TLC control). Water was added, and the reaction mixture was
extracted with ethyl acetate.The organic phase was washed with
saturated NH4Cl, NaHCO3 and NaCl solutions, dried, and
removed in vacuo. The crude product was purified by cc.
Method C (Symmetric Anhydride Method). An amount of
2.20 equiv of acid was dissolved in dichloromethane and cooled
to 0 ꢀC. Then an amount of 1.10 equiv of DCC was added and
the mixture was stirred for 1 h. Precipitated dicyclohexylurea
was filtered off, and 1.00 equiv of C-protected amine was added
to the filtrate (in the case of hydrochlorides or tosylates 1.00
equiv of TEA was added additionally) followed by 0.10 equiv of
DMAP. The reaction mixture was stirred at 0 ꢀC for 6-10 h and
at rt for 1 day (TLC control). The reaction mixture was washed
with saturated NaHCO3, KHSO4 solutions, and water, dried,
and removed in vacuo. The crude product was purified by cc.
Method D (Coupling with IBCF). An amount of 1 equiv of
acid was dissolved at -15 ꢀC in THF, and 1 equiv of NMM and
1 equiv of IBCF were added successively. Then 1 equiv of amino
acid dissolved in DMF was added, and the reaction mixture was
stirred at -15 ꢀC for 1 h, at 0 ꢀC for 1 - 2 h, and at rt for 10-18 h
(TLC control). The mixture was filtered off, and the solvent was
removed in vacuo. The remaining residue was dissolved in ethyl
actetate. The organic phase was washed with saturated NH4Cl,
NaHCO3, and NaCl solutions, dried, and removed in vacuo.
The crude product was purified by cc.
Method E (Coupling with PyBOP). Amounts of 1 equiv of
acid, 3 equiv of NMM or DIPEA, and 1 equiv of PyBOP were
dissolved in DMF and stirred at rt for 15-30 min. The amine
(1 equiv) was added, and the solution was stirred for 1-3 days
(TLC control). The reaction mixture was poured into ethyl
acetate and washed with saturated NaCl solution. The organic
phase was dried, filtered off, and removed in vacuo. The crude
product was purified by cc.
Method F (Michael Addition of Alcohols or Amines to Ethyl
Propiolate). An amount of 1.00 mmol (1.00 equiv) of alcohol or
amino acid was dissolved in CHCl3 and cooled to 0 ꢀC. Catalytic
amounts of TEA (0.05-0.1 mL) and 1.20 mmol (1.20 equiv) of
ethyl propiolate were added successively. The solution was stirred
for 1 day at rt and quenched by addition of saturated NH4Cl
solution. The mixture was poured into 70 mL of CHCl3, and the
organic phase was washed with saturated NH4Cl and NaCl solu-
tions, dried, and evaporated. The crude product was purified by cc.
Hydrochlorides or tosylates of amino acids were first reacted with
NaHCO3 and extracted with ethyl acetate to yield the free amines.
1516, 1492, 1446, 1367, 1297, 1260, 1171, 1094, 1032 cm-1
.
(S)-2-[(E)-3-((S)-1-Benzyloxycarbonyl-2-phenylethylcarbamoyl)-
acryloylamino]-3-phenylpropionic Acid Benzyl Ester (14). Amounts
of 584 mg (2.00 mmol) of PheOBn HCl and 0.29 mL (2.10 mmol) of
3
TEA were dissolved in DMF, and 0.11 mL (1.00 mmol) of fumaric
acid dichloride was added dropwise at -15 ꢀC. The mixture was
stirred at -15 ꢀC for 1 h, at 0 ꢀC for 1 h, and at rt for an additional 1
h. The solution was poured into an ethyl acetate/water mixture (1/1).
The aqueous phase was extracted with ethyl actetate, and the
combined organic phases were washed with brine, dried, and
evaporated in vacuo. The crude product was recrystallized from
dichoromethane/cyclohexane. Yield: 61 mg (0.27 mmol, 27%),
colorless solid. Mp: 215 ꢀC. [R]2D2 þ5.40ꢀ (c 0.50, CHCl3). LOOP-
ESI-MS: calcd for C36H34N2O6, 590.68; found, [M þ Na]þ 613.7.
1H NMR (CDCl3): δ = 3.10-3.21 (m, 4 H, J = 5.8, 8.4 Hz),
4.88-4.93 (dt, 2 H, J = 5.8, 7.9 Hz), 5.14, 5.16 (2d, 4 H, J =
12.1 Hz), 6.29 (d, 2 H, J = 7.9 Hz), 6.83 (s, 2 H), 7.18-7.39 (m,
20 H) ppm. IR (neat): ν~ = 3296, 3031, 2941, 1728, 1677, 1627, 1535,
1443, 1352 cm-1
.
(S,E)-Ethyl 4-(tert-Butoxycarbonylamino)-7-oxo-7-(tritylamino)-
hept-2-enoate (15).46 Reduction with DIBAL-H was done with
300 mg (600 μmol) of Boc-Gln(Trt)-OMe and 6.0 mL (6.00 mmol)
of DIBAL-H (1.0 M in hexane) in 10 mL of CH2Cl2abs for 3 h. The
crude aldehyde was subjected to olefination: 268 mg (567 μmol) of
aldehyde, 27.5 mg (624 μmol) of anhydrous LiCl, 135 μL (153 mg,
680 μmol) of triethylphosphonoacetate, and 96.4 μL (73.3 mg,
567 μmol) of DIPEA in 6-8 mL of CH3CNabs were mixed at 0
ꢀC and stirred at rt for 1 day. An amount of 10 mL of NaCl solution
was added. The reaction mixture was acidified to pH 6 with 2 M HCl
and extracted with ethyl acetate. The combinded organic phases
were washed with saturated NaHCO3, NH4Cl, and NaCl solutions,
dried, and evaporated in vacuo. The crude product was purified by
cc (silica gel 60, gradient cyclohexane/ethyl actetate
4/1 f 3/1). Yield: 130 mg (240 μmol, 42%), colorless solid.
Analytical data corresponded to literature values.46 Mp: 147-
1
150 ꢀC. H NMR (CDCl3): δ (ppm) = 7.18-7.30 (m, 15 H),
6.87 (br s, 1 H, NH), 6.79 (dd, 1 H, J = 5.3 Hz, J = 15.7 Hz), 5.88
(dd, 1 H, J = 1.5 Hz, J = 15.7 Hz), 4.76 (br d, 1 H, J = 6.6 Hz),
4.29 (br s, 1 H), 4.18 (q, 2 H, J = 7.1 Hz), 2.36 (mc, 2 H),
1.89-1.99 (m, 1 H), 1.71-1.80 (m, 1 H), 1.42 (s, 9 H), 1.27 (t, 3 H,
J = 7.1 Hz). IR (neat): ν~ = 3306, 2926, 1687, 1656, 1518, 1492,
1447, 1366, 1249 cm-1
.
(S,E )-Ethyl 4-((S/R)-2-(tert-Butoxycarbonylamino)-2-phenyl-
acetamido)-7-oxo-7-(tritylamino)hept-2-enoate (16a, 16b). Reduc-
tion with DIBAL-H was done with 500 mg (787 μmol) of Boc-Phg-
Gln(Trt)-OMe in 20 mL of CH2Cl2abs and 7.9 mL (7.87 mmol) of

1962 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Breuning et al.
DIBAL-H (1.0 M in hexane) for 7 h. The crude aldehyde was
subjected to olefination. An amount of 41.0 mg (944 μmol) of
anhydrous LiCl was suspended in 15 mL of CH3CNabs at rt.
Successively 187 μL (212 mg, 944 μmol) of triethylphosphonoace-
tate, 118 μL (120 mg, 787 μmol) of DBU, and a solution of 497 mg
(787 μmol) of Boc-Phg-Gln(Trt)-H in 20 mL of CH3CNabs were
added. The reaction mixture was worked up as described for 15.
Purification was done by cc (silica gel 60, gradient n-hexane/EtOAc
= 2:1 f 1.5:1). Yield: 16a, 57 mg (84.3 μmol, 11%); 16a þ 16b,
122 mg (181 μmol, 23%); 16b, 20 mg (29.6 μmol, 4%). The
Horner-Wadsworth-Emmons olefination also yielded the dia-
stereomeric mixture (16b/16a = 20/80). Compound 16b was
resynthesized using Boc-Phg-(R)-Gln(Trt)-OMe. The purification
was performed by preparative HPLC. Yield: 110 mg, 10%. Mp:
(16a þ 16b) 159-162 ꢀC. [R]D21 þ6.65 (16a, c 0.29, MeOH). ESI-
MS (16a) (m/z): calcd for C41H45N3O6, 675.33 [M]; found,
Schad and Cornelia Heindl for their support in syntheses and
enzyme testings.
Supporting Information Available: Synthetic methods and
analytical data (NMR, IR, MS, purity, optical rotation, melting
points) for the synthesized compounds and 13C NMR data for
compounds 11, 14, 16a, 16b, 42, 43. This material is available
free of charge via the Internet at http://pubs.acs.org.
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676.4 [M þ 1], 698.3 [M þ Naþ]. H NMR (CDCl3/D2O, 16a):
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1035 cm-1
.
(S )-Benzyl-2-[(Z)-3-((S)-1-benzyloxycarbonyl-2-phenylethyl-
carbamoyl)acryloylamino]-3-phenylpropionate (42). Method E
with 354 mg (1.00 mmol) of 37, 292 mg (1.00 mmol) of Phe-
OBn HCl, 521 mg (1.00 mmol) of PyBOP in 10 mL of dichlor-
3
methane/DMF (1/1), and 0.52 mL (3.00 mmol) of DIPEA was
used. Reaction time: 3 h, 0 ꢀC, 5 days, rt. Purification was done
by cc (silica gel 60, cyclohexane/EtOAc 2/1). Yield: 175 mg (0.30
mmol, 30%) yellowish resinous solid. [R]2D2 þ57.53ꢀ (c 0.73,
CHCl3). LOOP-ESI-MS: calcd for C36H34N2O6, 590.68; found,
[M þ Na]þ 613.7. 1H NMR (CDCl3): δ = 3.10-3.21 (m, 4 H, J
= 6.3 Hz), 4.88-4.93 (dt, 2 H, J = 6.3, 7.0 Hz), 5.15, 5.16 (2d, 4
H, J = 12.1 Hz), 6.06 (s, 2 H), 7.08-7.34 (m, 20 H), 8.51 (d, 2 H,
J = 7.0 Hz) ppm. IR (neat): ν~ = 3250, 3031, 2955, 1740, 1671,
1616, 1537, 1454, 1347, 1260 cm-1
.
(S )-Benzyl-2-{(Z)-3-[(S)-1-((S)-1-benzyloxycarbonylethyl-
carbamoyl)-2-phenylethylcarbamoyl]acryloylamino}-3-phenylpro-
pionate (43). Method E with 211 mg (0.55 mmol) of 37, 242 mg
(0.55 mmol) of Phe-Ala-OBn TFA and 286 mg (0.55 mmol) of
3
PyBOP in 20 mL of dichlormethane/DMF (1/1), and 0.28 mL
(1.65 mmol) of DIPEA was used. Reaction time: 5 h, 0 ꢀC, 5 days,
rt. Purification was done by cc (silica gel 60, cyclohexane/ethyl
acetate 1/1). Yield: 147 mg (0.22 mmol, 40%) yellowish resinous
solid. [R]2D2 þ1.28ꢀ (c 0.47, CHCl3). LOOP-ESI-MS: calcd for
C39H39N3O7, 661.76; found, [M þ Na]þ 684.7. 1H NMR
(CDCl3): δ = 1.35 (d, 3 H, J = 7.3 Hz), 3.08-3.18 (m, 4 H,
J = 6.0, 6.8 Hz), 4.51-4.58 (dt, 1 H, J = 7.1, 7.3 Hz), 4.67-4.74
(dt, 1 H, J = 6.8, 7.8 Hz), 4.84-4.91 (dt, 1 H, J = 6.0, 7.3 Hz),
5.13, 5.15 (2d, 4 H, J = 12.1 Hz), 6.03 (d, 1 H, J = 13.2 Hz), 6.08
(d, 1 H, J = 13.2 Hz), 6.81 (d, 1 H, J = 7.3 Hz), 7.16-7.40 (m, 20
H), 7.94 (d, 1 H, J = 7.3 Hz), 8.41 (d, 1 H, J = 7.8 Hz) ppm. IR
(neat): ν~ = 3273, 3063, 3032, 2929, 1741, 1658, 1621, 1536, 1497,
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Acknowledgment. We thank the DFG (Deutsche For-
schungsgemeinschaft) for ongoing financial support of our
work (Grants SFB 630, TP A3, and TP A4). We also thank
Prof. Dr. Heidrun Moll, Molecular Infection Biology, Uni-
€
versity of Wurzburg, for financial support of the trypanosome
and macrophage testings. We give many thanks to Caroline
€
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