Synthesis and Evaluation of Non-peptidic Cysteine Protease Inhibitors of P. falciparum Derived from Etacrynic Acid

Article abstract of DOI:10.3390/molecules14010019

A series of etacrynic acid derivatives was synthesized and screened for their in vitro activity against Plasmodium falciparum, as well as their activity against recombinantly expressed falcipain-2 and -3. The two most active compounds of the series displayed IC₅₀ values of 9.0 and 18.8 μM against Plasmodia.

Full text of DOI:10.3390/molecules14010019

Molecules 2009, 14, 19-35; doi:10.3390/molecules14010019
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Evaluation of Non-peptidic Cysteine Protease
Inhibitors of P. falciparum Derived from Etacrynic Acid
Marie-Adrienne Dude 1, Ulrich Kaeppler 2, Monika Herb 2, Markus Schiller 2, Franziska
Schulz 3, Birgit Vedder 2, Saskia Heppner 3, Gabriele Pradel 1, Jiri Gut 4, Philip J. Rosenthal 4,
Tanja Schirmeister 2, Matthias Leippe 3 and Christoph Gelhaus 3,*
1
Research Center for Infectious Diseases, University of Würzburg, Röntgenring 11, 97070
Würzburg, Germany; E-mail: adrienne.dude@uni-wuerzburg.de (M-A. D.),
gabriele.pradel@mail.uni-wuerzburg.de (G. P.)
2
Institute of Pharmacy and Food Chemistry, Am Hubland, D-97074 Würzburg, Germany
E-mail: schirmei@pharmazie.uni-wuerzburg.de (T. S.)
3
Zoological Institute, University of Kiel, Olshausenstr. 40, D-24098 Kiel, Germany
E-mail: mleippe@zoologie.uni-kiel.de (M. L.)
4
Department of Medicine, San Francisco General Hospital, University of California, San Francisco,
CA 94143-0811, USA; E-mail: philip.rosenthal@ucsf.edu (P-J. R.)
* Author to whom correspondence should be addressed; E-mail: cgelhaus@zoologie.uni-kiel.de.
Received: 20 November 2008; in revised form: 12 December 2008 / Accepted: 19 December 2008 /
Published: 23 December 2008
Abstract: A series of etacrynic acid derivatives was synthesized and screened for their in
vitro activity against Plasmodium falciparum, as well as their activity against
recombinantly expressed falcipain-2 and -3. The two most active compounds of the series
displayed IC50 values of 9.0 and 18.8 μM against Plasmodia.
Keywords: Malaria; Cysteine protease inhibitor; Etacrynic acid.
Introduction
Malaria is still a major cause of global human morbidity and mortality, with Plasmodium
falciparum being the deadliest among the four Plasmodium species that infect humans. Annually 350–
500 million clinical malaria episodes occur, causing more than 1 million deaths each year [1]. The

Molecules 2009, 14
20
increasing spread of P. falciparum due to inadequate vector control, increasing drug resistance, and the
lack of effective vaccine strengthens the need for the development of novel drugs for the treatment of
malaria [2, 3]. New therapeutic strategies are therefore urgently needed. Cysteine proteases of
P. falciparum represent attractive antiplasmodial drug targets due to their essential functions for the
parasite erythrocytic cycle [4, 5, 6]. Among them are falcipain-2 and falcipain-3, which are located in
the acidic food vacuole of the parasite, play a pivotal role in hemoglobin hydrolysis, and may also
participate in erythrocyte rupture [5, 7]. Disruption of the hemoglobin degradation pathway is lethal
for the parasite. Development of compounds targeting falcipain-2 and falcipain-3 is therefore currently
a focus of research. A recent study has further shown that individual cysteine protease inhibitors
significantly reduce microgametogenesis in P. falciparum, pointing to a potential role of these
inhibitors for transmission blocking strategies [8]. So far several peptidic and peptidomimetic as well
as non-peptidic cysteine protease inhibitors have been synthesized and tested against malaria parasites
[9-12]. In the present work a set of non-peptidic cysteine protease inhibitors derived from the loop
diuretic etacrynic acid as a lead compound were evaluated for their potency to inhibit recombinantly
expressed falcipain-2 and falcipain-3 [13-15]. Furthermore, activities against chloroquine-sensitive P.
falciparum strain 3D7 and the chloroquine-resistant strain W2 were investigated. Several of these
compounds have initially been tested against papain, the prototype cysteine protease of the CAC1
family, and against the SARS coronavirus main protease Mpro, as described elsewhere [16, 17].
Results and Discussion
In these previous studies compounds possessing an activated double bond revealed covalent, but
reversible binding to the cysteine residue of the respective protease. In addition to the structural
modifications implemented in previous work [16, 17], namely within the compounds 1, 2, 8-21, we
now included fluoro-substituted compounds 3-7, several analogues without an activated double bond
25-32, as well as derivatives with polar side chains 22-23, and finally, a biotin-labeled inhibitor 24. In
summary, the structure of etacrynic acid was modified as follows (Scheme 1): A: substitution pattern
of the aromatic ring, B: esters and amides, C: ortho-position of the double bond containing side chain
(cpd. 2), D: removal of the double bond.
Scheme 1. Sites of modification of the etacrynic acid lead.
A
Cl
Cl
O
CO2H
B
O
C
D
A: substitution pattern of the
aromatic ring; B: esters, amides, acids; C: ortho-position; D:
removal of the double bond.

Molecules 2009, 14
21
The inhibitors were synthesized according to previously described pathways [16,17] which are
summarized in Scheme 2. Halogen substituted anisoles were subjected to Friedel-Crafts acylation
yielding the corresponding phenolic ketones. Further alkylation of the phenolic hydroxyl functions
yielded amides 28, 30 – 32, and esters 1, 9 and 29. Introduction of the double bond was performed
either by Mannich reaction with TMDM (Æ 3, 6, 7) or by aldol condensation with formaldehyde (Æ
4, 8). The latter yielded the free acids 4 and 8 due to concurrent hydrolysis of the ester function. The
free acids were coupled to various amides to give the amides 5, 10 – 24. Amides 26 and 27 without
activated double bond were also synthesized by standard amide coupling methods.
Scheme 2. Synthetic pathways to the etacrynic acid derivatives. HOSuc, N-
hydroxysuccinimide;
DCC,
dicyclohexylcarbodiimide,
TMDM,
N,N,N’,N’
tetramethyldiaminomethane; EEDQ, ethyl 1,2-dihydro-2-ethoxyquinoline-1-carboxylate.
O
X, Y
X, Y
O
CO2H
O
R
N
H
25
6, 7
O
O
TMDM
Ac2O
O
acyl chloride
AlCl3
X, Y
X, Y
Br-CH2-(C=O)-NHR
K2CO3, KI
O-CH3
OH
X, Y
R
O
N
H
O
28, 30 - 32
O
Br-CH2-CO2R
K2CO3, KI
X, Y
X, Y
O
CO2R
X, Y
O
CO2R
O
CO2H
HCHO, K2CO3
TMDM, Ac2O
O
O
O
4, 8
1, 9, 29
3
HOSuc, DCC
or EEDQ
R-NH2
O
O
X, Y
X, Y
O
R
O
CO2H
X, Y
N
O
R
H
N
H
HOSuc, DCC
R-NH2
O
O
26, 27
O
5, 10 - 24
Recombinant falcipain-2 and falcipain-3 were produced as previously described [14, 18]. Inhibitory
activities against recombinant falcipain-2 and falcipain-3 were evaluated in fluorometric microplate
assays using the substrates Cbz-Phe-Arg-AMC and Cbz-Leu-Arg-AMC (AMC, 7-amino-4-methyl-
coumarin) [19]. The cysteine protease inhibitor E-64 was used as a positive control [20]. The solvent
DMSO was used as negative control. Compounds 1-6, 8-11 and 13-28 were tested in vitro against the
CQ-sensitive 3D7 P. falciparum strain or the CQ-resistant W2 P. falciparum strain. The corresponding

Molecules 2009, 14
22
IC50 values are shown in Table 1. The in vitro data for the etacrynic acid derivatives are compared to
those of the well known drug chloroquine and to E-64. In addition, the cytotoxicity of the inhibitor 23
was studied on human kidney epithelium cell-line 293T, as described previously [21, 22], resulting in
an IC50 value of >160 μM.
Table 1. Inhibition of falcipain-2 / -3 (FP-2 / -3) as well as antiplasmodial activity of non-
peptidic Michael-acceptors derived from etacrynic acid.
Y
X
1
R
2
R
O
P. f.
3D7/W2,
IC50
FP-2
IC50,
(μM)
FP-3
IC50,
(μM)
Cpd.
R1
X
Y
R2
(µM)
1
2
3
4
H5C2
H
Cl
H
F
O-CH2-CO2Et
Cl
498±8
110±5
ni
346±14
381±21
nd
nic
H5C2 O-CH2-CO2Et
nic
O
O
H5C2
H5C2
H
H
142±5b
79.8±6b
O
F
O-CH2CO2H
nd
nd
H
N
5
6
O
H5C2
H5C2
H
H
F
F
80±5
nd
nd
205±9b
141±4b
O
H
N
O
ni
O
H
N
CO2Me
O
7
O
H5C2
H
F
ni
nd
nd
nic
nic
8
9
H5C2
H5C2
Cl
Cl
Cl
Cl
O-CH2-CO2H
443±17
ni
O-CH2-CO2Et
60.6±4.2
163±5.6
H
N
CO2Bn
O
nic
10
H5C2
Cl
Cl
178±14
56.7±5.7
O
H
N
O
O
11
12
H5C2
H5C2
Cl
Cl
Cl
Cl
333±5
158±11
nic
nd
O
H
N
CO2Bn
O
165±12
nd

Molecules 2009, 14
23
Table 1. Cont.
H
N
O
13
H5C2
Cl
Cl
Cl
Cl
269±21
318±14
87.2±6
nic
O
O
H
N
14
H5C2
ni
29.3±3.4c
O
NH2
O
O
H
N
NH2
nic
nic
15
O
N
H
H5C2
Cl
Cl
Cl
Cl
212±17
242±19
ni
ni
O
O
O
16
H
N
H5C2
NH2
O
N
H
O
O
O
N
17
O
H5C2
Cl
Cl
Cl
Cl
Cl
Cl
305±30
255±4
479±23
153±16
nic
nic
O
H
N
O
18
H5C2
O
N
N
H5C2
144±11
557±23 27.4±4.1c
19
O
O
O
H
N
20
H5C2
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
184±17
182±9
ni
158±17
123±8
ni
nic
nic
nic
O
H
N
O
21
H5C2
O
H
N
CO2H
O
22
H5C2
O
CO2H
H
N
CO2H
O
H5C2
57.1±13
3.0±1.1
96.5±0.6 18.8±0.9c
11.9±1.1 9.0±0.4c
23
O
H
H
N
N
O
biotin
24
6
H5C2
O
Cl
Cl
Cl
531a
484a
713a
ni
ni
ni
nic
nic
nic
25
O
O
O
CO2H
Cl
O
26
O
N
H
Cl
Cl
O
O
27
O
N
H
F
66.4±2.2c
O
O
28
80±6
nd
O
N
H

Molecules 2009, 14
24
Table 1. Cont.
F
O
O
O
29
80±6
ni
nd
nd
nd
nd
nd
nd
O
F
F
O
O
O
30
O
N
H
O
31
ni
O
OH
N
H
F
O
O
O
CO2Me
32
ni
nd
nd
N
H
0.015±0. 0.075±0.
5.3±1.05c
E-64
008
02
CQ
(W2)
CQ
nd
nd
0.24[19]
0.01±0.0
nd
nd
(3D7)
048
a Only one experiment; ni, no inhibition; nd, not determined; CQ, chloroquine; b 3D7 strain;
c W2 strain.
Inspection of the data in Table 1 allows the following conclusions to be drawn: in general, the
etacrynic acid derivatives are weak or moderate inhibitors of falcipains and P. falciparum.
Nevertheless, some structure-activity relationship can be found:
The ethyl ester derivative (9) is more potent than the etacrynic acid (8), both against falcipain-2
and -3. This is in accordance with the results for papain and the SARS-CoV Mpro. The dichloro-
substituted compounds (e.g. 9, 20) are better inhibitors than the mono-chloro- (e.g. 1) or fluoro-
substituted (6) compounds, with one exception, namely inhibitor 5 (compared to 18). The α,ß-
unsaturated system appears to be favourable for activity against falcipains when comparing the
analogous dichloro-compounds 20 and 27 or compounds 18 and 26. Short voluminous moieties such
as the tertbutyl moiety of compounds 5, 28, 29, seem to be advantageous if combined with the fluoro-
substituted aromate. In these cases, the activated double bond is apparently not essential for inhibition.
Nearly all etacrynic acid amides show better inhibition properties than the free acid (8). A longer
acidic side-chain significantly enhances the activity (23, compared to 20, 21), making 23 the most
active inhibitor of falcipains and P. falciparum within the series. However, insertion of an additional
acidic group (e.g. 22) diminishes the inhibiting activity. The cytotoxicity/antiplasmodial ratio for the
most active compound 23 is >8.5, indicating selectivity against the parasite. As the data against the
target enzymes and the parasites do not correlate in all cases (e.g. 14), the question arises whether
there are additional or other targets. In order to allow further affinity binding studies the biotinylated
dichloro-substituted etacrynic acid amide 24 was included and synthesized according to the methods

Molecules 2009, 14
25
recently described [17,23] (Scheme 2). Notably, this compound emerged as the most potent inhibitor
of falcipains and P. falciparum within the series.
Conclusions
In summary, this paper describes a comprehensive screening of non-peptidic Michael acceptors
using etacrynic acid as lead structure. The best inhibition against recombinantly synthesized falcipain-
2 and falcipain-3 revealed the compound 24. Moreover, this etacrynic acid amide as well as compound
23 displayed modest antiplasmodial activity in vitro with IC50 values of 9 and 18.8 μM, respectively,
which are in the range of the standard cysteine protease inhibitor E-64. In addition, the high IC50 value
of >160 μM for compound 23 obtained from cytotoxicity assays using the human kidney epithelium
cell-line 293T indicates selectivity against the parasite. These results provide basic information for the
development of further non-peptidic irreversible cysteine protease inhibitors with etacrynic acid
amides as lead compounds. In addition, the good inhibitory properties of 24 allow further affinity
binding studies.
Experimental
General
Melting points were determined in open capillary on a melting point apparatus, model 530, from
Büchi, Switzerland. NMR spectra were recorded on an AVANCE 400 MHz spectrometer from Bruker
Biospin GmbH, Germany [solvent CDCl3 (unless otherwise noted); 1H-NMR, 400.13 MHz; 13C-NMR,
100.61 MHz]. The optical rotation values were determined on a Perkin-Elmer 241 polarimeter. ESI
mass spectra were recorded on an Agilent 1100 ion trap equipped with an Agilent HPLC system.
Hydrostatic column chromatography was performed with silica gel 60 (0.063-0.2 mm). All solvents
were purified and dried prior to use according to standard literature procedures. Chloroquine, E64 and
DMSO (dimethyl sulfoxide) were obtained from Sigma-Aldrich, Deisenhofen, Germany. Alamar
Blue® was purchased from Trinova Biochem, Giessen, Germany. DMEM (Dulbecco’s Modified
Eagle’s Medium) high glucose was delivered from Gibco/Sigma-Aldrich, Deisenhofen, Germany.
Hygromycin was obtained from Merck, Darmstadt, Germany.
Syntheses of inhibitors
The syntheses of compounds 1, 2, 8-21, and 25 are described in [17].
Method A: Introduction of the double bond. To substances 29, 30 or 32 (1 equiv.) and TMDM
(N,N,N’,N’-tetramethyldiaminomethane, 20 equiv.), acetic anhydride (20 equiv.) was added slowly.
1
The mixture was heated under reflux at 85 °C. The reaction is followed by H-NMR spectroscopy.
After completion of the reaction the mixture was cooled to room temperature and saturated K2CO3
solution was added until gas evolution stopped. The product was extracted with Et2O, washed with
water and brine and dried with Na2SO4. The solvent was removed in vacuo and the product purified by
column chromatography.

Molecules 2009, 14
26
Method B: Syntheses of bromoacetamides. Bromoacetyl bromide (1 equiv.) in absolute CH2Cl2 was
cooled to -30 °C. Amine (1 equiv.) and triethylamine (1 equiv.) in absolute CH2Cl2 were added
dropwise. After the addition, the reaction mixture was warmed up to room temperature and stirred for
1-2 h further. The solvent was removed in vacuo, the product was dissolved in acetone and filtered off.
The bromoacetamides were used directly without further purification.
Method C: Coupling of the bromoacetamide or bromoacetic acid ester with 1-(3-Fluoro-4-
hydroxyphenyl)butan-1-one. 1-(3-Fluoro-4-hydroxyphenyl)butan-1-one (1 equiv.), K2CO3 (1.5 equiv.)
and KI (0.1 equiv.) were refluxed in dry acetone for 1 h. Bromoacetamide (method B) or bromoacetic
acid ester (2 equiv.) were added and the mixture heated to reflux for a further 4 h. The mixture was
cooled to room temperature, filtered and the solvent was removed in vacuo. The product was dissolved
in Et2O, washed with brine, KOH solution (5%) and water, and dried with Na2SO4. The solvent was
removed in vacuo and the product was purified by column chromatography.
Tert-butyl 2-(2-fluoro-4-(2-methylenebutanoyl)phenoxy)acetate (3). Method A: Tert-butyl-2-(4-
butyryl-2-fluorophenoxy)acetate (29, 558 mg, 1.89 mmol), TMDM (5.14 mL, 37.7 mmol), acetic
anhydride (3.56 mL, 37.7 mmol). Rf: 0.73 (cyclohexane/ethyl acetate: 1/1); yield 180 mg (0.58 mmol,
31 %); yellow oil; LOOP-ESI-MS: calcd. for C16H21FO4 308.35, found [M+H]+ 309.2; LC-MS: Rt =
1
3
38.2 min, purity 100 %; H-NMR: δ 1.09 (t, 3H, J = 7.5 Hz, CH2CH2CH3), 1.47 (s, 9H, Cq(CH3)3),
3
2.45 (q, 2H, J = 7.4 Hz, CH2CH2CH3), 4.64 (s, 2H, OCH2C=O), 5.50 (s, 1H, Cq=CH2), 5.74 (s, 1H,
Cq=CH2), 6.88 (dd, 1H, J = 8.4 Hz, CHarom.), 7.54–7.59 (m, 2H, CHarom.); 13C-NMR: δ 12.30
(CqCH2CH3), 25.51 (CqCH2CH3), 28.01 (Cq(CH3)3), 66.48 (OCH2C=O), 82.95 (Cq(CH3)3), 114.09
(CHarom.), 117.98 (CHarom.), 122.82 (Cq=CH2), 126.52 (CHarom.), 131.64 (CqC=O), 149.39 (Cq=CH2),
150.58 (CqF or CqOCH2C=O), 153.06 (CqF or CqOCH2C=O), 166.96 (OCH2C=O), 196.12 (CqC=O).
2-(2-fluoro-4-(2-methylenebutanoyl)phenoxy)acetic acid (4): 1-(3-fluoro-4-hydroxyphenyl)butan-1-
one. 2-Fluoroanisole (4.00 g, 31.7 mmol) and butyric acid chloride (5.07 g, 47.6 mmol) were dissolved
under a N2 atmosphere in absolute CH2Cl2 (50 mL) and the mixture was cooled to 0-10 °C. AlCl3 (6.35
g, 47.6 mmol) was added within 30 minutes, and the mixture was stirred for 2-3 h. An additional
amount of AlCl3 (6.35 g, 47.6 mmol) was added, and the mixture was heated under reflux for 2 h. The
mixture was poured on ice and acidified with concentrated HCl to pH 1. Tartaric acid was added for
complexation of aluminum until the solution was clear. The solution was extracted with Et2O and the
organic layer was washed with KOH solution (10%) and brine. The organic layer was dried with
Na2SO4 and solvent was removed in vacuo. Yield 4.97 g (27.3 mmol, 86 %); white solid; mp. 91 °C
(water); 1H-NMR: δ 0.98 (t, 3H, 3J = 7.3 Hz, CH2CH2CH3), 1.74 (sext, 2H, 3J = 7.3 Hz, CH2CH2CH3),
3
2.87 (t, 2H, J = 7.3 Hz, CH2CH2CH3), 6.04 (bs, 1H, OH), 7.04 (dd, 1H, J = 8.5 Hz, CHarom.), 7.67–
7.73 (m, 2H, CHarom.).
Ethyl-2-(4-butyryl-2-fluorophenoxy)acetate. 1-(3-Fluoro-4-hydroxyphenyl)butan-1-one (2.00 g, 11.0
mmol), bromoacetic acid ethyl ester (3.67 g, 22.0 mmol), K2CO3 (2.28 g, 16.5 mmol) and KI (183 mg,
1.10 mmol) were refluxed in dry acetone for 5-6 h. The reaction mixture was cooled to room
temperature and filtered off. The solvent was removed in vacuo and the product was extracted with

Molecules 2009, 14
27
Et2O, washed with NaOH solution (10%), water and brine, dried with Na2SO4, and the solvent was
1
removed in vacuo. Yield 2.30 g (8.60 mmol, 78%); white solid; mp. 79-80 °C (Et2O). H-NMR
(DMSO-d6): δ 0.90 (t, 3H, 3J = 7.5 Hz, CH2CH2CH3), 1.20 (t, 3H, 3J = 7.2 Hz, OCH2CH3), 1.60 (sext,
3
3
3
2H, J = 7.3 Hz, CH2CH2CH3), 2.93 (t, 2H, J = 7.1 Hz, CH2CH2CH3), 4.17 (q, 2H, J = 7.1 Hz,
OCH2CH3), 4.99 (s, 2H, OCH2C=O), 7.20 (dd, 1H, J = 8.5, CHarom.), 7.74–7.78 (m, 2H, CHarom.).
2-(2-fluoro-4-(2-methylenebutanoyl)phenoxy)acetic acid (4). Ethyl-2-(4-butyryl-2-fluorophenoxy)-
acetate (2.11 g, 7.87 mmol), formaldehyde solution (40%, 1.08 mL, 15.7 mmol) and K2CO3 (2.17 g,
15.7 mmol, dissolved in water) were refluxed in ethanol for 24 h. The reaction mixture was cooled to
room temperature, acidified with concentrated HCl to pH 1 and the product was extracted with Et2O.
The solvent was removed in vacuo. Yield 556 mg (2.20 mmol, 28%); white solid; mp 99-101 °C
(Et2O); LOOP-ESI-MS: calcd. for C13H13FO4 252.24, found [M+H]+ 253.1; LC-MS: Rt = 23.8 min,
purity 100 %; 1H-NMR: δ 1.10 (t, 3H, 3J = 7.5 Hz, CqCH2CH3). 2.46 (q, 2H, 3J = 7.4 Hz, CqCH2CH3),
4.81 (s, 2H, OCH2C=O), 5.52 (s, 1H, Cq=CH2), 5.78 (s, 1H, Cq=CH2), 6.95 (dd, 1H, J = 8.4 Hz,
CHarom.), 7.56–7.60 (m, 2H, CHarom.); 13C-NMR: δ 12.30 (CqCH2CH3), 25.44 (CqCH2CH3), 65.65
(OCH2C=O), 114.50 (CHarom.), 118.19 (CHarom.), 123.35 (Cq=CH2), 126.59 (CHarom.), 132.35 (CqC=O),
148.87 (Cq=CH2), 149.33 (CqF or CqOCH2C=O), 153.11 (CqF or CqOCH2C=O), 172.29 (OCH2C=O),
196.16 (CqC=O).
N-tert-butyl-2-(2-fluoro-4-(2-methylenebutanoyl)phenoxy)acetamide (5). 2-(2-Fluoro-4-(2-methylene-
butanoyl)phenoxy)acetic acid (4, 767 mg, 3.00 mmol) and N-hydroxysuccinimide (336 mg, 3.00
mmol) were dissolved under a N2 atmosphere in absolute CH2Cl2 (10 mL). Dicyclohexylcarbodiimide
(DCC) (619 mg, 3.00 mmol) was added in small portions and the mixture was stirred overnight. The
mixture was filtered into a flask with tert-butylamine (219 mg, 3.00 mmol) in CH2Cl2 (10 mL), stirred
for 3 h at room temperature and filtered. The reaction mixture was washed with brine and KOH
solution (5%), dried with Na2SO4 and the solvent was removed in vacuo. The product was purified by
column chromatography. Rf: 0.43 (cyclohexane/ethyl acetate: 2/1); yield 314 mg (1.02 mmol, 34 %);
white solid; mp 85 °C (cyclohexane/ethyl acetate); LOOP-ESI-MS: calcd. for C17H22FNO3 307.37,
1
3
found [M+H]+ 308.4; LC-MS: Rt = 18.0 min, purity 100 %; H-NMR: δ 1.10 (t, 3H, J = 7.5 Hz,
3
CqCH2CH3), 1.40 (s, 9H, Cq(CH3)3), 2.46 (q, 2H, J = 7.4 Hz, CqCH2CH3), 4.46 (s, 2H, OCH2C=O),
5.51 (s, 1H, Cq=CH2), 5.78 (s, 1H, Cq=CH2), 6.43 (bs, 1H, NH), 6.96 (dd, 1H, J = 8.2 Hz, CHarom.),
13
7.56–7.62 (m, 2H, CHarom.); C-NMR: δ 12.30 (CqCH2CH3), 25.45 (CqCH2CH3), 28.71 (Cq(CH3)3),
51.46 (Cq(CH3)3), 68.50 (OCH2C=O), 114.05 (CHarom.), 117.73 (CHarom.), 123.23 (Cq=CH2), 126.92
(CHarom.), 132.23 (CqC=O), 146.73 Cq=CH2), 148.87 (CqF or CqOCH2C=O), 149.35 (CqF or
CqOCH2C=O), 165.95 (OCH2C=O), 196.01 (CqC=O).
N-butyl-2-(2-fluoro-4-(2-methylenebutanoyl)phenoxy)acetamide (6). Method A: N-butyl-2-(4-butyryl-
2-fluorophenoxy)acetamide (30) (105 mg, 0.36 mmol), TMDM (0.97 mL, 7.11 mmol), acetic
anhydride (0.67 mL, 7.11 mmol). Rf: 0.59 (cyclohexane/ethyl acetate: 1/2); yield 55.5 mg (0.18 mmol,
51 %); white solid; mp 73 °C (cyclohexane/ethyl acetate); LOOP-ESI-MS: calcd. for C17H22FNO3
1
3
307.37; found [M+H]+ 308.4; LC-MS: Rt = 15.8 min, purity 100 %; H-NMR: δ 0.93 (t, 3H, J = 7.4
3
3
Hz, NHCH2CH2CH2CH3), 1.11 (t, 3H, J = 7.4 Hz, CqCH2CH3), 1.36 (sext, 2H, J = 7.4 Hz,

Molecules 2009, 14
28
3
3
NHCH2CH2CH2CH3), 1.53 (sext, J = 7.3 Hz, NHCH2CH2CH2CH3), 2.46 (q, 2H, J = 7.5 Hz,
3
CqCH2CH3), 3.37 (q, 2H, J = 6.7 Hz, NHCH2CH2CH2CH3), 4.57 (s, 2H, OCH2C=O), 5.51 (s,
Cq=CH2), 5.78 (s, Cq=CH2), 6.60 (s, 1H, NH), 6.96 (dd, 1H, J = 8.1 Hz, CHarom.), 7.57–7.62 (m, 2H,
13
CHarom.); C-NMR: δ 12.31 (CqCH2CH3), 13.69 (NHCH2CH2CH2CH3), 20.00 (NHCH2CH2CH2CH3),
25.45 (CqCH2CH3), 31.53 (NHCH2CH2CH2CH3), 38.90 (NHCH2CH2CH2CH3), 68.21 (OCH2C=O),
113.90 (CHarom.), 117.77 (CHarom.), 123.22 (Cq=CH2), 126.92 (CHarom.), 149 (CqF or CqOCH2C=O),
166.77 (OCH2C=O).
Methyl-2-(2-(2-fluoro-4-(2-methylenebutanoyl)phenoxy)acetamido)-3-methylpentanoate (7). Method
A: Methyl-2-(2-(4-butyryl-2-fluorophenoxy)acetamido)-3-methylpentanoate (32, 58.0 mg, 0.16
mmol), TMDM (0.43 mL, 3.16 mmol), acetic anhydride (0.30 mL, 3.16 mmol). Rf: 0.75
(cyclohexane/ethyl acetate: 1/1); yield 41.5 mg (0.11 mmol, 68 %); colorless oil; LOOP-ESI-MS:
calcd. for C20H26FNO5 379.43; found [M+H]+ 380.4; LC-MS: Rt = 18.9 min, purity 100 %; [α]22 : 61.1
D
(CHCl3, c = 0.1). 1H-NMR: δ 0.90–0.94 (m, 6H, Ile-CH2CH3 and Ile-CHCH3), 1.11 (t, 3H, 3J = 7.5 Hz,
CqCH2CH3), 1.14–1.22 (m, 2H, Ile-CH2CH3), 1.40–1.48 (m, 1H, Ile-C=O), 1.93–1.99 (m, 1H, Ile-
3
CHCH3), 2.47 (t, 2H, J = 7.3 Hz, CqCH2CH3), 3.74 (s, 3H, OCH3), 4.62 (s, 2H, OCH2C=O), 5.51 (s,
3
Cq=CH2), 5.78 (s, Cq=CH2), 6.98 (dd, 1H, J = 8.2 Hz, CHarom.), 7.08 (d, 1H, J = 8.5 Hz, NH), 7.57–
7.63 (m, 2H, CHarom.); 13C-NMR: δ 11.51 (Ile-CH2CH3 or Ile-CHCH3), 12.31 (CqCH2CH3), 15.50 (Ile-
CH2CH3 or Ile-CHCH3), 25.08 (CqCH2CH3 or Ile-CHC=O), 25.43 (CqCH2CH3 or Ile-NCHC=O), 37.79
(Ile-CHCH3), 52.23 (OCH3), 68.32 (OCH2C=O), 114.25 (CHarom.), 117.86 (CHarom.), 123.26 (Cq=CH2),
126.81 (CHarom.), 132.46 (CqC=O), 149.37 (CqOCH2C=O), 150.66 (Cq=CH2), 153.14 (CqF), 166.83
(OCH2C=O), 171.68 (CqOCH3), 195.98 (CqC=O).
2-{2-[2,3-Dichloro-4-(2-methylene-butyryl)-phenoxy]acetylamino}pentanedioic acid (22). Compound
22 was synthesized according to [17] starting from etacrynic acid (240 mg, 0.79 mmol), N-hydroxy-
succinimide (91 mg, 0.79 mmol), DCC (163 mg, 0.79 mmol) in THF. The solvent was removed and a
solution of glutamic acid (116 mg, 0.79 mmol) and KOH (133 mg, 2.37 mmol) in water was added.
After stirring 7 days at room temperature the mixture was acidified with conc. HCl and extracted with
dichloromethane. The crude product, which still contained etacrynic acid, was suspended in hot
benzene and filtered. The solid residue yielded the pure amide as a white powder. Yield: 55 mg (0.127
1
3
mmol, 16 %). H-NMR (d4-MeOH): δ = 1.14 (t, 3H, J = 8.0 Hz, CH2CH3), 2.05 (m, 1H), 2.27 (m,
1H), 2.38 – 2.47 (m, 4H, 2 CH2), 4.57 (m, 1H, NHCH), 4.75 (s, 2H, OCH2CO), 5.60 (s, 1H, C=CH),
3
3
6.02 (s, 1H, C=CH), 7.10 (d, 1H, J = 8.0 Hz, Ar-H), 7.24 (d, 1H, J = 8.0 Hz, Ar-H); LC-MS
(negative mode): m/z: 431.2 (100) [M-H]-, Rt = 2.3 min.
6-{2-[2,3-Dichloro-4-(2-methylene-butyryl)-phenoxy]acetylamino}-hexanoic acid (23). Compound 23
was synthesized according to [17] starting from etacrynic acid (201 mg, 0.66 mmol), N-hydroxy-
succinimide (76 mg, 0.66 mmol), DCC (137 mg, 0.66 mmol) in dichloromethane (20 mL). The solvent
was removed and the residue taken up in THF. This solution was added to a solution of 6-amino-
hexanoic acid (130 mg, 0.99 mmol) and KOH (111 mg, 1.98 mmol) in water (10 mL). After 3 days
stirring at room temperature the mixture was acidified with conc. HCl and extracted with dichloro-
methane. After removal of the organic solvent the crude product yielded a yellow oil which was

Molecules 2009, 14
purified by column chromatography (MeOH/CHCl3 10:2). Product: colourless oil, which solidified to
29
1
3
a white solid. Yield: 93 mg (0.22 mmol, 34 %); H-NMR (d4-MeOH): δ = 1.14 (t, 3H, J = 8.0 Hz,
3
CH2CH3), 1.33 – 1.40 (m, 2H, CH2), 1.53 – 1.66 (m, 4H, 2 CH2), 2.28 (t, 2H, J = 8.0 Hz, CH2), 2.44
3
(q, 2H, J = 8.0 Hz, CH2CH3), 3.30 (m, 2H, NHCH2), 4.68 (s, 2H, OCH2CO), 5.59 (s, 1H, C=CH),
3
3
13
6.03 (s, 1H, C=CH), 7.07 (d, 1H, J = 8.0 Hz, Ar-H), 7.24 (d, 1H, J = 8.0 Hz, Ar-H); C-NMR (d4-
MeOH): δ = 13.0 (CH3), 24.5 (CH2), 25.7 (CH2), 27.4 (CH2), 30.1 (CH2), 34.9 (CH2), 40.0 (CH2), 69.5
(OCH2CO), 112.9 (Ar-CH), 124.0 (Ar-qC), 128.5 (Ar-CH), 129.9 (C=CH2), 131.8 (Ar-qC), 135.0 (Ar-
qC), 151.7 (qC, C=CH2), 156.9 (Ar-qC), 169.7 (qC, NHCONH), 177.6 (qC, COOH), 197.3 (qC, C=O);
LC-MS: m/z: 418.0 (15), Rt = 0.5 min.
5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)pentanoic acid (6-{2-[2,3-dichloro-4-(2-methylene-
butyryl)-phenoxy]acetylamino}-hexyl)-amide (24)
(A) Coupling of Boc-diaminohexane and D-biotin: Boc-diaminohexane (443 mg, 2.05 mmol) and D-
biotin (500 mg, 2.05 mmol) were suspended in absolute DMF (45 mL) and cooled to 0 °C. DPPA
(620 mg, 2.25 mmol) and triethylamine (229 mg, 2.25 mmol) were added and the mixture was stirred
at 4 °C for 8 days. Dichloromethane (25 mL) was added and the organic phase was washed twice with
citric acid (10%). Upon addition of water to the organic phase the amide precipitated as a white solid
which was filtered off.
(B) Removal of the Boc-protecting group: The solid obtained in step (A) was suspended in
dichloromethane (9 mL) and treated with TFA (3 mL) at 0 °C. Excess TFA was repeatedly removed in
vacuo. For better evaporation dichloromethane was added to the residue. Quantitative removal of the
protecting group was verified by 1H-NMR.
(C) Amide coupling to etacrynic acid: Biotin-(6-amino)-hexylamide (TFA-salt) (175 mg, 0.383 mmol),
triethylamine (39 mg, 0.383 mmol) and etacrynic acid (232 mg, 0.767 mmol) were dissolved in DMF
(2 mL). EEDQ (190 mg, 0.767 mmol) was added and the mixture was stirred at room temperature for
14 days. Ethyl acetate (50 mL) was added. Treatment of the organic phase with Na2CO3-solution (2%,
25 mL) yielded the desired product as a white precipitate which was filtered off. Overall yield: 62 mg
1
3
(0.0988 mmol) as a white solid; H-NMR (DMSO-d6): δ = 1.07 (t, 3H, J = 7.4 Hz, CH2CH3), 1.23 –
1.57 (m, 14H, CH2), 2.03 (t, 2H, 3J = 7.2 Hz, CH2), 2.36 (q, 2H, J = 7.4 Hz, CH2CH3), 2.56 (d, 1H,
3
3J = 12.3 Hz, SCH), 2.80 (dd, 1H, J = 12.4 Hz, J = 4.8 Hz, SCH), 2.97 – 3.12 (m, 5H, 2 NHCH2,
SCH), 4.11 (m, 1H, NHCH), 4.29 (m, 1H, NHCH), 4.70 (s, 2H, OCH2CO), 5.55 (s, 1H, C=CH), 6.06
(s, 1H, C=CH), 6.33 (s, 1H, biotin-NH), 6.39 (s, 1H, biotin-NH), 7.07 (d, 1H, 3J = 8.6 Hz, Ar-H), 7.32
(d, 1H, 3J = 8.6 Hz, Ar-H), 7.70 (br s, 1H, CONH), 8.01 (br s, 1H, CONH); 13C-NMR (DMSO-d6): δ =
12.3 (CH3), 22.9 (CH2CH3), 25.3 (CH2), 26.0 (CH2), 26.0 (CH2), 28.0 (CH2), 28.2 (CH2), 28.9 (CH2),
29.1 (CH2), 35.2 (CH2), 38.2 (CH2), 38.3 (CH2), 39.8 (SCH2), 55.4 (SCH), 59.2 (NHCH), 61.0
(NHCH), 67.9 (OCH2CO), 111.9 (Ar-CH), 121.2 (Ar-qC), 127.4 (Ar-CH), 129.3 (C=CH2), 129.9 (Ar-
qC), 132.4 (Ar-qC), 149.3 (qC, C=CH2), 155.5 (Ar-qC), 162.6 (qC, NHCONH), 166.3 (qC, CONH),
171.7 (qC, CONH), 195.1 (qC, C=O); LC-MS: m/z 628.0 (100) [M+H]+, Rt = 16.3 min.
3
2

Molecules 2009, 14
30
N-tert-butyl-2-(4-butyryl-2,3-dichloro-phenoxy)-acetamide (26). Compound 26 was synthesized
according to [17] starting from 2,3-dichloro-4-butyrylphenoxyacetic acid (500 mg, 1.72 mmol),
N-hydroxysuccinimide (198 mg, 1.72 mmol), DCC (354 mg, 1.72 mmol) and t-butylamine (251 mg,
3.43 mmol) in dichloromethane (15 mL). Reaction time: 21 d stirring at room temperature. The crude
product was purified by column chromatography (SiO2, Cy/EtOAc 1:1). Yield: 374 mg (1.08 mmol, 63
1
3
%) as white solid; H-NMR: δ = 0.97 (t, 3H, J = 7.4 Hz, CH2CH3), 1.41 (s, 9H, (CH3)3), 1.72 (sext,
2H, 3J = 7.3 Hz, CH2CH3), 2.88 (t, 2H, 3J = 7.2 Hz, COCH2CH2CH3), 4.44 (s, 2H, OCH2), 6.62 (br s,
3
3
1H, NH), 6.84 (d, 1H, J = 8.6 Hz, Ar-H), 7.38 (d, 1H, J = 8.6 Hz, Ar-H). 13C-NMR: δ = 13.7
(CH2CH3), 17.8 (CH2CH3), 28.7 (C(CH3)3), 44.7 (COCH2), 51.5 (C(CH3)3), 68.4 (OCH2), 111.1 (Ar-
CH), 123.2 (Ar-qC), 127.6 (Ar-CH), 131.4 (Ar-qC), 134.9 (Ar-qC), 155.2 (Ar-qC), 165.5 (qC,
CONH), 201.8 (C=O).
N-butyl-2-(4-butyryl-2,3-dichloro-phenoxy)-acetamide (27). Compound 27 was synthesized according
to [17] starting from 2,3-dichloro-4-butyrylphenoxyacetic acid (500 mg, 1.72 mmol), N-hydroxy-
succinimide (198 mg, 1.72 mmol), DCC (354 mg, 1.72 mmol) and n-butylamine (251 mg, 3.43 mmol)
in dichloromethane (15 mL). Reaction time: 21 d stirring at room temperature. The crude product was
purified by column chromatography (SiO2, Cy/EtOAc 1:1). Yield: 270 mg (0.880 mmol, 45 %) as a
1
white solid; H-NMR: δ = 0.92 – 0.98 (m, 6H, NH(CH2)3CH3, COCH2CH2CH3), 1.38 (sext, 2H,
3
3J = 7.4 Hz, NHCH2CH2CH2CH3), 1.55 (quint, 2H, J = 7.3 Hz, NHCH2CH2CH2CH3), 1.72 (sextett,
2H, 3J = 7.3 Hz, COCH2CH2CH3), 2.88 (t, 2H, 3J = 7.3 Hz, COCH2CH2CH3), 3.37 (q, 2H, 3J = 6.7 Hz,
3
3
NHCH2), 4.55 (s, 2H, OCH2), 6.72 (br s, 1H, NH), 6.85 (d, 1H, J = 8.8 Hz, Ar-H), 7.38 (d, 1H, J =
8.6 Hz, Ar-H); 13C-NMR:
13.7 (n-butyl-CH3, CH2CH3), 17.8 (CH2CH3), 20.0
δ
=
(NHCH2CH2CH2CH3), 31.5 (NHCH2CH2CH2CH3), 38.9 (NHCH2), 44.8 (COCH2CH2CH3), 68.2
(OCH2), 111.0 (Ar-CH), 123.3 (Ar-qC), 127.6 (Ar-CH), 131.4 (Ar-qC), 135.0 (Ar-qC), 155.1 (Ar-qC),
166.4 (qC, CONH), 201.8 (C=O).
N-tert-butyl-2-(4-butyryl-2-fluorophenoxy)acetamide (28). Method B: Bromoacetyl bromide (2.94 g,
14.6 mmol), tert-butylamine (1.07 mg, 14.6 mmol), triethylamine (2.05 mL, 14.6 mmol); Method C: 1-
(3-fluoro-4-hydroxyphenyl)butan-1-one (856 mg, 4.70 mmol), K2CO3 (974 mg, 7.05 mmol), KI (78.0
mg, 0.47 mmol), 2-bromo-N-tert-butylacetamide (see method B). Rf: 0.31 (cyclohexane/ethyl acetate:
1
2/1); yield 1.00 g (3.38 mmol, 72%); white solid; mp 80 °C (cyclohexane/ethyl acetate). H-NMR: δ
3
3
0.99 (t, 3H, J = 7.5 Hz, CH2CH2CH3), 1.37 (s, 9H, Cq(CH3)3), 1.75 (sext, 2H, J = 7.3 Hz,
3
CH2CH2CH3), 2.88 (t, 2H, J = 7.2 Hz, CH2CH2CH3), 4.46 (s, 2H, OCH2C=O), 6.41 (bs, 1H, NH),
6.97 (dd, 1H, J = 8.4 Hz, CHarom.), 7.72–7.76 (m, 2H, CHarom.).
Tert-butyl-2-(4-butyryl-2-fluorophenoxy)acetate (29). Method C: 1-(3-Fluoro-4-hydroxyphenyl)butan-
1-one (1.00 g, 5.49 mmol), K2CO3 (1.19 g, 8.64 mmol), KI (91.0 mg, 0.55 mmol), bromoacetic acid
tert-butylester (1.50 mL, 10.2 mmol); Rf: 0.69 (cyclohexane/ethyl acetate: 1/1); yield 1.46 g (4.93
mmol, 90 %); white solid; mp 62 °C (cyclohexane/ethyl acetate); 1H-NMR: δ 0.98 (t, 3H, 3J = 7.3 Hz,
CH2CH2CH3), 1.47 (s, 9H, Cq(CH3)3), 1.74 (sext, 2H, 3J = 7.3 Hz, CH2CH2CH3), 2.86 (t, 2H, 3J = 7.2
Hz, CH2CH2CH3), 4.64 (s, 2H, OCH2C=O), 6.89 (dd, 1H, J = 8.5 Hz, CHarom.), 7.68–7.72 (m, 2H,
CHarom.).

Molecules 2009, 14
31
N-butyl-2-(4-butyryl-2-fluorophenoxy)acetamide (30). Method B: Bromoacetyl bromide (2.08 g, 10.3
mmol), n-butylamine (753 mg, 10.3 mmol), triethylamine (1.45 mL, 10.3 mmol). Method C: 1-(3-
Fluoro-4-hydroxyphenyl)butan-1-one (750 mg, 4.12 mmol), K2CO3 (853 mg, 6.17 mmol), KI (68.4
mg, 0.41 mmol), 2-bromo-N-n-butylacetamide (see method B). Rf: 0.32 (cyclohexane/ethyl acetate:
1/2); yield 1.00 g (3.39 mmol, 82 %); white solid; mp 75 °C (cyclohexane/ethyl acetate); 1H-NMR: δ
0.93 (m, 3H, NHCH2CH2CH2CH3), 0.99 (t, 3H, 3J = 7.4 Hz, CH2CH2CH3), 1.36 (sext, 2H, 3J = 7.3 Hz,
3
3
NHCH2CH2CH2CH3), 1.53 (sext, J = 7.3 Hz, NHCH2CH2CH2CH3), 1.75 (sext, 2H, J = 7.3 Hz,
CH2CH2CH3), 2.88 (t, 2H, 3J = 7.3 Hz, CH2CH2CH3), 3.36 (q, 2H, 3J = 6.8 Hz, NHCH2CH2CH2CH3),
4.57 (s, 2H, OCH2C=O), 6.59 (s, 1H, NH), 6.97 (dd, 1H, J = 8.3 Hz, CHarom.), 7.72–7.76 (m, 2H,
CHarom.); 13C-NMR: δ 13.68 und 13.82 (CH2CH2CH3 und NHCH2CH2CH2CH3), 17.76 (CH2CH2CH3),
19.98 (NHCH2CH2CH2CH3), 31.52 (NHCH2CH2CH2CH3), 38.89 (NHCH2CH2CH2CH3), 40.24
(CH2CH2CH3), 68.15 (OCH2C=O), 114.04 (CHarom.), 116.03 (CHarom.), 125.31 (CHarom.), 131.22
(CqC=O), 149.12 (CqF or CqOCH2C=O), 153.30 (CqF or CqOCH2C=O), 166.69 (OCH2C=O), 197.84
(CqC=O).
2-(4-Butyryl-2-fluorophenoxy)-N-(1-hydroxy-2-methylpropan-2-yl)acetamide (31). Method B:
Bromoacetyl bromide (0.76 mL, 8.75 mmol), 2-amino-2-methyl-1-propanol (780 mg, 8.75 mmol),
triethylamine (1.23 mL, 8.75 mmol). Method C: 1-(3-Fluoro-4-hydroxyphenyl)butan-1-one (495 mg,
2.72 mmol), K2CO3 (563 mg, 4.08 mmol), KI (45.2 mg, 0.27 mmol), 2-bromo-N-(1-hydroxy-2-
methylpropan-2-yl)acetamide (see method B). Rf: 0.28 (cyclohexane/ethyl acetate: 1/2); yield 455 mg
3
(1.46 mmol, 54 %); white solid; mp 84 °C (cyclohexane/ethyl acetate); 1H-NMR: δ 0.99 (t, 3H, J =
3
7.5 Hz, CH2CH2CH3), 1.35 (s, 6H, Cq(CH3)2), 1.75 (sext, 2H, J = 7.4 Hz, CH2CH2CH3), 2.88 (t, 2H,
3J = 7.3 Hz, CH2CH2CH3), 3.65 (s, 2H, CqCH2OH), 4.52 (s, 2H, OCH2C=O), 6.66 (s, 1H, NH), 6.98
13
(dd, 1H, J = 8.1 Hz, CHarom.), 7.73–7.76 (m, 2H, CHarom.); C-NMR: δ 13.83 (CH2CH2CH3), 17.74
(CH2CH2CH3), 24.66 (Cq(CH3)2), 40.26 (CH2CH2CH3), 56.50 (Cq(CH3)2), 68.24 (OCH2C=O), 70.17
(CqCH2OH), 114.38 (CHarom.), 116.30 (CHarom.), 125.28 (CHarom.), 132.21 (CqC=O), 148.87 (CqF or
CqOCH2C=O), 153.34 (CqF or CqOCH2C=O), 167.42 (OCH2C=O), 197.85 (CqC=O).
Methyl 2-(2-(4-butyryl-2-fluorophenoxy)acetamido)-3-methylpentanoate (32). Method B: Bromoacetyl
bromide (2.00 g, 9.92 mmol), isoleucine methylester HCl (1.80 mg, 9.92 mmol), triethylamine (2.79
mL, 19.8 mmol). Method C: 1-(3-Fluoro-4-hydroxyphenyl)butan-1-one (600 mg, 3.29 mmol), K2CO3
(683 mg, 4.94 mmol) KI (54.6 mg, 0.33 mmol), methyl-2-(2-bromoacetamido)-3-methyl-pentanoate
(see method B)). Rf: 0.65 (cyclohexane/ethyl acetate: 1/1); yield 616 mg (1.68 mmol, 51 %); white
solid; mp 74 °C (cyclohexane/ethyl acetate); [α] 2D2 = 61.1 (CHCl3, c = 0.1); 1H-NMR: δ 0.90–0.94 (m,
3
6H, Ile-CH2CH3 and Ile-CHCH3), 0.99 (t, 3H, J = 7.5 Hz, CH2CH2CH3), 1.12–1.23 (m, 2H, Ile-
3
CH2CH3), 1.40–1.49 (m, 1H, Ile-CHC=O), 1.75 (sext, J = 7.3 Hz, CH2CH2CH3), 1.92–1.99 (m, 1H,
Ile-CHCH3), 2.88 (t, 2H, 3J = 7.3 Hz, CH2CH2CH3), 3.74 (s, 3H, OCH3), 4.62 (s, 2H, OCH2C=O), 6.99
(dd, 1H, 3J = 8.5 Hz, CHarom.), 7.08 (d, 1H, J = 8.5 Hz, NH), 7.72–7.77 (m, 2H, CHarom.).

Molecules 2009, 14
32
Protein expression and purification
Falcipain-2 and falcipain-3 were recombinantly expressed in E. coli and refolded to active enzyme
as previously described [13,24].
Enzyme assays, in-vitro assays
IC50 values for inhibition of falcipain-2 and falcipain-3 were determined as described previously
using the fluorogenic substrates Cbz-Phe-Arg-AMC and Cbz-Leu-Arg-AMC, respectively (AMC, 7-
amino-4 methylcoumarin) [7, 13, 14]. As a positive control the well-known cysteine protease inhibitor
E-64 [7, 25, 26] and as a negative control the solvent (DMSO) was used. Dose-dependent effects of
compounds on parasite development (P. falciparum strain W2) were quantified by flow cytometry
according to a previously published method [27]. First, a screening with inhibitor concentrations of
100, 10 and 1 mM was performed and the percentage activity of infected red blood cells (RBCs)
relative to the negative control was determined. Compounds showing concentration dependent
inhibition in these assays were selected for determination of IC50 values (Table 1). Chloroquine [20]
and E-64 were used as positive controls, and the solvent DMSO was used as a negative control. For
selected compounds assays with the P. falciparum strain 3D7 were performed using a fluorometric
assay with Hoechst-33258 [28], or the microculture tetrazolium test measuring parasite lactate
dehydrogenase activity [12, 29]. The parasites were cultivated in vitro as described previously [30].
Compounds were screened at concentrations between 1 nM and 100 μM. Synchronized ring stages
were plated in 96-well plates at a parasitemia of 1%, in the presence of the compounds (dissolved in
DMSO). Incubation of parasites with DMSO alone at a concentration of 0.5% v/v was used as a
negative control. IC50 values were calculated by non-linear regression analyses using the programs
GaphPad® Prism 4 and GraFit® [31, 32].
In vitro cytotoxicity assay
The human kidney epithelium-cell line 293T was cultured in DMEM medium supplemented with
20% fetal bovine serum and 1% l-glutamine (200 mM) at 37 °C in a humidified atmosphere containing
5% CO2. For the experimental procedures, cells were detached from the flasks with a rubber
policeman, washed twice with PBS (phosphate-buffer saline) containing 1 mL trypsin-EDTA (1 x),
and suspended at 2 x 106 cells mL-1 in complete medium. Inhibitor assays were carried out as
described elsewhere except that the cell concentration used in this study was 2 x 104 mL-1 well-1 [33].
Acknowledgements
This work was supported by the DFG (Deutsche Forschungsgemeinschaft) (TS: SFB 630, ML: SFB
617); PJR: NIH grants AI035800 and AI35707; SH: Studienstiftung des Deutschen Volkes. We thank
Prof. Dr. Heidrun Moll, Dr. Tobias Öhlschläger, and Elena Katzowitsch (Institute for Molecular
Infection Biology, University of Würzburg) for performing the cytotoxicity assays on human kidney
cells, and Astrid Evers (Zoological Institute, University of Kiel) for assistance.

Molecules 2009, 14
33
References and Notes
1. WHO. World Malaria Report. World Health Organization: Geneva, Switzerland, 2005.
2. Talisuna, A. O.; Okello, P. E.; Erhart, A.; Coosemans, M.; D'Alessandro, U. Intensity of malaria
transmission and the spread of Plasmodium falciparum resistant malaria: a review of
epidemiologic field evidence. Am. J. Trop. Med. Hyg. 2007, 77, 170-180.
3. White, N. J. Antimalarial drug resistance. J. Clin. Invest. 2004, 113, 1084-1092.
4. Otto, H.-H.; Schirmeister, T. Cysteine proteases and their inhibitors. Chem. Rev. 1997, 97, 133-
172.
5. Rosenthal, P. J. Cysteine proteases of malaria parasites. Int. J. Parasitol. 2004, 34, 1489-1499.
6. Rosenthal, P. J.; Sijwali, P. S.; Singh, A.; Shenai, B. R. Cysteine proteases of malaria parasites:
targets for chemotherapy. Curr. Pharm. Des. 2002, 8, 1659-1672.
7. Dahl, E. L.; Rosenthal, P. J. Biosynthesis, localization, and processing of falcipain cysteine
proteases of Plasmodium falciparum. Mol. Biochem. Parasitol. 2005, 139, 205-212.
8. Rupp, I.; Bosse, R.; Schirmeister, T.; Pradel, G. Effect of protease inhibitors on exflagellation in
Plasmodium falciparum. Mol. Biochem. Parasitol. 2008, 158, 208-212.
9. Lavrado. J.; Paulo A.; Gut, J.; Rosenthal, P. J; Moreira, R. Cryptolepine analogues containing
basic aminoalkyl side-chains at C-11: synthesis, antiplasmodial activity, and cytotoxicity. Bioorg.
Med. Chem. Lett. 2008, 18, 1378-1381.
10. Vale, N.; Matos, J.; Gut, J.; Nogueira, F.; do Rosário, V.; Rosenthal, P. J.; Moreira, R.; Gomes, P.
Imidazolidin-4-one peptidomimetic derivatives of primaquine: synthesis and antimalarial activity.
Bioorg. Med. Chem. Lett. 2008, 18, 4150-4153.
11. Verissimo, E.; Berry, N.; Gibbons, P.; Cristiano, M. L.; Rosenthal, P. J.; Gut, J.; Ward, S. A.;
O'Neill, P. M. Design and synthesis of novel 2-pyridone peptidomimetic falcipain 2/3 inhibitors.
Bioorg. Med. Chem. Lett. 2008, 18, 4210-4214.
12. Ettari, R.; Nizi, E.; Di Francesco, M. E.; Dude, M. A.; Pradel, G.; Vicík, R.; Schirmeister, T.;
Micale, N.; Grasso, S.; Zappalà, M. Development of peptidomimetics with a vinyl sulfone
warhead as irreversible falcipain-2 inhibitors. J. Med. Chem. 2008, 51, 988-996.
13. Sijwali, P. S.; Shenai, B. R..; Gut, J.; Singh, A.; Rosenthal, P. J. Expression and characterization
of the Plasmodium falciparum haemoglobinase falcipain-3. Biochem. J. 2001, 360, 481-489.
14. Desai, P. V.; Patny, A.; Gut, J.; Rosenthal, P. J.; Tekwani, B.; Srivastava, A.; Avery, M.
Identification of novel parasitic cysteine protease inhibitors by use of virtual screening. 2. The
available chemical directory. J. Med. Chem. 2006, 49, 1576-1584.
15. Semenov, A.; Olson, P. J.; Rosenthal, P. J. Antimalarial synergy of cysteine and aspartic protease
inhibitors. Antimicrob. Agents Chemother. 1998, 42, 2254-2258.
16. Kaeppler, U.; Schirmeister, T. New non-peptidic inhibitors of papain derived from etacrynic acid.
Med. Chem. 2005, 1, 361-370.
17. Kaeppler, U.; Stiefl, N.; Schiller, M.; Vicik, R.; Breuning, A.; Schmitz, W.; Rupprecht, D.;
Schmuck, C.; Baumann, K.; Ziebuhr, J.; Schirmeister, T. J. A new lead for nonpeptidic active-
site-directed inhibitors of the severe acute respiratory syndrome coronavirus main protease
discovered by a combination of screening and docking methods. J. Med. Chem. 2005, 48, 6832-
6842.

Molecules 2009, 14
34
18. Shenai, B. R.; Sijwali, P. S.; Singh, A.; Rosenthal, P. J. Stage-specific antimalarial activity of
cysteine protease inhibitors. J. Biol. Chem. 2000, 275, 29000-29010.
19. Schulz, F.; Gelhaus, C.; Degel, B.; Vicik, R.; Heppner, S.; Breuning, A.; Leippe, M.; Rosenthal,
P. J.; Schirmeister, T. Screening of protease inhibitors as antiplasmodial agents. Part I: Aziridines
and epoxides. ChemMedChem. 2007, 2, 1214-1224
20. Sijwali, P. S.; Koo, J.; Singh, N.; Rosenthal, P. J. Gene disruptions demonstrate independent roles
for the four falcipain cysteine proteases of Plasmodium falciparum. Mol. Biochem. Parasitol.
2006, 150, 96-106.
21. Mikus, J.; Steverding, D. A simple colorimetric method to screen drug cytotoxicity against
Leishmania using the dye Alamar Blue. Parasitol. Int. 2000, 48, 265-269.
22. Ponte-Sucre, A.; Faber, J. H.; Gulder, T.; Kajahn, I.; Pedersen, S. E.; Schultheis, M.; Bringmann,
G.; Moll, H. Activities of naphthylisoquinoline alkaloids and synthetic analogs against
Leishmania major. Antimicrob. Agents Chemother. 2007, 51, 188-194.
23. Gelhaus, C.; Vicik, R.; Hilgenfeld, R.; Schmidt, C. L.; Leippe, M.; Schirmeister, T. Synthesis and
antiplasmodial activity of a cysteine protease-inhibiting biotinylated aziridine-2,3-dicarboxylate.
Biol. Chem. 2004, 385, 435-438.
24. Pandey, K. C.; Wang, S. X.; Sijwali, P. S.; Lau, A. L.; McKerrow, J. H.; Rosenthal, P. J. The
Plasmodium falciparum cysteine protease falcipain-2 captures its substrate, hemoglobin, via a
unique motif. Proc. Natl. Acad. Sci. USA 2005, 102, 9138-9143.
25. Ramjee, M. K.; Flinn, N. S.; Pemberton, T. P.; Quibell, M.; Wang, Y. ; Watts, J. P. Substrate
mapping and inhibitor profiling of falcipain-2, falcipain-3 and berghepain-2: implications for
peptidase anti-malarial drug discovery. Biochem. J. 2006, 399, 47-57.
26. Sijwali, P. S.; Rosenthal, P. J. Gene disruption confirms a critical role for the cysteine protease
falcipain-2 in hemoglobin hydrolysis by Plasmodium falciparum. Proc. Natl. Acad. Sci. USA
2004, 101, 4384-4389.
27. Musonda, C. C.; Gut, J.; Rosenthal, P. J.; Yardley, V.; Carvalho de Souza, R. C.; Chibale, K.
Application of multicomponent reactions to antimalarial drug discovery. Part 2: New
antiplasmodial and antitrypanosomal 4-aminoquinoline gamma- and delta-lactams via a 'catch and
release' protocol. Bioorg. Med. Chem. 2006, 14, 5605-5615.
28. Smeijsters, L. J.; Zijlstra, N. M.; Franssen, F. F.; Overdulve, J. P. Simple, fast, and accurate
fluorometric method to determine drug susceptibility of Plasmodium falciparum in 24-well
suspension cultures. Antimicrob. Agents Chemother. 1996, 40, 835-838.
29. Makler, M. T.; Hinrichs, D. J. Measurement of the lactate dehydrogenase activity of Plasmodium
falciparum as an assessment of parasitemia. Am. J. Trop. Med. Hyg. 1993, 48, 205-210.
30. Ifediba, T.; Vanderberg, J. P. Complete in vitro maturation of Plasmodium falciparum
gametocytes. Nature 1981, 294, 364-366.
31. GraphPad® Prism 4; GraphPad Software, Inc.: La Jolla, CA, USA, 2003.
32. GraFit® 5.0.13; Erithacus Software Ltd.: Horley, Surrey, UK, 2006.
33. Ahmed, S. A.; Gogal, R. M.; Walsh, J. E. A new rapid and simple non-radioactive assay to
monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine
incorporation assay. J. Immunol. Methods 1994, 170, 211-224.

Molecules 2009, 14
35
Sample Availability: Samples of compound 24 are available from the authors.
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).