2-Amido-3-(1H-Indol-3-yl)-N-substitued-propanamides as a new class of falcipain-2 inhibitors. 1. Design, synthesis, biological evaluation and binding model studies

Article abstract of DOI:10.3390/molecules14010494

The Plasmodium falciparum cysteine protease falcipain-2 (FP-2) is an important cysteine protease and an essential hemoglobinase of erythrocytic P. falciparum trophozoites. The discovery of new FP-2 inhibitors is now a hot topic in the search for potential malaria treatments. In this study, a series of novel small molecule FP-2 inhibitors have been designed and synthesized based on three regional optimizations of the lead (R)-2-phenoxycarboxamido-3-(1H-indol- 3-yl)-N-benzylpropanamide (1), which was identified using structure-based virtual screening in conjunction with surface plasmon resonance (SPR)-based binding assays. Four compounds - 1, 2b, 2k and 2l - showed moderate FP-2 inhibition activity, with IC₅₀ values of 10.0-39.4 μM, and the inhibitory activity of compound 2k was ～3-fold better than that of the prototype compound 1 and may prove useful for the development of micromolar level FP-2 inhibitors. Preliminary SAR data was obtained, while molecular modeling revealed that introduction of H-bond donor or/and acceptor atoms to the phenyl ring moiety in the C region would be likely to produce some additional H-bond interactions, which should consequently enhance molecular bioactivity.

Full text of DOI:10.3390/molecules14010494

Molecules 2009, 14, 494-508; doi:10.3390/molecules14010494
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
2-Amido-3-(1H-Indol-3-yl)-N-Substitued-Propanamides as a
New Class of Falcipain-2 Inhibitors. 1. Design, Synthesis,
Biological Evaluation and Binding Model Studies
Jin Zhu 1, †, Tong Chen 1, †, Lili Chen 2, Weiqiang Lu 1, Peng Che 1, Jin Huang 1,*, Honglin Li 1,*,
Jian Li 1,* and Hualiang Jiang 1,2
1
School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road,
Shanghai 200237, P.R. China. E-Mails: jinzh@mail.ecust.edu.cn (J. Z.); libaolu04@126.com (T. C.);
luweiqiangnb@126.com (W. L.); magean83@163.com (P. C.); hljiang@ecust.edu.cn (H. J.)
2
Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, P.R. China; E-Mail: lilichen@mail.shcnc.ac.cn
(L. C.)
* Authors to whom correspondence should be addressed; E-mails: jianli@ecust.edu.cn (J. L.);
huangjin@ecust.edu.cn (J. H.); hlli@ecust.edu.cn (H. L.); Tel. +86-21-64252584;
Fax: +86-21-64252584.
†
These authors contributed equally to this work
Received: 7 December 2008; in revised form: 11 January 2009 / Accepted: 13 January 2009 /
Published: 21 January 2009
Abstract: The Plasmodium falciparum cysteine protease falcipain-2 (FP-2) is an
important cysteine protease and an essential hemoglobinase of erythrocytic P. falciparum
trophozoites. The discovery of new FP-2 inhibitors is now a hot topic in the search for
potential malaria treatments. In this study, a series of novel small molecule FP-2
inhibitors have been designed and synthesized based on three regional optimizations of
the lead (R)-2-phenoxycarboxamido-3-(1H-indol-3-yl)-N-benzylpropanamide (1), which
was identified using structure-based virtual screening in conjunction with surface
plasmon resonance (SPR)-based binding assays. Four compounds – 1, 2b, 2k and 2l –
showed moderate FP-2 inhibition activity, with IC50 values of 10.0-39.4 μM, and the
inhibitory activity of compound 2k was ~3-fold better than that of the prototype
compound 1 and may prove useful for the development of micromolar level FP-2

Molecules 2009, 14
inhibitors. Preliminary SAR data was obtained, while molecular modeling revealed that
495
introduction of H-bond donor or/and acceptor atoms to the phenyl ring moiety in the C
region would be likely to produce some additional H-bond interactions, which should
consequently enhance molecular bioactivity.
Keywords: 3-(1H-Indol-3-yl)-propanamide derivatives; Falcipain-2 inhibitor; Malaria;
SAR
1. Introduction
Malaria, which accounts for 300-500 million clinical cases and up to 2.7 million deaths each year.
remains one of the deadliest diseases on the planet. About 90 % of these casualties occur in tropical
Africa, the great majority being children under the age of 5 [1]. Malaria is caused by protozoan
parasites of the genus Plasmodium. Of the four species of Plasmodium responsible for the disease,
Plasmodium falciparum is the most lethal. Development of effective vaccines has been significantly
hampered by the high mutability of the genome of Plasmodium falciparum [2]. The resistance of
malaria parasites to available conventional drug therapy is an increasingly serious problem [3-5].
Accordingly, the discovery of effective drugs to counter the spread of malaria parasites that are
resistant to existing agents, especially those acting on new targets, is an urgent need.
Among various potential new targets, the cysteine protease falcipain-2 (FP-2) of P. falciparum is an
attractive and most promising target enzyme [6-7]. FP-2 is a principal cysteine protease and essential
hemoglobinase of erythrocytic P. falciparum trophozoites. Many in vitro studies have all confirmed
that inhibitors of FP-2 can blocked parasite hemoglobin hydrolysis and halt the development of culture
parasites [8-22]. Some of them were also effective in vivo against murine malaria [15, 21-22].
However, FP-2 inhibitors described in the literature are mainly derived from peptide analogues [8-10,
12, 20], which tend to form covalent bonds with the thiolate of the catalytic cysteine and commonly
have nanomolar IC50 values. Obviously, it is desirable to design non-peptidic inhibitors that would
bind non-covalently to the target enzyme, in order to minimize toxicity while retaining the potential
for high in vivo activity and selectivity.
Computer-aided drug design has contributed to the introduction of ~50 compounds into clinical
trials and to numerous drug approvals [23]. Using Virtual Screening or rational drug design based on
homology models, some non-peptidic inhibitors of FP-2 with IC50 values in the micromolar range have
been reported [14, 16, 24-25]. Recently, crystal structures for FP-2 have been reported [26-27], and the
reservoir of structural and functional information of FP-2 has offered a solid starting point for the
rational structure-based design of novel antimalarial drugs targeting for FP-2. By using docking based
virtual screening approach in conjunction with a binding assay based on the surface plasmon
resonance (SPR), a novel small molecule inhibitor of FP-2 (compound 1) featuring a 2-amido-3-(1H-
indol-3-yl)-N-aubstitued-propanamide framework has been discovered [28]. After the identification of
compound 1 as a possible prototype to design selective inhibitor of FP-2, three regions of the molecule
were selected to perform chemical modifications suitable to provide expedient and significant SAR
information and improve inhibitory activity. Noticeably, the processes of chemical modifications were

Molecules 2009, 14
496
achieved step by step from region A to C on the basis of bioassay results. In total, thirteen new
compounds including 1 and twelve analogs 2a-l have been synthesized and tested against FP-2.
Finally, four compounds – 1, 2b, 2k and 2l – were found to show moderate inhibitory effects against
FP-2, with the inhibitory activity of compound 2k being ~3 times greater than that of the lead
compound 1. This encouraging result proves the validity of our chemical modification methods. Our
molecular modeling results also provided information about the binding between inhibitors and FP-2
and should be helpful for future inhibitor design.
2. Results and Discussion
2.1. Identification of Binders (Hits) by Virtual Screening
Targeting the crystal structure of FP-2 (PDB entry 2GHU) [26], a total of 80,000 compounds were
subsequently docked and ranked according to the Glide and GAsDock software [28]. Finally, 81
compounds were purchased and submitted to biological evaluations against FP-2. Because enzymatic
assays are time-consuming, surface plasmon resonance (SPR) measurements were used for the primary
screening to determine the binding affinity of these 81 candidate molecules to FP-2. Immobilization of
FP-2 on the Biacore biosensor chip resulted in a resonance signal of 9,300 resonance units (RUs).
Among the 81 compounds, the biosensor RU of compound 1 was concentration-dependent. The
collected data indicated that compound 1 can bind to FP-2 in vitro, with a binding affinity towards FP-
2 in the micromolar range (KD = 5.88 μM). The substrate Z-Phe-Arg-pNA (Bachem AG), which
binding affinity to FP-2 is in the submicromolar range (KD = 32.1 μM), was used as a positive control.
Thus, compound 1 could be designated as a binder (or hit) of FP-2. The binding affinities of
compounds 1 and Z-Phe-Arg-pNA are shown in Figures 1a-b, respectively.
Figure 1. Sensorgrams for binding of compounds 1 (a) and Z-Phe-Arg-pNA (b) to a FP-2
surface on the CM5 sensor chip. Binding responses are shown for 1 and Z-Phe-Arg-pNA
injected at concentrations of 0, 1.7, 2.4, 3.3, 4.9, 7 and 10 μM (from bottom to top).
(a)
(b)
80
70
60
50
40
30
20
10
0
20
15
10
5
0
100
150
Time (s)
200
250
100
150
Time (s)
200
250
Z-Phe-Arg-pNA
Compound 1

Molecules 2009, 14
497
2.2. Analog Design and Synthesis
Compound 1 (Figure 2) with the higher binding affinities towards FP-2 and identified by surface
plasmon resonance (SPR) technology, was used as a lead compound to design new FP-2 inhibitors.
Retaining as the common moiety of compound 1 the 2-amido-3-(1H-indol-3-yl)-N-substituted-
propanamide framework, three regions: (A) C2 stereo-configuration, (B) the phenoxycarbonyl group,
and (C) the N-benzyl substitution (Figure 2), of this molecule were selected to perform chemical
modifications that would provide expedient and significant SAR information and improve inhibitory
activity. First, we changed the C2 stereo-configuration (R) in region A to its enantiomer (S) and thus
obtained analogue 2a. Secondly, maintaining region A (R configuration) and replacing the
phenoxycarbonyl group in region B with phenylacetyl and benzyl groups, respectively, we designed
compounds 2b-c (Table 1). Finally, compounds 2d-l were prepared by replacing the benzyl moiety in
region C with substituted aryl or aralkyl groups with different electronic and hydrophobic properties..
Figure 2. The structure of compound 1 and corresponding three structural modification
regions.
B
O
NH
O
O
NH
A
1
3
NH
C
Scheme 1 shows the sequence of reactions that led to the preparation of compounds 1 and 2a-l
using N-Boc-D-tryptophan or N-Boc-L-tryptophan (3) as the starting material.
Scheme 1a
.HCl
NH2
Boc
O
Boc
O
NH
NH
NH
NH
NH
R2NH2
b
O
a
NH
HO
NH
R2
R2
5
3
4
O
Ph
c
X
Cl
d
X
NH
O
O
NH
NH
NH
O
NH
NH
R2
1, 2a-b and 2d-l
R2
2c
a Reagents and conditions: (a) EDCI, Et3N, CH2Cl2, 25 °C; (b) saturated HCl in methanol, 25 °C;
(c) (i) benzaldehyde, MgSO4, K2CO3, CH2Cl2, 25 °C, (ii) NaBH4, 0 °C; (d) pyridine, 25 °C; R2 is as
indicated in Table 1.

Molecules 2009, 14
498
. Compound 4 was prepared by coupling 3 with substituted primary amine using EDCI and Et3N in
CH2Cl2. The key intermediate 5 was obtained by deprotection of the tert-butyloxycarbonyl group of 4
with a saturated methanolic solution of hydrogen chloride. Then 5 was acylated using different acyl
chlorides or benzylated by reductive amination, giving the target compounds 1 and 2a-l. Synthetic
procedures and characterization data are given in the Experimental, while the inhibition rates and IC50
values are given in Table 1.
Table 1. Chemical Structures of Compounds 1 and 2a-m and Their Inhibitory Activities
against FP-2.
R1
NH
NH
2
O
NH
R2
C2
Inhibition rate
IC50
Compd
R1
R2
configuration
at 10 μM (%)
(μM)
O
1
R
S
29.4
14.3
29.7
-
O
O
O
O
2a
2b
R
25.7
39.4
2c
R
R
13.0
11.8
-
-
O
O
O
O
O
O
2d
O
O
O
O
O
O
2e
2f
R
R
R
R
R
3.1
13.6
3.1
-
-
-
-
-
-
F
C2H5O
O
2g
2h
2i
O
S
N N
S
2.2

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499
Table 1. Cont.
O
O
O
2j
2k
2l
R
R
R
6.6
-
O
O
O
49.5
32.9
10.0
13.3
O2N
H3COOC
2.3. Enzyme Inhibition Assay
For the primary assay, the percent inhibitions of the compounds were measured at 10 μM. The
results are listed in Table 1. Four compounds, i.e. 1, 2b, 2k and 2l, showed moderate inhibitory
activity (percent inhibition at 10 μM > 20%) and therefore we determined their IC50 values, which
were 29.7, 39.4, 10.0 and 13.3 μM, respectively. It is clear that the inhibitory activity of compound 2k
was ~3 times higher than that of the lead compound 1, proving the validity of our chemical
modifications. The anti-malarial potential of compound 1 in mice was investigated with Plasmodium
berghei ANKA using a standard 4-day suppression test [29]. Daily intragastric administration of
compound 1 at a dose of 9 mg/kg body weight for the 4 days immediately following infection resulted
in an indistinctive (< 50%) reduction in parasitemia relative to normal control (unpublished data). The
enzymatic activity of compound 1 does not consistently correlate with the animal level efficiency,
which may be caused by metabolic instability of the amide groups of compound 1.
2.4. Structure Activity Relationship Correcting with the Binding Models
To gain structural information for further structural optimization, the 3D binding of the designed
compounds to FP-2 were superimposed onto the docked conformation of compound 1 by shape
similarity.
Figure 3 shows that R configuration series of inhibitors reported here (Table 1, 1 and 2b-l) interact
with FP-2 in a similar way: the common indole ring locates on the edge of S1’ and S1 sub-pockets, the
phenoxycarbonyl (1 and 2d-m), phenacetyl (2b) or benzyl moietes (2c) fill large hydrophobic region
of the S1’ sub-pocket; and the R2-substituents (see Table 1) form strong hydrophobic interactions with
several hydrophobic residues of the S2 sub-pocket [28]. The inhibitory activities of the less active
compounds 2d-j (Table 1) evidently decrease because of the shorter or unsuitable hydrophobic R2
groups which separate from the hydrophobic S2 pocket and immerse in the solvent.

Molecules 2009, 14
Figure 3. Superimposition of the matched conformations of compounds (2a-2l, cyan) on
500
compound 1 (green) in the active site of FP-2. Hydrogen atoms have been omitted for
clarity. Key residues of the binding pocket are shown as lines. The FP-2 surface was
colored by electrostatic potential. The sub-sites are labeled as S1, S1’, and S2. The
structure figure was prepared using PyMol (http://pymol.sourceforge.net/).
More concretely, we compared the 3D binding models of prototype 1 to FP-2 with that of
compound 2k to FP-2 generated based on the docking simulation (Figure 4). Figure 4A shows the
nitrogen of the indole ring of compound 1 forms a H-bond with the nitrogen of Trp206, the two
nitrogens and three oxygen atoms of the carbamate and amide groups of compound 1 simultaneously
form five H-bond interactions with the residues Cys42 and Asn173. From the hydrogen-bond network
of compound 1, we can see that the carbamate moiety is important to maintain the inhibitory activity,
explaining why compounds 2b-c lose part of their inhibitory activity (Table 1). The same is true for
compound 2a, whose inverted configuration places it in another orientation. Compared with compound
1, the nitro group of compound 2k forms two additional H-bonds with the Ile85 at the end of the
subpocket S2 (Figure 4B), which substantially increases the inhibitory activity (Table 1).

Molecules 2009, 14
Figure 4. Detailed interactions of 1 (A) and 2k (B) to the binding sites of FP-2.
501
Compounds 1 and 2k are indicated by cyan and green thick sticks, respectively, and
hydrogen atoms have been omitted for clarity. Key residues of the binding pocket are
shown as lines. Hydrogen bonds are shown as red dotted lines with the distance between
donor and acceptor atoms. The structure figures were prepared using PyMol
(http://pymol.sourceforge.net/).
(A)
(B)
3. Experimental
Reagents were purchased from Alfa, Acros and the Shanghai Chemical Reagent Company, and
used without further purification. Analytical thin-layer chromatography (TLC) was perfomed on
HSGF 254 plates (150-200 µm thickness, Yantai Huiyou Company, P.R. China). Yields were not

Molecules 2009, 14
502
optimized. Melting points were measured in capillary tubes on a SGW X-4 melting point apparatus
and are reported without correction. Nuclear magnetic resonance (NMR) spectra were recorded on a
Brucker AVANCE 500 NMR instrument. Chemical shifts are given in parts per million (ppm, δ)
downfield from tetramethylsilane used as internal standard. Proton coupling patterns are described as
singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). Low- and high-resolution
mass spectra (LRMS and HRMS) were geberated by electrospray (ESI) produced by a LCQ-TOF
spectrometer.
3.1. Virtual Screening by Molecular Docking
The crystal structure of FP-2 (PDB entry 2GHU) [26] from Plasmodium falciparum was retrieved
from the Protein Database Bank [30]. Residues located within 14 Å from the catalytic thiolate of
Cys42 were defined as part of the binding site for docking studies. All crystallographic water
molecules were removed from the coordinate set. The pipeline of virtual screening based on molecular
docking method is presented in detail in [28].
3.2. Surface Plasmon Resonance-based FP-2/Ligand Binding Assay
The purification and refolding of recombinant protein FP-2 was performed as described by Shenai
et al [6]. The binding affinity of compound 1 to FP-2 in vitro was determined based on surface
plasmon resonance (SPR) technology. The measurement was performed using the dual flow cell
Biacore 3000 instrument (Biacore AB, Uppsala, Sweden). Immobilization of FP-2 to the hydrophilic
carboxymethylated dextran matrix of the sensor chip CM5 (Biacore) was carried out by the standard
primary amine coupling reaction wizard. The FP-2 to be covalently bound to the matrix was diluted in
10 mM sodium acetate buffer (pH 4.2) to a final concentration of 0.069 mg/mL. Equilibration of the
baseline was completed by a continuous flow of HBS-EP running buffer (10 mM Hepes, 150 mM
NaCl, 3 mM EDTA, and 0.005% (v/v) surfactant P20, pH 7.4) through the chip for 1–2 h. All the
Biacore data were collected at 25°C with HBS-EP as running buffer at a constant flow rate of 30
μL/min. All the sensorgrams were processed by using automatic correction for non-specific bulk
refractive index effects. The substrate Z-Phe-Arg-pNA (Benzoxycarbonyl-Phe-Arg-p-nitroanilide,
Bachem AG) was used for the positive control. The equilibrium constants (KD values) evaluating the
protein–ligand binding affinities were determined by the steady state affinity fitting analysis of the
Biacore data.
3.3. Enzyme Inhibition Assay
The purification and refolding of recombinant protein FP-2 was performed as described by Shenai
et al [6]. IC50 values against recombinant FP-2 were determined as described previously [8, 31]. FP-2
(30nM) was incubated for 30 min at room temperature in 100 mM sodium acetate, pH 5.5, 10 mM
DTT, with different concentrations of tested inhibitors. Inhibitor solutions were prepared from stock in
DMSO (maximum concentration of DMSO in the assay was 1%). After 30 min incubation, the
substrate Z-Leu-Arg-AMC (benzoxycarbonyl-Leu-Arg-7-amino-4-methylcoumarin, Bachem AG) in

Molecules 2009, 14
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the same buffer was added to a final concentration of 25 μM. The increase in fluorescence (excitation
at 355 nM and emission at 460 nM) was monitored for 30 min at room temperature with an automated
microtiter plate spectrofluorimeter (Molecular Devices, Flex Station). Half-maximal inhibitory
concentration (IC50) was determined from plots of percent activity over compound concentration using
GraphPad Prism software.
3.4. Preparation of (R)-2-phenoxycarboxamido-3-(1H-indol-3-yl)-N-benzylpropanamide (1).
Step A: (R)-2-(N-BOC-amino)-3-(1H-indol-3-yl)-N-benzylpropanamide (4a). To a 0 °C stirred solution
of the N-BOC derivative of (R)-tryptophan (0.62 g, 2 mmol) in CH2Cl2 (10 mL) was added
sequentially EDCI (0.58 g, 3 mmol ) and Et3N (0.3 g, 3 mmol). After 1 h, the solution was treated with
benzylamine (0.22 g, 2 mmol), and then the mixture was allowed to warm to 25 °C. After 2 d of
stirring, the mixture was washed sequentially with portions (30 mL) of 5% aqueous HOAc, H2O, 5%
aqueous NaHCO3, H2O, and brine, dried, and concentrated. The residue was purified by flash column
chromatography on silica gel, eluted with a mixture of EtOAc/petroleum ether (1:2, v/v), to afford 4a
1
(0.68 g, 84%) as a white solid: mp 138-141 °C; H-NMR (CDCl3) δ 1.39 (s, 9H), 3.16 (m, 1H), 3.33
(m, 1H), 4.26 (m, 2H), 4.41 (m, 1H), 6.96 (m, 3H), 7.13 (t, 1H), 7.18-7.21 (m, 4H), 7.35 (d, 1H), 7.66
(d, 1H).
Step B: (R)-2-Amino-3-(1H-indol-3-yl)-N-benzylpropanamide hydrochloride (5b). Compound 4a (0.68
g) was dissolved in saturated methanol solution of hydrogen chloride (10 mL). The reaction mixture
was stirred at 25 °C for 12 h. Concentration of the reaction mixture to dryness, the residue was washed
by Et2O, afforded the desired amine hydrochloride (5b) (0.50 g, 88%) as a yellow solid: mp 214-218
°C; 1H-NMR (CDCl3) δ 3.16 (m, 1H), 3.25 (m, 1H), 4.02 (s, 1H), 4.27 (m, 2H), 7.00 (t, 1H), 7.07-7.10
(m, 3H), 7.19-7.27 (m, 4H), 7.38 (d, 1H), 7.66 (d, 1H).
Step C: (R)-2-Phenoxycarboxamido-3-(1H-indol-3-yl)-N-benzylpropanamide (1). A mixture of phenyl
chloroformate (82 mg, 0.52 mmol) and compound 5b (150 mg, 0.46 mmol) in pyridine (5 mL) was
stirred 12 h at 25 °C, poured into H2O (50 mL), extracted with EtOAc. The combined organic layer
was washed, dried, filtered and condensed. The residue was purified by flash column chromatography
on silica gel, eluted with a mixture of EtOAc/petroleum ether (1:2, v/v), to afford 1 (95 mg, 50%) as a
1
white solid: mp 176-177 °C; H-NMR (CDCl3) δ 3.14 (m, 1H), 3.38 (m, 1H), 4.32 (m, 2H), 4.48 (m,
1H), 5.71 (s, 1H), 5.84 (d, 1H), 6.89 (m, 3H), 7.03 (d, 2H), 7.08 (t, 1H), 7.12-7.18 (m, 5H), 7.29 (m,
3H), 7.68 (d, 1H), 7.91 (s, 1H); ESI-MS m/z 414 [M+H]+; HRMS (ESI) m/z calcd. C25H24N3O3
[M+H]+ 414.1818, found 414.1813.
3.5 Preparation of 2-amide-3-(1H-indol-3-yl)-N-substitued-propanamide compounds 2a,b and 2d-l
These compounds were prepared by the procedure described for the preparation of 1, with the
indicated modifications.

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(S)-2-Phenoxycarboxamido-3-(1H-indol-3-yl)-N-benzylpropanamide (2a). Replacing N-Boc-D-
tryptophan with N-Boc-L-tryptophan (Step A), compound 2a was prepared as a white solid: mp 175-
1
176 °C; H-NMR (CDCl3) δ 3.21 (q, 1H), 3.46 (q, 1H), 4.26 (q, 1H), 4.34 (q, 1H), 4.55 (q, 1H), 5.74
(s, 1H), 5.90 (d, 1H), 6.95 (s, 1H), 6.98 (m, 2H), 7.10 (d, 2H), 7.15 (t, 1H), 7.19-7.26 (m, 5H), 7.36 (m,
3H), 7.76 (d, 1H), 7.97 (s, 1H); MS (ESI) m/z 414 [M+H]+; HRMS (ESI) m/z calcd. C25H24N3O3
[M+H]+ 414.1818, found 414.1804.
(R)-2-(2-Phenylacetamido)-3-(1H-indol-3-yl)-N-benzylpropanamide
(2b).
Replacing
phenyl
chloroformate with 2-phenylacetyl chloride (Step C), compound 2b was prepared as a yellow solid:
1
mp 202-205 °C; H-NMR (CDCl3) δ 3.07 (q, 1H), 3.28 (q, 1H), 3.54 (s, 2H), 4.24 (m, 2H), 4.72 (q,
1H), 5.85 (s, 1H), 6.22 (d, 1H), 6.76 (s, 1H), 6.95 (m, 2H), 7.13 (m, 3H), 7.20-7.33 (m, 7H), 7.36 (m,
1H), 7.67 (d, 1H), 7.89 (s, 1H); MS (ESI) m/z 412 [M+H]+; HRMS (ESI) m/z calcd. C26H26N3O2
[M+H]+ 412.2025, found 412.2022.
(R)-2-Phenoxycarboxamido-3-(1H-indol-3-yl)-N-phenylpropanamide (2d). Replacing benzylamine
with aniline (Step A), compound 2d was prepared as a white solid: mp 195-197 °C; 1H-NMR (CDCl3)
δ 3.28 (m, 1H), 3.53 (q, 1H), 4.71 (m, 1H), 6.00 (d, 1H), 7.07-7.16 (m, 6H), 7.21-7.26 (t, 6H), 7.35-
7.42 (m, 3H), 7.80 (d, 1H), 8.12 (s, 1H); MS (ESI) m/z 400 [M+H]+; HRMS (ESI) m/z calcd.
C24H22N3O3 [M+H]+ 400.1661, found 400.1669.
(R)-2-Phenoxycarboxamido-3-(1H-indol-3-yl)-N-(4-fluorophenyl)-propanamide
(2e).
Replacing
benzylamine with 4-fluorobenzenamine (Step A), compound 2e was prepared as a yellow solid: mp
185-189 °C; 1H-NMR (CDCl3) δ 3.28 (m, 1H), 3.50 (q, 1H), 4.70 (m, 1H), 6.00 (d, 1H), 6.93 (t, 2H),
7.05-7.17 (m, 6H), 7.21-7.30 (m, 3H), 7.35-7.47 (m, 3H), 7.77 (d, 1H), 8.12 (s, 1H); MS (ESI) m/z 418
[M+H]+; HRMS (ESI) m/z calcd. C24H21N3O3 [M+H]+ 418.1567, found 418.1572.
(R)-2-Phenoxycarboxamido-3-(1H-indol-3-yl)-N-(4-ethoxyphenyl)-propanamide
(2f).
Replacing
benzylamine with 4-ethoxybenzenamine (Step A), compound 2f was prepared as a white solid: mp
180-184 °C; 1H-NMR (CDCl3) δ 1.39 (t, 3H), 3.27 (m, 1H), 3.53 (q, 1H), 3.98 (q, 2H), 4.68 (m, 1H),
6.00 (d, 1H), 6.77 (d, 2H), 7.08-7.17 (m, 7H), 7.20-7.25 (m, 2H), 7.35-7.41 (m, 3H), 7.80 (d, 1H), 8.12
(s, 1H); MS (ESI) m/z 444 [M+H]+; HRMS (ESI) m/z calcd. C26H26N3O4 [M+H]+ 444.1923, found
444.1938.
(R)-2-Phenoxycarboxamido-3-(1H-indol-3-yl)-N-(benzo[1,3]dioxol-5-yl)-propanamide
(2g).
Replacing benzylamine with benzo[1,3]dioxol-5-amine (Step A), compound 2g was prepared as a
white solid: mp 186-188 °C; 1H-NMR (CDCl3) δ 3.26 (q, 1H), 3.51 (q, 1H), 4.67 (q, 1H), 5.93 (s, 2H),
6.00 (d, 1H), 6.44 (d, 1H), 6.64 (d, 1H), 6.92 (s, 1H), 7.05-7.11 (m, 3H), 7.13-7.24 (m, 4H), 7.35-7.41
(m, 3H), 7.60 (d, 1H), 8.13 (s, 1H); MS (ESI) m/z 466 [M+Na]+; HRMS (ESI) m/z calcd.
C25H21N3O5Na [M+Na]+ 466.1379, found 466.1372.
(R)-2-Phenoxycarboxamido-3-(1H-indol-3-yl)-N-(5-cyclopropyl-1,3,4-thiadiazol-2-yl)-propanamide
(2h). Replacing benzylamine with 5-cyclopropyl-1,3,4-thiadiazol-2-amine (Step A), compound 2h was

Molecules 2009, 14
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1
prepared as a white solid: mp 95-101 °C; H-NMR (CDCl3) δ 1.00-1.08 (m, 2H), 1.12-1.14 (m, 2H),
2.21 (m, 1H), 3.38-3.44 (m, 2H), 4.95 (q, 1H), 6.97 (d, 2H), 7.04-7.08 (m, 2H), 7. 15 (q, 2H), 7.20 (d,
1H), 7.30 (m, 3H), 7.61 (d, 1H), 8.02 (s, 1H), 8.12 (s, 1H); MS (ESI) m/z 448 [M+H]+; HRMS (ESI)
m/z calcd. C23H22N5O3S [M+H]+ 448.1443, found 448.1423.
(R)-2-Phenoxycarboxamido-3-(1H-indol-3-yl)-N-[(thiophen-2-yl)methyl]-propanamide (2i). Replacing
benzylamine with (thiophen-2-yl)methanamine (Step A), compound 2i was prepared as a white solid:
1
mp 184-186 °C; H-NMR (DMSO-d6) δ 2.98 (q, 1H), 3.16 (q, 1H), 4.29 (q, 1H), 4.46 (m, 2H), 6.91-
6.95 (m, 4H), 6.98 (t, 1H), 7.06 (t, 1H), 7. 13-7.22 (m, 2H), 7.29-7.38 (m, 4H), 7.66 (d, 1H); MS (ESI)
m/z 442 [M+Na]+; HRMS (ESI) m/z calcd. C23H21N3O3NaS [M+Na]+ 442.1201, found 442.1200.
(R)-2-Phenoxycarboxamido-3-(1H-indol-3-yl)-N-(prop-2-yn-1-yl)-propanamide
(2j).
Replacing
benzylamine with prop-2-yn-1-amine (Step A), compound 2j was prepared as a white solid: mp 173-
177 °C; 1H-NMR (DMSO-d6) δ 2.93-2.99 (m, 1H), 3.09-3.16 (m, 2H), 3.88-3.91 (m, 2H), 4.27 (q, 1H),
6.92 (d, 2H), 6.98 (t, 1H), 7.06 (t, 1H), 7. 13-7.22 (m, 2H), 7.29-7.35 (m, 3H), 7.66 (d, 1H); MS (ESI)
m/z 384 [M+Na]+; HRMS (ESI) m/z calcd. C21H19N3O3Na [M+Na]+ 384.1324, found 384.1330.
(R)-2-Phenoxycarboxamido-3-(1H-indol-3-yl)-N-[(4-nitrophenyl)methyl]-propanamide
(2k).
Replacing benzylamine with (4-nitrophenyl)methanamine (Step A), compound 2k was prepared as a
1
white solid: mp 182-185 °C; H-NMR (DMSO-d6) δ 3.26 (q, 1H), 3.46 (dd, 1H), 4.32 (dd, 1H), 4.42
(dd, 1H), 4.62 (m, 1H), 5.82 (d, 1H), 6.06 (s, 1H), 7.01-7.10 (m, 5H), 7.14-7.24 (m, 3H), 7.36 (t, 2H),
7.42 (d, 1H), 7.73 (d, 1H), 8.04 (d, 2H), 8.10 (s, 1H); MS (ESI) m/z 459 [M+H]+; HRMS (ESI) m/z
calcd. C25H23N4O5 [M+H]+ 459.1668, found 459.1677.
(R)-2-Phenoxycarboxamido-3-(1H-indol-3-yl)-N-(4-methoxycarbonyl-phenylmethyl)-propanamide (2l).
Replacing benzylamine with methyl 4-(aminomethyl)benzoate, (Step A) compound 2l was prepared as
a white solid: mp 210-213 °C; 1H-NMR (DMSO-d6) δ 3.45 (dd, 1H), 3.48 (dd, 1H), 3.92 (s, 3H), 4.30
(dd, 1H), 4.37 (dd, 1H), 4.59 (m, 1H), 5.86 (m, 2H), 6.98-7.06 (m, 3H), 7.09-7.24 (m, 5H), 7.35-7.45
(m, 3H), 7.75 (d, 1H), 7.90 (d, 2H), 8.04 (s, 1H)；MS (ESI) m/z 494 [M+Na]+; HRMS (ESI) m/z
calcd. C27H25N3O5Na [M+Na]+ 494.1692, found 494.1682.
(R)-2-Benzylamino-3-(1H-indol-3-yl)-N-benzyl-propanamide (2c). A mixture of compound 5b (250
mg, 0.76 mmol), benzaldehyde (80 mg, 0.76 mmol), K2CO3 (315 mg, 2.28 mmol), and magnesium
sulfate (160 mg) in CH2Cl2 (5 mL) was stirred at 25 °C for 6 h. Filtration and concentration gave an
imine, which was dissolved in 5 mL of methanol. At 0 °C, sodium borohydride (32 mg, 0.84 mmol)
was added. After 1 h at 0 °C, the solvent was evaporated and the residue was purified by flash column
chromatography on silica gel, eluted with a mixture of EtOAc/petroleum ether (1:2, v/v), to afford 2c
(80 mg, 34%) as yellow solid: mp 38-40 °C; 1H-NMR (CDCl3) δ 3.03 (q, 1H), 3.38 (q, 1H), 3.56-3.60
(m, 2H), 3.70 (d, 1H), 4.44 (d, 2H), 6.95 (d, 1H), 7.00-7.02 (m, 2H), 7.11 (t, 1H), 7.17-7.23 (m, 6H),
7.26-7.33 (m, 4H), 7.37 (d, 1H), 7.60 (t, 1H), 7.66 (d, 1H), 8.04 (s, 1H); MS (ESI) m/z 384 [M+H]+;
HRMS (ESI) m/z calcd. C25H26N3O [M+H]+ 384.2076, found 384.2072.

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4. Conclusions
In this study, using a structure-based virtual screening approach in conjunction with chemical
synthesis and bioassay we have discovered four moderately active FP-2 inhibitors (1, 2b, 2k and 2l).
The preliminary SAR data was obtained, which shows that an R configuration of C2 in A region, a
phenoxycarboxamido moiety in the B region and a 4-nitrobenzyl moiety in the C region are essential
for the inhibitory activity against FP-2. Meanwhile, analysis of binding positions revealed that
introduction of H-bond donor or/and acceptor atom to phenyl ring part in C region would be likely to
produce some additional H-bond interactions to enhance molecular bioactivity. Although the most
potent compound 2k at this stage has activity in the submicromolar range, the preliminary structure-
activity relationships (SARs) of compounds 2a-l give some valuable clues for further structural
optimization. The anti-malarial activities of these FP-2 inhibitors presented here need to be further
investigated.
Acknowledgements
We gratefully acknowledge the financial support from the National Natural Science Foundation of
China (Grants 90813005, 30672539, and 20803022), the 863 Hi-Tech Program of China (Grants
2006AA020404, 2007AA02Z147, and 2007AA02Z304), the Shanghai Committee of Science and
Technology (Grant 08JC1407800), and the Shanghai Rising-Star Program (A type) of Shanghai
Ministry of Science and Technology (Grant 07QA14013).
References and Notes
1. Vangapandu, S.; Jain, M.; Kaur, K.; Patil, P.; Patel, S.R.; Jain, R. Recent advances in antimalarial
drug development. Med. Res. Rev. 2007, 27, 65-107.
2. Fidock, D.A.; Rosenthal, P.J.; Croft, S.L.; Brun, R.; Nwaka, S. Antimalarial drug discovery:
Efficacy models for compound screening. Nat. Rev. Drug Discov. 2004, 3, 509-520.
3. Wellems, T.E.; Plowe, C.V. Chloroquine-resistant malaria. J. Infect. Dis. 2001, 184, 770-776.
4. Trape, J.F. The public health impact of chloroquine resistance in Africa. Am. J. Trop. Med. Hyg.
2001, 64, 12-17.
5. Plowe, C.V. Monitoring antimalarial drug resistance: making the most of the tools at hand. J. Exp.
Biol. 2003, 206, 3745-3752.
6. Shenai, B.R.; Sijwali, P.S.; Singh, A.; Rosenthal, P.J. Characterization of native and recombinant
falcipain-2, a principal trophozoite cysteine protease and essential hemoglobinase of Plasmodium
falciparum. J. Biol. Chem. 2000, 275, 29000-29010.
7. Dua, M.; Raphael, P.; Sijwali, P.S.; Rosenthal, P.J.; Hanspal, M. Recombinant falcipain-2 cleaves
erythrocyte membrane ankyrin and protein 4.1. Mol. Biochem. Parasitol. 2001, 116, 95-99.
8. Rosenthal, P.J.; Olson, J.E.; Lee, G.K.; Palmer, J.T.; Klaus, J.L.; Rasnick, D. Antimalarial effects
of vinyl sulfone cysteine proteinase inhibitors. Antimicrob. Agents Chemother. 1996, 40, 1600-
1603.

Molecules 2009, 14
507
9. Shenai, B.R; Lee, B.J.; Alvarez-Hernandez, A.; Chong, P.Y.; Emal, C.D.; Neitz, R.J.; Roush,
W.R.; Rosenthal, P.J. Structure-activity relationships for inhibition of cysteine protease activity
and development of Plasmodium falciparum by peptidyl vinyl sulfones. Antimicrob. Agents
Chemother. 2003, 47, 154-160.
10. Lee, B.J.; Singh, A.; Chiang, P.; Kemp, S.J.; Goldman, E.A.; Weinhouse, M.I.; Vlasuk, G.P.;
Rosenthal, P.J. Antimalarial activities of novel synthetic cysteine protease inhibitors. Antimicrob.
Agents Chemother. 2003, 47, 3810-3814.
11. Domínguez, J.N.; López, S.; Charris, J.; Iarruso, L.; Lobo, G.; Semenov, A.; Olson, J.E.;
Rosenthal, P.J. Synthesis and antimalarial effects of phenothiazine inhibitors of a Plasmodium
falciparum cysteine protease. J. Med. Chem. 1997, 40, 2726-2732.
12. Huang, L.; Lee, A.; Ellman, J.A. Identification of potent and selective mechanism-based
inhibitors of the cysteine protease cruzain using solid-phase parallel synthesis. J. Med. Chem.
2002, 45, 676-684.
13. Greenbaum, D.C.; Mackey, Z.; Hansell, E.; Doyle, P.; Gut, J.; Caffrey, C.R.; Lehrman, J.;
Rosenthal, P.J.; McKerrow, J.H.; Chibale, K. Synthesis and structure-activity relationships of
parasiticidal thiosemicarbazone cysteine protease inhibitors against Plasmodium falciparum,
Trypanosoma brucei, and Trypanosoma cruzi. J. Med. Chem. 2004, 47, 3212-3219.
14. Desai, P.V.; Patny, A.; Sabnis, Y.; Tekwani, B.; Gut, J.; Rosenthal, P.; Srivastava, A.; Avery, M.
Identification of novel parasitic cysteine protease inhibitors using virtual screening. 1. The
ChemBridge database. J. Med. Chem. 2004, 47, 6609-6615.
15. Domínguez, J.N.; León, C.; Rodrigues, J.; Gamboa de Domínguez, N.; Gut, J.; Rosenthal, P.J.
Synthesis and evaluation of new antimalarial phenylurenyl chalcone derivatives. J. Med. Chem.
2005, 48, 3654-3658.
16. 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.
17. Batra, S.; Sabnis, Y.A.; Rosenthal, P.J.; Avery, M.A. Structure-based approach to falcipain-2
inhibitors: Synthesis and biological evaluation of 1,6,7-trisubstituted dihydroisoquinolines and
isoquinolines. Bioorg. Med. Chem. 2003, 11, 2293-2299.
18. Chiyanzu, I.; Hansell, E.; Gut, J.; Rosenthal, P.J.; Mckerrow, J.H.; Chibale, K. Synthesis and
evaluation of isatins and thiosemicarbazone derivatives against cruzain, falcipain-2 and rhodesain.
Bioorg. Med. Chem. Lett. 2003, 13, 3527-3530.
19. Chipeleme, A.; Gut, J.; Rosenthal, P.J.; Chibale, K. Synthesis and biological evaluation of
phenolic Mannich bases of benzaldehyde and (thio)semicarbazone derivatives against the cysteine
protease falcipain-2 and a chloroquine resistant strain of Plasmodium falciparum. Bioorg. Med.
Chem. 2007, 15, 273-282.
20. Rosenthal, P.J.; Wollish, W.S.; Palmer, J.T.; Rasnick, D. Antimalarial effects of peptide inhibitors
of a Plasmodium falciparum cysteine proteinase. J. Clin. Invest. 1991, 88, 1467-1472.
21. Rosenthal, P.J.; Lee, G.K.; Smith, R.E. Inhibition of a Plasmodium vinckei cysteine proteinase
cures murine malaria. J. Clin. Invest. 1993, 91, 1052-1056.
22. Sajid, M.; McKerrow, J. Cysteine proteases of parasitic organisms. Mol. Biochem. Parasitol.
2002, 120, 1-21.

Molecules 2009, 14
508
23. Jorgensen, W.L. The many roles of computation in drug discovery. Science 2004, 303, 1813-
1818.
24. Sabnis, Y.; Rosenthal, P.J.; Desai, P.; Avery, M.A. Homology modeling of falcipain-2:
Validation, de novo ligand design and synthesis of novel inhibitors. J. Biomol. Struct. Dyn. 2002,
19, 765-774.
25. Goh, L.L.; Sim, T.S. Homology modeling and mutagenesis analyses of Plasmodium falciparum
falcipain 2A: Implications for rational drug design. Biochem. Biophys. Res. Commun. 2004, 323,
565-572.
26. Hogg, T.; Nagarajan, K.; Herzberg, S.; Chen, L.; Shen, X.; Jiang, H.; Wecke, M.; Blohmke, C.;
Hilgenfeld, R.; Schmidt, C.L. Structural and functional characterization of Falcipain-2, a
hemoglobinase from the malarial parasite Plasmodium falciparum. J. Biol. Chem. 2006, 281,
25425-25437.
27. Wang, S.X.; Pandey, K.C.; Somoza, J.R.; Sijwali, P.S.; Kortemme, T.; Brinen, L.S.; Fletterick,
R.J.; Rosenthal, P.J.; Mckerrow, J.H. Structural basis for unique mechanisms of folding and
hemoglobin binding by a malarial protease. Proc. Natl. Acad. Sci. USA 2006, 103, 11503-11508.
28. Li, H.; Chen, L.; Huang, J.; Zhu, J.; Shen, X.; Li, J.; Hilgenfeld, R.; Jiang, H. Identification of
novel Falcipain-2 inhibitors as potential antimalarial agents through structure-based virtual
screening. Submitted to J. Med. Chem.
29. Peters, W. Drug resistance in Plasmodium berghei Vincke and Lips, 1948. I. Chloroquine
resistance. Expl. Parasite 1965, 17, 80-89.
30. Berman, H.M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.N.; Weissig, H.; Shindyalov, I.N.;
Bourne, P.E. The Protein Data Bank. Nucleic Acids Res. 2000, 28, 235-242.
31. Rosenthal, P.J.; McKerrow, J.H.; Rasnick, D.; Leech, J.H. Plasmodium falciparum: Inhibitors of
lysosomal cysteine proteinases inhibit a trophozoite proteinase and block parasite development.
Mol. Biochem. Parasitol. 1989, 35, 177-183.
Sample Availability: Samples of the compounds reported in this paper are available from authors.
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