2-(3,4-dihydro-4-oxothieno[2,3-d]pyrimidin-2-ylthio) acetamides as a new class of falcipain-2 inhibitors. 3. design, synthesis and biological evaluation

Article abstract of DOI:10.3390/molecules14020785

The cysteine protease falcipain-2 (FP-2) of Plasmodium falciparum is a principal cysteine protease and an essential hemoglobinase of erythrocytic P. falciparum trophozoites, making it become an attractive target enzyme for developing anti-malarial drugs.

Full text of DOI:10.3390/molecules14020785

Molecules 2009, 14, 785-797; doi:10.3390/molecules14020785
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
2-(3,4-Dihydro-4-Oxothieno[2,3-d]pyrimidin-2-ylthio)
Acetamides as a New Class of Falcipain-2 Inhibitors. 3. Design,
Synthesis and Biological Evaluation
Jin Zhu 1,†, Tong Chen 1,†, Jie Liu 1, Ruoqun Ma 1, Weiqiang Lu 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
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
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed. E-mail: jianli@ecust.edu.cn (J.L.),
huangjin@ecust.edu.cn (J.H.), hlli@ecust.edu.cn (H.L.).
Received: 15 December 2008; in revised form: 18 January 2009 / Accepted: 13 February 2009 /
Published: 16 February 2009
Abstract: The cysteine protease falcipain-2 (FP-2) of Plasmodium falciparum is a
principal cysteine protease and an essential hemoglobinase of erythrocytic P. falciparum
trophozoites, making it become an attractive target enzyme for developing anti-malarial
drugs. In this study, a series of novel small molecule FP-2 inhibitors have been designed
and synthesized based on compound 1, which was identified by using structure-based
virtual screening in conjunction with an enzyme inhibition assay. All compounds showed
high inhibitory effect against FP-2 with IC50s of 1.46-11.38 μM, and the inhibitory activity
of compound 2a was ~2 times greater than that of prototype compound 1. The preliminary
SARs are summarized and should be helpful for future inhibitor design, and the novel
scaffold presented here, with its potent inhibitory activity against FP-2, also has potential
application in discovery of new anti-malarial drugs.
Keywords: 2-(3,4-Dihydro-4-oxothieno[2,3-d]pyrimidin-2-ylthio)acetamide derivatives;
Falcipain-2 inhibitor; Malaria; SAR.

Molecules 2009, 14
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Introduction
Malaria remains one of the most important infectious disease problems in the world, accounting for
300-500 million clinical cases and up to 2.7 million deaths each year. About 90% of these casualties
occur in tropical Africa, and the great majority are children under the age of 5 [1]. Plasmodium
falciparum, one of the four species of Plasmodium, is the most lethal protozoan parasite of the genus,
which is responsible for malaria. At present no effective vaccines are available due to the high
mutability of the genome of Plasmodium falciparum [2], meanwhile, resistance of malaria parasites to
available conventional drug therapy is an increasingly serious problem [3-5]. Accordingly, the
discovery of new effective drugs to counter the spread of malaria parasites that are resistant to existing
agents, especially 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 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 confirmed that
inhibitors of falcipain-2 can block parasite hemoglobin hydrolysis and halt the development of culture
parasites [8-22]. Some of them were also effective against murine malaria in vivo [15, 21-22].
However, FP-2 inhibitors reported 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.
Recently, crystal structures for falcipain-2 have been reported [23-24], 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 FP-2. By using a docking-based virtual
screening approach in conjunction with an enzyme inhibition assay, a novel small molecule inhibitor
of FP-2 featuring the 2-(3,4-dihydro-4-oxothieno[2,3-d]pyrimidin-2-ylthio) acetamide framework
(compound 1) has been discovered [25]. After the identification of compound 1 as a possible prototype
for the design of selective inhibitors of FP-2, fifteen new compounds, including 1 and its fourteen
analogs (2a-e, 3a-c, and 4a-f), have been synthesized and tested against FP-2. All of the compounds
show high inhibitory effect against FP-2 with IC50s of 1.46-11.38 μM, and inhibitory activity of
compound 2a increases ~2 times than that of prototype compound 1. The preliminary SARs are
summarized and should be helpful for future design of inhibitors.
Results and Discussion
Identification of Prototype (Hit) 1 by Virtual Screening
Targeting the crystal structure of falcipain-2 (PDB entry 2GHU) [23], a total of 80,000 compounds
were subsequently docked and ranked according to the software Glide and GAsDock [25]. Finally, 81
compounds were purchased and submitted to biological evaluations against falcipain-2. Among the 81
compounds, the inhibitory activity of compound 1 was concentration-dependent. The collected data

Molecules 2009, 14
787
indicated that compound 1 can inhibit FP-2 in vitro, with the inhibitory effect against FP-2 is in the
micromolar range (IC50 = 2.81 μM). The substrate (L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl-
agmatin (E-64), whose inhibitory effect against FP-2 is in the subnanomolar range (IC50 = 18.1 nM),
was used for the positive control. Compound 1 could be designated as a hit of FP-2. The inhibitory
rates (%) off compounds 1 and E-64 plotted against the common logarithm of the compound
concentrations are shown in Figures 1a and 1b, respectively.
Figure 1. Concentration dependence of inhibitory activity by 1 and E-64, the concentration
of FP-2 was kept constant at 30 nM while the concentration of compounds ranged from
0.001 to10 μM.
(a)
(b)
100
80
60
40
20
0
100
80
60
40
20
0
IC50 = 2.81 μM
IC = 18.1 nM
50
4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4
6
8
10
12
1 concentration(-lg dose/M)
E-64 concentration(-lg dose/M)
Compou
E-64
nd 1
Design and Synthesis of Compounds 1-4
On the basis of the framework of compound 1, fourteen compounds (2a-e, 3a-c, and 4a-f) were
designed and synthesized. Their chemical structures are shown in Table 1. Retaining the common 2-
(3,4-dihydro-4-oxothieno[2,3-d]pyrimidin-2-ylthio) acetamide framework of compound 1, we first
changed the allyl group on the pyrimidine ring into cyclohexyl, aryl, and benzyl groups, and obtained
analogs 2a-e (Table 1). We then replaced the phenyl group on the thiophene ring with cyclohexyl or
substituted phenyl groups to prepare compounds 3a-c (Table 1). Finally, compounds 4a-f (Table 1)
were achieved by replacing the amide N-substituents with other aryl groups.
Compounds 1-4 were synthesized through the route outlined in Scheme 1. Using our previous
method [26], using basic aluminium oxide as solid support and morpholine as base catalyst, ketones,
cyanoacetates, and sulphur were mixed and subjected to microwave irradiation for several minutes,
which afforded 2-aminothiophenes 5a-d. Compounds 5a-d were converted to the corresponding
thioureas 6a-i by reaction with isothiocyanates, then 6a-i were cyclized under alkaline conditions,
giving the key intermediates 7a-i (Scheme 1). Various aromatic amines were chloracetylated with
chloroacetic chloride to give compounds 8a-g, which were reacted with compounds 7a-i to produce
the target compounds 1-4 smoothly. The details of the synthetic procedures and product
characterizations are described in the Experimental section.

Molecules 2009, 14
788
Table 1. Chemical Structures of Compounds 1, 2a-e, 3a-c and 4a-f and Their Inhibitory
Activities against FP-2.
O
R2
R3
S
N
H
N
R1
S
N
R4
O
Inhibition rate
at 10 μM (%)
IC50
(μM)
Compd. R1
R2
R3
R4
S
1
H
H
H
H
H
H
88.7
92.7
79.0
85.4
84.7
90.6
82.2
2.81
1.46
2.05
2.77
4.30
5.74
5.77
O
O
O
O
O
O
O
NH2
S
2a
2b
2c
2d
2e
3a
NH2
S
F
NH2
S
O2N
NH2
S
Cl
NH2
S
NH2
S
—(CH2)4—
NH2

Molecules 2009, 14
789
Table 1. Cont.
S
3b
3c
4a
H
H
H
85.7
53.0
93.3
2.95
11.8
6.63
O
O
O
Cl
NH2
S
O2N
NH2
S
O
O
O
4b
4c
4d
H
H
H
94.3
90.3
93.2
5.70
3.31
2.49
S
N
S
O
NH2
O
S
O
4e
4f
H
H
72.0
92.0
5.58
5.43
O
NH2
F
Cl
Enzyme Inhibition Assay
For the primary assay, the percent inhibitions of the compounds at 10 μM were measured. The
results are listed in Table 1. All compounds can remarkably inhibit the activity of FP-2 (Percent
inhibition at 10 μM > 50%), indicating that these fifteen compounds (including compound 1) are FP-2
inhibitors. Therefore, we determined their IC50 values (Table 1). From the data in Table 1, we can see
that the percent inhibitory rates at 10 μM of compounds 2a, 2e, 4a-d, and 4f obviously increased (more
than 90%, Table 1). Among five anologs varied on R3 substituents (2a-2e, Table 1), it is remarkable
that the inhibitory activity of compound 2a increased ~2 times than that of compound 1 (IC50 decreases
from 2.81 μM down to 1.46 μM, Table 1), the former replace the allyl group on pyrimidine ring with a

Molecules 2009, 14
790
cyclohexyl group. The percent inhibitory rates at 10 μM of analogs 3a-c, which vary on R1 and R2
substituents, were decreased in different extent, especially analogue 3c which showed a marked
decrease from 88.7% to 53.0% (Table 1). Changes of R4 substituents did not have a distinct
improvement for the IC50s of analogs 4a-f (Table 1).
Scheme 1a The synthetic route to compounds 1-4.
O
O
O
N
R2
R2
O
O
R2
O
R3
O
O
c
R3NCS
a
N
R1
R2
+
+
S
NH
R1
b
NH2
S
R3
S
R1
SH
NC
S
R1
N
6
S
7
5
H
5a: R1 = H, R2 = phenyl
6a and 7a: R1 = H, R2 = phenyl, R3 = allyl
6b and 7b: R1 = H, R2 = phenyl, R3 = cyclohexyl
5b: R1 = R2 = -(CH2)4-
5c: R1 = H, R2 = 4-Cl-phenyl
5d: R1 = H, R2 = 3-NO2-phenyl
6c and 7c: R1 = H, R2 = phenyl, R3 = 3-F-phenyl
6d and 7d: R1 = H, R2 = phenyl, R3 = 4-NO2-phenyl
6e and 7e: R1 = H, R2 = phenyl, R3 = 4-Cl-phenyl
6f and 7f: R1 = H, R2 = phenyl, R3 = benzyl
6g and 7g: R1 = R2 = -(CH2)4-, R3 = allyl
6h and 7h: R1 = H, R2 = 4-Cl-phenyl, R3 = allyl
6i and 7i: R1 = H, R2 = 3-NO2-phenyl, R3 = allyl
O
O
d
R4
Cl
Cl
R4NH2
+
N
Cl
H
8
O
S
S
O
8a:
8b:
8c:
O
O
NH2
S
O
S
O
R2
8e:
8d:
8f:
N
R3
S
O
e
N
NH2
O
H
R1
S
N
F
S
O
N
R4
8g:
O
Cl
O
NH2
1, 2a-e, 3a-c, and 4a-f
a Reagents and conditions: (a) MWI, morpholine, basic Al2O3, 140 W, 15 min; (b) pyridine, 45 °C,
5 h; (c) CH3ONa, CH3OH, 25 °C, 10 h; (d) THF, Et3N, 25 °C, 10 h; (e) THF, K2CO3, 40 °C, 10 h.
Structure and Activity Relationships
After testing the inhibitory activities of compounds 1, 2a-e, 3a-c, and 4a-f against FP-2, from the
data in Table 1, we can deduce the preliminary structure-activity relationships (SARs) as follows: (1)
Replacing the allyl group of compound 1 with proper substituent, such as a cyclohexyl (2a) group,
moderately improves potency, as seen with the IC50 of compound 2a which is decreased to 1.46 μM
(Table 1); (2) Replacing the phenyl group on the thiophene ring of 1 with cyclohexyl (3a) or
substituted phenyl (3b-3c) can not be tolerated and leads to the loss of inhibitory activity; (3) N-
substituents of amide of compound 1 have considerable adaptability and alterability. This encouraging
result and primary SARs are helpful for future inhibitors design.

Molecules 2009, 14
791
Conclusions
In this study, we have discovered a new kind of scaffold of FP-2 inhibitor by using a structure-
based virtual screening approach in conjunction with chemical synthesis and bioassay. The preliminary
SARs were obtained, which show changes of substituents on 3, 5, and 6-positions of thieno[2,3-
d]pyrimidine moiety have very important influence on inhibitory activity, and appropriate structural
optimizations on the above regions can substantially improve potency. These primary SARs are
helpful for future inhibitors design, and the novel scaffold presented here with potent inhibitory
activity against FP-2 also provides potential application in discovery of anti-malarial drugs.
Experimental
Reagents were purchased from Alfa, Acros and Shanghai Chemical Reagent Company, and used
without further purification. Analytical thin-layer chromatography (TLC) was performed on HSGF
254 plates (150-200 µm thickness, Yantai Huiyou Company, P.R. China). Yields were not optimized.
Melting points were measured in capillary tube on a SGW X-4 melting point apparatus without
correction. Nuclear magnetic resonance (NMR) spectra were given on a Bruker AVANCE 500 NMR
(TMS as internal standard). Chemical shifts were reported in parts per million (ppm, δ) downfield from
tetramethylsilane. Proton coupling patterns were 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
given with electrospray (ESI) produced by LCQ-TOF spectrometer. Microwave experiments were
carried out in a domestic microwave oven (Haier MA-2270EGC)
Virtual Screening by Molecular Docking
The crystal structure of falcipain-2 (PDB entry 2GHU) [23] from Plasmodium falciparum was
retrieved from the Protein Database Bank [27]. 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 the reference 25.
Enzyme Inhibition Assay
The purification and refolding of recombinant protein Falcipain-2 was performed as described by
Shenai et al [6]. IC50 values against recombinant Falcipain-2 were determined as described previously
[8, 28]. FP-2 (30 nM) 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 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).

Molecules 2009, 14
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Half-maximal inhibitory concentration (IC50) was determined from plots of percent activity over
compound concentration using GraphPad Prism software.
General procedures for the preparation of 2-(3,4-dihydro-4-oxothieno[2,3-d]pyrimidin-2-ylthio)-
acetamides 1, 2a-e, 3a-c, and 4a-f, exemplified by the preparation of compound
1
Ethyl 2-amino-4-phenylthiophene-3-carboxylate (5a). A one-neck 50-mL flask containing
acetophenone (2.4 g, 20 mmol), ethyl cyanoacetate (3.4 g, 30 mmol), sulphur (0.96 g, 30 mmol), basic
Al2O3 (1.8 g), and morpholine (2.6 g, 30 mmol) was placed into a microwave oven and irradiated at
the power of 140W for 15 min. After cooling, the residue was separated by column chromatography
with silica gel using petroleum ether/ethyl acetate (10/1) as eluting solution, to afford 5a (2.0 g, 27%)
as a yellow solid: mp 86-90 °C; 1H-NMR (CDCl3) δ 0.92 (t, 3H), 4.03 (q, 2H), 6.06 (s, 1H), 7.30-7.32
(m, 5H). Compounds 5b-d were prepared in a similar manner.
3-Allyl-2-mercapto-5-phenylthieno[2,3-d]pyrimidin-4(3H)-one (7a). A mixture of 5a (1 g, 4 mmol)
and allyl isocyanate (0.5 g, 5 mmol) in THF (5 mL) was stirred at 45 °C for 5 h. Concentration of the
reaction mixture gave the compound 6a, which was suspended in MeOH (5 mL). To this suspension
was added MeONa (0.55 g, 10 mmol).After being stirred at room temperature for 10 h, the mixture
was adjusted to pH 1 with 2 N HCl at 0 °C. The solution was extracted with EtOAc. The combined
organic layer was washed, dried, filtered and concentrated. The residue was separated by column
chromatography with silica gel using petroleum ether/ethyl acetate (5/1) as eluting solution to afford
1
7a (0.26 g, 22%) as a yellow solid: mp 194-197 °C; H-NMR (CDCl3) δ 5.11 (d, 2H), 5.25 (d, 1H),
5.37 (d, 1H), 5.96-6.01 (m, 1H), 6.82 (s, 1H), 7.38-7.43 (m, 3H), 7.47-7.49 (m, 2H), 11.85 (s, 1H).
Compounds 7b-i were prepared in a similar manner.
2-(2-Chloroacetamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (8a). To a stirred,
room-temperature solution of 2-amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (180
mg, 1 mmol, which was prepared in the same manner as described for 5a, triethylamine (0.1 mL) and
THF (2 mL) was added dropwise chloroacetic chloride (170 mg, 1.5 mmol). After 10 hours, poured
into H2O (10 mL), and 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
1
mixture of EtOAc/petroleum ether (1:2, v/v), to afford 8a (129 mg, 50%) as white solid. H-NMR
(CDCl3) δ 2.33-2.36 (m, 2H), 2.80 (t, 2H), 2.91 (t, 2H), 4.52 (s, 2H). Compounds 8b-g were similarly
prepared.
2-(2-(3-Allyl-3,4-dihydro-4-oxo-5-phenylthieno[2,3-d]pyrimidin-2-ylthio)acetamido)-5,6-dihydro-4H-
cyclopenta[b]thiophene-3-carboxamide (1) A mixture of 7a (132 mg, 0.44 mmol), 8a (114 mg, 0.44
mmol), and K2CO3 (0.6 g, 4.4 mmol) in THF (5 mL) was stirred at 40 °C for 10 h. poured into H2O (10
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 (68 mg, 30%) as yellow solid: mp 218-221 °C; 1H-NMR
(DMSO) δ 2.32-2.36 (m, 2H), 2.76 (t, 2H), 2.89 (t, 2H), 4.26 (s, 2H), 4.74 (d, 2H), 5.17-5.25 (m, 2H),

Molecules 2009, 14
793
5.91-5.97 (m, 1H), 7.34-7.39 (m, 4H), 7.47-7.49 (m, 2H); ESI-MS m/z 545 [M+Na]+; HRMS (ESI)
m/z calcd C25H22N4O3S3Na [M+Na]+ 545.0752, found 545.0745.
2-(2-(3-Cyclohexyl-3,4-dihydro-4-oxo-5-phenylthieno[2,3-d]pyrimidin-2-ylthio)acetamido)-5,6-
dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (2a) Replacing allyl isocyanate with cyclohexyl
isocyanate, 2a was prepared as a yellow solid: mp 225-229 °C; 1H-NMR (DMSO) δ 1.35 (m, 2H), 1.83
(m, 4H), 2.30-2.36 (m, 2H), 2.50-2.56 (m, 4H), 2.78 (t, 2H), 2.89 (t, 2H), 4.16 (m, 1H), 4.21 (s, 2H),
7.31 (s, 1H), 7.34-7.39 (m, 3H), 7.44 (m, 2H); ESI-MS m/z 565 [M+H]+; HRMS (ESI) m/z calcd
C28H29N4O3S3 [M+H]+ 565.1402, found 565.1411.
(2-(2-(3-(3-Fluorophenyl)-3,4-dihydro-4-oxo-5-phenylthieno[2,3-d]pyrimidin-2-ylthio)acetamido)-
5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (2b) Replacing allyl isocyanate with 3-fluoro-
phenyl isocyanate, 2b was prepared as a pale yellow solid: mp 234-239 °C; 1H-NMR (DMSO) δ 2.31-
2.36 (m, 2H), 2.78 (t, 2H), 2.90 (t, 2H), 4.10 (s, 2H), 7.30-7.35 (m, 2H), 7.43 (s, 1H), 7.46-7.48 (m,
4H), 7.58-7.68 (m, 3H); ESI-MS m/z 599 [M+Na]+; HRMS (ESI) m/z calcd C28H21N4O3FS3Na
[M+Na]+ 599.0658, found 599.0667.
(2-(2-(3-(4-Nitrophenyl)-3,4-dihydro-4-oxo-5-phenylthieno[2,3-d]pyrimidin-2-ylthio)acetamido)-5,6-
dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (2c) Replacing allyl isocyanate with 4-
nitrophenyl isocyanate, 2c was prepared as an orange solid: mp 271-274 °C; 1H-NMR (CDCl3) δ 2.47-
2.50 (m, 2H), 2.86-2.90 (m, 4H), 4.08 (s, 2H), 7.04 (s, 1H), 7.25-7.36 (m, 3H), 7.47-7.50 (m, 2H), 7.71
(d, 2H), 8.40 (d, 2H); ESI-MS m/z 626 [M+Na]+; HRMS (ESI) m/z calcd C28H21N5O5S3Na [M+Na]+
626.0603, found 626.0605.
(2-(2-(3-(4-Chlorophenyl)-3,4-dihydro-4-oxo-5-phenylthieno[2,3-d]pyrimidin-2-ylthio)acetamido)-
5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (2d) Replacing allyl isocyanate with 4-
1
chloro-phenyl isocyanate, 2d was prepared as a yellow solid: mp 247-249 °C; H-NMR (CDCl3) δ
2.47-2.54 (m, 2H), 2.89-2.93 (m, 4H), 4.08 (s, 2H), 7.04 (s, 1H), 7.32-7.36 (m, 3H), 7.41-7.43 (m, 2H),
7.52 (d, 2H), 7.54 (d, 2H); ESI-MS m/z 615 [M+Na]+; HRMS (ESI) m/z calcd C28H21N4O3ClS3Na
[M+Na]+ 615.0362, found 615.0351.
(2-(2-(3-Benzyl-3,4-dihydro-4-oxo-5-phenylthieno[2,3-d]pyrimidin-2-ylthio)acetamido)-5,6-dihydro-
4H-cyclopenta[b]thiophene-3-carboxamide (2e) Replacing allyl isocyanate with benzyl isocyanate, 2e
1
was prepared as a gray solid: mp 163-167 °C; H-NMR (CDCl3) δ 2.48-2.53 (m, 2H), 2.89-2.96 (m,
4H), 4.16 (s, 2H), 4.26 (s, 2H), 7.00 (s, 1H), 7.30-7.43 (m, 8H), 7.54-7.56 (m, 2H); ESI-MS m/z 595
[M+Na]+; HRMS (ESI) m/z calcd C29H24N4O3S3Na [M+Na]+ 595.0908, found 595.0908.
(2-(2-(3-Allyl-3,4,5,6,7,8-hexahydro-4-oxo-benzo[4,5]thieno[2,3-d]pyrimidin-2-ylthio)
acetamido)-
5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (3a) Replacing acetophenone with cyclo-
hexanone, 3a was prepared as a yellow solid: mp 247-250 °C; 1H-NMR (CDCl3) δ 1.82-1.87 (m, 4H),
2.48-2.52 (m, 2H), 2.71 (t, 2H), 2.88-2.91 (m, 4H), 2.97 (t, 2H), 4.15 (s, 2H), 4.83 (d, 2H), 5.31 (m,
2H), 5.92 (m, 1H); ESI-MS m/z 523 [M+Na]+; HRMS (ESI) m/z calcd C23H24N4O3S3Na [M+Na]+
523.0908, found 523.0911.

Molecules 2009, 14
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2-(2-(3-Allyl-3,4-dihydro-4-oxo-5-(4-chlorophenyl)thieno[2,3-d]pyrimidin-2-ylthio) acetamido)-5,6-
dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (3b) Replacing acetophenone with 4-
1
chloroacetophenone, 3b was prepared as a gray solid: mp 236-239 °C; H-NMR (CDCl3) δ 2.49-2.54
(m, 2H), 2.89-2.93 (m, 4H), 4.20 (s, 2H), 4.83 (d, 2H), 5.28-5.35 (m, 2H), 5.90-5.98 (m, 1H), 6.97 (s,
1H), 7.38 (d, 2H), 7.47 (d, 2H); ESI-MS m/z 579 [M+Na]+; HRMS (ESI) m/z calcd
C25H21N4O3S3ClNa [M+Na]+ 579.0362, found 579.0350.
2-(2-(3-Allyl-3,4-dihydro-4-oxo-5-(3-nitrophenyl)thieno[2,3-d]pyrimidin-2-ylthio)acetamido)-5,6-
dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (3c) Replacing acetophenone with 3-
1
nitroacetophenone, 3c was prepared as a yellow solid: mp 157-161 °C; H-NMR (CDCl3) δ 2.47-2.51
(m, 2H), 2.87-2.91 (m, 4H), 4.19 (s, 2H), 4.83 (d, 2H), 5.26-5.31 (m, 2H), 5.88-5.97 (m, 1H), 7.08 (s,
1H), 7.56 (q, 1H), 7.88 (d, 1H), 8.20 (d, 1H), 8.37 (s, 1H); ESI-MS m/z 590 [M+Na]+; HRMS (ESI)
m/z calcd C25H21N5O5S3Na [M+Na]+ 590.0603, found 590.0616.
Ethyl
2-(2-(3-allyl-3,4-dihydro-4-oxo-5-phenylthieno[2,3-d]pyrimidin-2-ylthio)acetamido)-5,6-
(4a) Replacing 2-amino-5,6-dihydro-4H-
dihydro-4H-cyclopenta[b]thiophene-3-carboxylate
cyclopenta[b]thiophene-3-carboxamide with ethyl 2-amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-
carboxylate, 4a was prepared as a white solid: mp 166-170 °C; 1H-NMR (DMSO) δ 1.28 (t, 3H), 2.28-
2.33 (m, 2H), 2.77-2.83 (m, 4H), 4.28 (q, 2H), 4.32 (s, 2H), 4.73 (d, 2H), 5.20-5.24 (m, 2H), 5.90-5.97
(m, 1H), 7.20 (s, 1H), 7.35-7.38 (m, 3H), 7.46-7.48 (m, 2H); ESI-MS m/z 574 [M+Na]+; HRMS (ESI)
m/z calcd C27H25N3O4S3Na [M+Na]+ 574.0905, found 574.0907.
Ethyl 4-(2-(3-allyl-3,4-dihydro-4-oxo-5-phenylthieno[2,3-d]pyrimidin-2-ylthio)acetamido) benzoate
(4b) Replacing 2-amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide with ethyl 4-amino-
benzoate, 4b was prepared as a white solid: mp 162-164 °C; 1H-NMR (CDCl3) δ 1.40 (t, 3H), 4.05 (s,
2H), 4.37 (q, 2H), 4.79 (d, 2H), 5.30-5.35 (m, 2H), 5.86-5.96 (m, 1H), 7.09 (s, 1H), 7.41-7.45 (m, 3H),
7.54-7.57 (m, 2H), 7.62 (d, 2H), 8.02 (d, 2H); ESI-MS m/z 528 [M+Na]+; HRMS (ESI) m/z calcd
C26H23N3O4S2Na [M+Na]+ 528.1028, found 528.1033.
2-(3-Allyl-3,4-dihydro-4-oxo-5-phenylthieno[2,3-d]pyrimidin-2-ylthio)-N-(benzo[d]thiazol-2-yl)-
acetamide (4c) Replacing 2-amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide with
1
benzo[d]thiazol-2-amine, 4c was prepared as a white solid: mp 192-194 °C; H-NMR (CDCl3) δ 4.20
(s, 2H), 4.79 (d, 2H), 5.31-5.35 (m, 2H), 5.86-5.96 (m, 1H), 7.07 (s, 1H), 7.33-7.48 (m, 5H), 7.53-7.55
(m, 2H), 7.80-7.85 (m, 2H); ESI-MS m/z 513 [M+Na]+; HRMS (ESI) m/z calcd C24H18N4O2S3Na
[M+Na]+ 513.0490, found 513.0502.
2-(2-(3-Alyl-3,4-dihydro-4-oxo-5-phenylthieno[2,3-d]pyrimidin-2-ylthio)acetamido)-4,5,6,7-tetra-
hydrobenzo[b]thiophene-3-carboxamide (4d) Replacing 2-amino-5,6-dihydro-4H-cyclopenta[b]-
thiophene-3-carboxamide with 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide, 4d was
1
prepared as a white solid: mp 212-214 °C; H-NMR (CDCl3) δ 1.86 (m, 4H), 2.71 (m, 4H), 4.20 (s,
2H), 4.84 (d, 2H), 5.27-5.37 (m, 2H), 5.90-6.02 (m, 1H), 6.97 (s, 1H), 7.38-7.43 (m, 3H), 7.52-7.54
(m, 2H); ESI-MS m/z 559 [M+Na]+; HRMS (ESI) m/z calcd C26H24N4O3S3Na [M+Na]+ 559.0908,
found 559.0915.

Molecules 2009, 14
Ethyl 5-(2-(3-allyl-3,4-dihydro-4-oxo-5-phenylthieno[2,3-d]pyrimidin-2-ylthio)acetamido)-4-
795
carbamoyl-3-methylthiophene-2-carboxylate (4e) Replacing 2-amino-5,6-dihydro-4H-cyclopenta[b]-
thiophene-3-carboxamide with ethyl 5-amino-4-carbamoyl-3-methylthiophene-2-carboxylate, 4e was
prepared as a white solid: mp 136-140 °C; 1H-NMR (CDCl3) δ 1.40 (t, 3H), 2.80 (s, 3H), 4.19 (s, 2H),
4.40 (q, 2H), 4.80 (d, 2H), 5.27-5.33 (m, 2H), 5.86-5.94 (m, 1H), 6.96 (s, 1H), 7.36-7.43 (m, 3H),
7.49-7.52 (m, 2H); ESI-MS m/z 591 [M+Na]+; HRMS (ESI) m/z calcd C26H24N4O5S3Na [M+Na]+
591.0807, found 591.0809.
2-(3-Allyl-3,4-dihydro-4-oxo-5-phenylthieno[2,3-d]pyrimidin-2-ylthio)-N-(3-chloro-4-fluorophenyl)-
acetamide (4f) Replacing 2-amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide with 3-
1
chloro-4-fluorobenzenamine, 4f was prepared as a yellow solid: mp 145-149 °C; H-NMR (CDCl3) δ
4.02 (s, 2H), 4.78 (d, 2H), 5.30-5.35 (m, 2H), 5.86-5.95 (m, 1H), 7.07-7.12 (m, 2H), 7.31-7.34 (m,
1H), 7.41-7.47 (m, 3H), 7.54-7.56 (m, 2H), 7.78 (q, 1H); ESI-MS m/z 508 [M+Na]+; HRMS (ESI) m/z
calcd C23H17N3O2FClS2Na [M+Na]+ 508.0332, found 508.0328.
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
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.

Molecules 2009, 14
796
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.
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.

Molecules 2009, 14
797
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.
23. 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.
24. 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.
25. Li, H.; Chen, Li.; 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.
26. Huang, W.; Li, J.; Tang, J.; Liu, H.; Shen, J.; Jiang, H. Microwave-assisted Synthesis of 2-Amino-
thiophene-3-carboxylic Derivatives under Solvent-Free Condition. Syn. Comm. 2005, 35, 1351-
1357.
27. 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.
28. 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 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
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