Synthesis of novel peptidomimetics as inhibitors of protozoan cysteine proteases falcipain-2 and rhodesain

Article abstract of DOI:10.1016/j.ejmech.2010.04.003

This paper describes the synthesis of novel peptidomimetics bearing a protected aspartyl aldeyde warhead leading to the thioacylals 2a,b and the acylals 3a,b. Compounds 2a and 3a proved to possess an increased antiplasmodial activity with respect to the parent molecule 1. Furthermore thioacylal 2a can be considered as a promising trypanocidal agent.

Full text of DOI:10.1016/j.ejmech.2010.04.003

European Journal of Medicinal Chemistry 45 (2010) 3228e3233
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of novel peptidomimetics as inhibitors of protozoan cysteine proteases
falcipain-2 and rhodesain
,
a
a
b
c
Roberta Ettari a , Maria Zappalà , Nicola Micale , Tanja Schirmeister , Christoph Gelhaus ,
*
Matthias Leippe c, Astrid Evers c, Silvana Grasso a
a Dipartimento Farmaco-Chimico, University of Messina, Viale Annunziata, 98168 Messina, Italy
b Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany
c Institute of Zoology, University of Kiel, Olshausenstr. 40, D-24098 Kiel, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper describes the synthesis of novel peptidomimetics bearing a protected aspartyl aldeyde
warhead leading to the thioacylals 2a,b and the acylals 3a,b. Compounds 2a and 3a proved to possess an
increased antiplasmodial activity with respect to the parent molecule 1. Furthermore thioacylal 2a can be
considered as a promising trypanocidal agent.
Received 1 February 2010
Received in revised form
16 March 2010
Accepted 7 April 2010
Available online 14 April 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Peptidomimetics
Falcipain-2 inhibitors
Rhodesain inhibitors
Cysteine proteases
1. Introduction
Trypanosoma brucei rhodesiense [7] and, if not treated, can be fatal.
In 2007, WHO estimated that the number of HAT cases in sub-
Neglected tropical diseases are a symptom of poverty and
disadvantage. Those most affected are the poorest populations
often living in remote, rural areas, urban slums or in conflict zones.
Over 1 billion people, one sixth of the world’s population, suffer
from one or more neglected tropical diseases [1], two of which are
malaria and sleeping sickness.
Malaria remains an urgent problem in global public health: it is
currently the most widespread and severe tropical disease, causing
more than one million deaths eachyear, with 90% of the victims being
African childrenunder five years [2]. Inhumans, itiscaused byseveral
species of the Plasmodium genus, with Plasmodium falciparum being
the most dangerous species responsible, together with Plasmodium
vivax, for more than 95% of malaria cases in the world [3]. Since 2006,
WHO has recommended an artemisinin-based combination therapy
(ACT), such as artesunateeamodiaquine and artemetherelumefan-
trine, as first-line treatment for uncomplicated malaria [4,5].
However, there are first reports concerning a decreased sensitivity of
the malaria parasite to artemesinin drugs in South East Asia [6].
Human African trypanosomiasis (HAT) or sleeping sickness is
caused by protozoan parasites Trypanosoma brucei gambiense and
Saharan Africa had already reached 50,000, and that 60 million of
people are at risk of infection [8,9]. At present only four drugs are
available to treat HAT: two for the treatment of early haemolym-
phatic-stage (pentamidine and suramin) and two for late menin-
goencephalitic-stage (melarsoprol and eflornithine) of the disease
[10]. Unfortunately, these drugs suffer from severe side-effects
and are expensive to manufacture and administer [11].
Taking in consideration all these reasons there is an obvious and
urgent need to develop new chemotherapies to treat malaria and/
or HAT and in particular to find new targets for drug design.
In this context, it has been demonstrated that falcipain-2 (FP-2),
a papain-family (clan CA, family C1) cysteine protease isolated from
P. falciparum, is among the prime targets for the development of
novel antimalarial agents. FP-2 is involved in the hemoglobin
degradation, that is essential for the growth and maturation of the
parasite [12]. To digest this protein and other cytosolic proteins,
Plasmodia have developed a specialized acidic organelle, termed
the food vacuole, which contains a set of specific proteases,
including FP-2. FP-2 is also responsible for the erythrocyte rupture
by cleaving ankyrin and band protein 4.1, the cytoskeletal elements
vital to the stability of the red cell membrane [13].
On the other hand, rhodesain is a key enzyme of T. brucei rho-
desiense, belonging to the cathepsin L subfamily of the papain-like
* Corresponding author. Tel.: þ39 090 6766466; fax: þ39 090 6766404.
E-mail address: rettari@pharma.unime.it (R. Ettari).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.04.003

R. Ettari et al. / European Journal of Medicinal Chemistry 45 (2010) 3228e3233
3229
(clan CA, family C1) cysteine proteases. Rhodesain is involved in the
degradation of parasite proteins and intracellularly transported
host proteins in both the insect and the mammalial host [14]. This
enzyme revealed to be essential for the development of the parasite
and, in view of these reasons, it is considered a promising target for
the development of novel trypanocidal agents.
Our ongoing research in the field of malaria pertains to the design
and synthesis of peptidomimetics as FP-2 inhibitors [15e19]. For the
initial design, it has been employed a benzo[1,4]diazepin-2-one
scaffold introduced internally to a peptide sequence, which mimics
N-protected L-aspartic acid 4-tert-butyl ester, firstly converted into
the mixed anhydride and then reduced with NaBH4, according to
a previously reported procedure [21].
Compound 4 was oxidized under Swern conditions into the
corresponding aldehyde 5 which was immediately reacted with
tert-butyl mercaptan, in the presence of catalytic amount of TiCl4,
a useful catalyst for thioacetalization. The successive treatment
with TFA caused the deprotection of the ester function and the
subsequent cyclization to the desired thioacylal 6 as diastereoiso-
meric mixture which was easily separated, affording compounds 6a
(2R,3S) and 6b(2S,3S) in a 3:2 ratio. The stereochemistry of thio-
acylals 6a and 6b was assigned, in agreement with literature data
for similar compounds [22], on the basis of the multiplicity of H-2
which resonates as a doublet with a 6.3 Hz coupling constant in the
cis-diastereoisomer 6b and as a broad singlet in the trans one 6a.
The aspartyl aldehyde building block 7 (Scheme 2), synthesized
according to a previous described procedure [21], was employed in
the successive step as 1:1 mixture of the two diastereoisomers.
The synthesis of pseudotripeptides 2a,b and 3a,b (Scheme 2)
was accomplished through a coupling of compounds 6a,b and 7,
after removal of their N-protecting group, with 1,4-benzodiazepin-
2-one-1-acetic acid 8 [15]. Desilylation of the coupling products 9a,
b gave the alcohols 11a,b. After deprotection of compound 10, it
was possible to separate the two diastereoisomers (2S,3S)-12a and
(2R,3S)-12b. The subsequent treatment of the alcohols 11a,b and
12a,b with 4-chloro-2-trifluoromethyl-phenyl isocyanate afforded
carbamates 2a,b and 3a,b in high yields.
the fragment D-Ser-Gly, with the hydroxyl group of the serine func-
tionalized with various aryl isocyanates. In regard to the pharmaco-
phore portion, it has been elected the aspartyl aldehyde building
block able to interact with the thiol group of the cysteine active site by
forming a reversible covalent bond [15]. Among all the synthesized
compounds, themostactiveFP-2inhibitorrevealedtobecompound 1
(Fig.1) which, when testedagainst culturedP. falciparum, provedto be
inactive (IC50 >100 mM). The lack of the antiplasmodial activity might
be explained in terms of the difficulty for this inhibitor to cross the
biological membranes of the parasites and reach the target enzyme.
On these basis, we planned the synthesis of novel peptidomi-
metics in which the aspartyl aldehyde moiety has been function-
alized in such a way to increase the lipophilicity of compound 1 and
its permeability through the parasite cell membrane. This approach
has already successfully been applied to the development of pral-
nacasan, an orally bioavailable caspase-1 inhibitor [20]. The tert-
butylthio and the benzyloxy group have been selected as protecting
groups, leading to the thioacylals 2a,b and the acylals 3a,b (Fig. 1).
Herein, we report the synthesis and the biological evaluation of
compounds 2 and 3 against the FP-2 and rhodesain enzymes, and
against their respective cultured parasites, P. falciparum and
T. brucei brucei. Furthermore, selectivity against the target enzymes
was estimated by testing the new compounds against human
cysteine proteases of the papain-family, i.e. cathepsins B and L.
2.2. Biological activity
Compounds 2a,b and 3a,b were tested in vitro to evaluate their
potency in the inhibition of FP-2 using the pure recombinant active
P. falciparum enzyme expressed in E. coli and Cbz-Phe-Arg-AMC as
fluorogenic substrate [23]. An equivalent volume of DMSO was
used as negative control, and E-64 [24], the irreversible standard
inhibitor of clan CA family C1 cysteine proteases, was used as
2. Results and discussion
positive control. The preliminary screening at 30 mM showed an
inhibition percentage less than 30% for cis-diastereoisomers 2b and
3b, whereas trans diastereoisomers 2a and 3a, which showed more
than 50% inhibition, were screened in details. Compounds 2a and
3a showed to abolish enzyme activity in a reversible manner with
2.1. Chemistry
The synthesis of the aspartyl aldehyde synthon 6 has been
realized starting from the alcohol 4 (Scheme 1), obtained from
a Ki value of 8.2
3a proved to be nearly equipotent to the lead compound 1
M), putting in evidence that a bulkiness increase at
mM and 8.9 mM, respectively (Table 1). Both 2a and
(Ki ¼ 4.0
m
position 2 of the aspartyl aldehyde warhead did not affect the
binding affinity for the target enzyme.
Compounds 2a and 3a were also tested against the P. falciparum
strain FCBR (Table 1). Dose-dependent effects of compounds on
parasite development were quantified using a previously published
assay [25], at variance with the enzymatic assay, where derivatives
2a and 3a turned out to be almost equiactive with lead compound 1,
the ability of both derivatives to inhibit the parasite development
resulted significantly increased. As
compounds 2a and 3a possess an IC50 in the micromolar range
(28.5 and 49.5 M, respectively), the parent compound 1 turned out
to be inactive (IC50 > 100 M). Such a different outcome in the
a matter of fact, while
m
m
functional assay can reasonably be attributed to an increase in
lipophilicity as reported in Table 1.
Compounds 1, 2a and 3a were also tested against rhodesain to
evaluate their potential trypanocidal activity. Again, the presence of
hydrophobic groups seems to be favourable for the interaction with
the target enzyme. As a matter of fact, both compounds 2a and 3a
proved to inhibit this enzyme in the submicromolar range, with a Ki
value of 0.7 and 0.8
showed only a 30% enzyme inhibition at 100
m
M, respectively. On the contrary, compound 1
M.
Fig. 1. Structure of peptidomimetics 1e3.
m

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R. Ettari et al. / European Journal of Medicinal Chemistry 45 (2010) 3228e3233
Scheme 1. (a) (COCl)2, DMSO, TEA, ꢁ65 ꢂC, 2 h; (b) t-BuSH, TiCl4, CHCl3, ꢁ10 ꢂC e room temperature, 1 h; (c) TFA, CH2Cl2, room temperature, 3 h.
The trypanocidal activity of the rhodesain inhibitor 2a was also
4. Experimental protocols
evaluated. This compound exhibited an IC50 value of 14.2 ꢀ 1.0
slightly better to that reported for eflornithine (IC50 ¼ 22.9
[26], the drug currently used as a first-line therapeutic agent.
m
M,
mM)
4.1. Chemistry
All reagents and solvents were obtained from commercial
suppliers and were used without further purification. Melting
points were determined on a Kofler hot stage apparatus and are
uncorrected Elemental analyses were carried out on a C. Erba
Model 1106 (Elemental Analyzer for C, H and N) and the results are
within ꢀ0.4% of the theoretical values. Merck Silica Gel 60 F254
plates were used as analytical TLC; flash column chromatography
was performed on Merck Silica Gel (200e400 mesh) or was con-
ducted using prepacked cartridges on a MP-LC BUCHI system. 1H
3. Conclusion
In conclusion, in view of the obtained results, compounds 2a
and 3a proved to possess an increased antiplasmodial activity with
respect to the parent compound 1. Furthermore, thioacylal 2a can
be considered as a promising trypanocidal agent. Further work is
required to improve the inhibitory properties and to enhance
selectivity against the rhodesain enzyme.
Scheme 2. (a) PdCl2(PPh3)2, n-Bu3SnH, CH2Cl2, room temperature, 30 min; (b) EDCI, HOBt, DMF/CH2Cl2 (1/1), rt, 15 h; (c) TBAF, THF, room temperature, 5 h; (d) 2-CF3-4-Cl-
C6H3NCO, CH2Cl2, room temperature, 12 h.

R. Ettari et al. / European Journal of Medicinal Chemistry 45 (2010) 3228e3233
3231
Table 1
(triphenylphosphine)palladium (II) (2 mol%), and after keeping the
mixture under argon, n-Bu3SnH (200 L, 0.87 mmol) was added
Inhibition of FP-2, rhodesain and antiplasmodial activity of compounds 1e3a and
their cLog P values.b
m
dropwise. In a few minutes the solution became black orange color.
After 30 min the solution containing the deprotected amine was
diluted (1/1) with DMF, and HOBt (44 mg, 0.29 mmol) and EDCI
(66 mg, 0.35 mmol) were added and stirred for 15 h with an
equivalent of acid 8 [15] (125 mg, 0.29 mmol). The mixture was
diluted with CHCl3 and washed with NaHCO3 solution and brine.
The organic solution was dried and concentrated to give a residue.
The obtained residue was purified by flash column chromatography
(CHCl3/EtOAc 1:1) to afford the product 9a (169 mg, 96%).
Compd
Falcipain-2
Ki [ M]
P. falciparumc
IC50 M]
>100
28.5 ꢀ 0.8
49.5 ꢀ 0.1
9.3 ꢀ 0.3
Rhodesain
Ki [ M]
cLog P
m
[m
m
1
2a
3a
E-64
4.0 ꢀ 1.8
8.2 ꢀ 1.5
8.9 ꢀ 1.0
40.3 ꢀ 4.9
30% at 100
0.7 ꢀ 0.02
0.8 ꢀ 0.04
mM
2.98
5.81
4.80
a
All results include standard deviations from two independent measurements,
each performed in duplicate. ni: no inhibition.
b
Osiris Property Explorer, Actelion.
FCBR strain.
[a
]
20e20.8 (c ¼ 1.25, CHCl3). Rf ¼ 0.74 (CHCl3/EtOAc 1:1). 1H NMR
c
D
(300 MHz, CDCl3): 0.13 (s, 3H), 0.14 (s, 3H), 0.91 (s, 9H), 1.32 (s, 9H),
2.58 (dd, 2H, J ¼ 17.9 Hz, J ¼ 8.5 Hz, J ¼ 5.8 Hz, Δ ¼ 40.2 Hz), 3.83 (t,
n
and 13C NMR spectra were recorded in CDCl3 on a Varian Gemini
1H, J ¼ 6.3 Hz), 4.22e4.32 (m, 2H), 4.39 (d, 1H, J ¼ 15.7 Hz),
4.53e4.59 (m, 1H), 4.58 (d, 1H, J ¼ 15.7 Hz), 5.45 (d, 1H, J ¼ 5.2 Hz),
7.10 (d, 1H, J ¼ 7.1 Hz), 7.21e7.68 (m, 9H); 13C NMR (75 MHz, CDCl3):
e 5.24, 18.42, 25.88, 31.24, 34.22, 44.94, 51.98, 52.52, 63.80, 65.14,
86.65, 122.39, 125.02, 128.34, 129.53, 129.57, 130.17, 130.73, 131.89,
138.20, 142.04, 168.81, 169.27, 169.87, 173.68.
300 spectrometer. 1H chemical shifts are expressed in
d (ppm)
relative to TMS as internal standard and coupling constants (J) in
hertz. 13C chemical shifts are referenced to CDCl3 (central peak,
d
¼ 77.0 ppm).
4.1.1. [(2R,3S)-2-tert-Butylthio-5-oxo-tetrahydrofuran-3-yl]-
carbamic acid allyl ester (6a) and [(2S,3S)-2-tert-butylthio-5-oxo-
tetrahydrofuran-3-yl]-carbamic acid allyl ester (6b)
4.1.3. 2-[(3R)-3-(tert-Butyl-dimethyl-silanyloxymethyl)-2-oxo-5-
phenyl-2,3-dihydro-benzo[e][1,4]diazepin-1-yl]-N-[(2S,3S)-2-tert-
butylthio-5-oxo-tetrahydrofuran-3-yl]-acetamide (9b)
To a solution of oxalyl chloride 2 M in CH2Cl2 (1.20 mmol,
0.60 mL) at ꢁ65 ꢂC under N2 atmosphere was added a solution of
DMSO (2.78 mmol, 0.19 mL) in CH2Cl2 (2 mL) dropwise over 20 min.
The alcohol 4 [21] (182 mg, 0.70 mmol) in CH2Cl2 (6 mL) was added
dropwise to the mixture over 20 min. The reaction mixture was
stirred at ꢁ65 ꢂC for 45 min; then TEA (4.57 mmol, 0.63 mL) in
CH2Cl2 (5 mL) was added over 20 min. The reaction mixture was
stirred at ꢁ23 ꢂC for 40 min. The solution was poured into Et2O and
0.5 N KHSO4 solution (1/1200 mL). The organic layer was washed
with 0.5 N KHSO4 solution (2 ꢃ 100 mL), then concentrated to one-
half volume, diluted with Et2O, and washed again with H2O
(100 mL) and saturated NaCl (100 mL). The organic layer was dried
over MgSO4, filtered, and evaporated in vacuo. A solution of crude
aldehyde 5 in CHCl3 was treated with 2-methyl-2-propanethiol
(109 mg, 1.20 mmol) and the resulting mixture was cooled to
ꢁ10 ꢂC, then TiCl4 (12 mg, 0.06 mmol) was introduced under stir-
ring and the reaction temperature allowed to attain room
temperature during next few minutes. After being stirred for an
additional 1 h, water was introduced and CHCl3 layer was sepa-
rated. The aq. phase was extracted with CHCl3 (3 ꢃ 30 mL) and the
combined extracts washed with water, brine and dried (Na2SO4).
After removal of solvent the crude was dissolved in anhydrous
CH2Cl2 and treated with TFA (25% in CH2Cl2). After 3 h the reaction
mixture was diluted with EtOAc, washed with NaHCO3, dried
(Na2SO4) and then evaporated in vacuo. The residue was purified on
flash column chromatography (20% EtOAc in CH2Cl2/hexane 1:1) to
give two diastereoisomer thioacylals (0.48 mmol, 68%, of which
0.29 mmol, 60% of 6a and 0.19 mmol, 40% of 6b). 6a: Rf ¼ 0.68 (20%
EtOAc in CH2Cl2/hexane 1:1). 1H NMR (300 MHz, CDCl3): 1.44 (s,
Compound 9b was obtained from 6b (53 mg, 0.19 mmol)
employing the procedure described for compound 9a. The obtained
residue was purified by flash column chromatography (CHCl3/
20
EtOAc 1:1). Yield: 110 mg, 94%. [
a
]
- 82.9 (c ¼ 2.45, CHCl3).
D
Rf ¼ 0.68 (CHCl3/EtOAc 1:1). 1H NMR (300 MHz, CDCl3): 0.15 (s, 3H),
0.17 (s, 3H), 0.93 (s, 9H), 1.31 (s, 9H), 2.55 (dd, 2H, J ¼ 17.3 Hz,
J ¼ 8.2 Hz, J ¼ 8.8 Hz, Δ ¼ 33.7 Hz), 3.82 (t, 1H, J ¼ 6.3 Hz), 4.14 (d,
n
1H, J ¼ 15.4 Hz), 4.30e4.39 (m, 1H), 4.52e4.61 (m, 1H), 4.76 (d, 1H,
J ¼ 15.4 Hz), 4.86e4.98 (m, 1H), 5.99 (d, 1H, J ¼ 6.6 Hz), 7.19e7.68
(m, 10H); 13C NMR (75 MHz, CDCl3): - 5.16, 17.48, 25.95, 31.33,
33.96, 44.43, 49.04, 52.28, 64.03, 65.20, 86.12, 122.46, 124.95,
128.27, 129.38, 129.62, 130.26, 130.62, 131.67, 138.37, 142.29, 169.08,
169.23, 170.01, 172.80.
4.1.4. N-[(3S)-2-Benzyloxy-5-oxo-tetrahydrofuran-3-yl]-2-[(3R)-3-
(tert-butyl-dimethyl-silanyloxymethyl)-2-oxo-5-phenyl-2,3-
dihydro-benzo[e][1,4]diazepin-1-yl]-acetamide (10)
Compound 10 was obtained from 7 [21] (117 mg, 0.40 mmol),
employing the procedure described for compound 9a. The obtained
residue was purified by flash column chromatography (CHCl3/
EtOAc 1:1). Yield: 176 mg, 70%. Rf ¼ 0.52 (CHCl3/EtOAc 1:1); 1H
NMR (300 MHz, CDCl3): 0.16 (s, 3H), 0.18 (s, 3H), 0.90 (s, 9H),
2.23e3.02 (m, 2H), 3.80 (t, 1H, J ¼ 6.6 Hz), 4.20 (d, 1H, J ¼ 16.3 Hz),
4.15e4.44 (m, 2H), 4.47e4.77 (m, 3H), 4.70e4.78 (m, 1H), 5.51 (d,
0.5H, J ¼ 5.5 Hz), 5.53 (bs, 0.5H), 6.91 (d, 0.5H, J ¼ 6.6 Hz,), 7.00 (bs,
0.5H), 7.12e7.78 (m, 14H).
4.1.5. N-[(2R,3S)-2-tert-Butylthio-5-oxo-tetrahydrofuran-3-yl]-2-
[(3R)-3-hydroxy methyl-2-oxo-5-phenyl-2,3-dihydro-benzo[e][1,4]
diazepin-1-yl]-acetamide (11a)
To a solution of protected alcohol 9a (168 mg, 0.28 mmol) in THF
(15 mL) was added tetrabutylammonium fluoride (1 M in THF,
0.4 mL) and stirred for 5 h at room temperature. The reaction
mixture was diluted with EtOAc and washed with water. The
organic layer was dried and concentrated to give a residue that was
9H), 2.72 (dd, 2H, J ¼ 17.3 Hz, J ¼ 7.7 Hz, J ¼ 7.4 Hz, Δ ¼ 41.6 Hz),
n
4.20e4.25 (m, 1H), 4.59 (d, 2H, J ¼ 4.4 Hz), 5.20e5.39 (m, 3H), 5.71
(bs, 1H), 5.85e5.98 (m, 1H). 6b: Rf ¼ 0.54 (20% EtOAc in CH2Cl2/
hexane 1:1). 1H NMR (300 MHz, CDCl3): 1.41 (s, 9H), 2.78 (dd, 2H,
J ¼ 17.6 Hz, J ¼ 8.0 Hz, J ¼ 3.8 Hz, Δ ¼ 40.8 Hz), 4.62 (d, 2H,
n
J ¼ 5.4 Hz), 4.73e4.85 (m, 1H), 5.24e5.47 (m, 3H), 5.94 (d, 1H,
J ¼ 6.3 Hz), 5.89e6.02 (m, 1H).
purified by flash column chromatography (CH2Cl2/MeOH 95:5) to
20
4.1.2. 2-[(3R)-3-(tert-Butyl-dimethyl-silanyloxymethyl)-2-oxo-5-
phenyl-2,3-dihydro-benzo[e][1,4]diazepin-1-yl]-N-[(2R,3S)-2-tert-
butylthio-5-oxo-tetrahydrofuran-3-yl]-acetamide (9a)
give the alcohol 11a (112 mg, 82%). [
a
]
D
- 41.1 (c ¼ 1.75, CHCl3).
Rf ¼ 0.67 (CH2Cl2/MeOH 9:1). 1H NMR (300 MHz, CDCl3): 1.33 (s,
9H), 2.68 (dd, 2H, J ¼ 17.9 Hz, J ¼ 8.2 Hz, J ¼ 4.7 Hz, Δ ¼ 34.1 Hz),
n
To a solution of 6a (79 mg, 0.29 mmol) in CH2Cl2 anhydrous
3.21 (bs, 1H), 3.84 (t, 1H, J ¼ 5.5 Hz), 4.18e4.27 (m, 1H), 4.24 (d, 1H,
J ¼ 15.7 Hz), 4.28e4.40 (m, 2H), 4.51 (d, 1H, J ¼ 15.7 Hz), 5.59 (d, 1H,
(10 mL), was added
a
catalytic amount of dichlorobis

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R. Ettari et al. / European Journal of Medicinal Chemistry 45 (2010) 3228e3233
J ¼ 4.1 Hz), 7.19e7.62 (m, 10H). 13C NMR (75 MHz, CDCl3): 31.29,
34.53, 45.06, 52.05, 52.71, 62.60, 63.70, 86.93, 122.48, 125.09,
128.37, 129.38, 129.68, 130.23, 130.91, 132.09, 138.13, 142.03, 168.51,
170.21, 171.02, 174.28.
J ¼ 6.6 Hz), 7.10 (bs, 1H), 7.30e7.71 (m, 10H), 8.04e8.15 (m, 2H). 13C
NMR (75 MHz, CDCl3): 31.26, 34.39, 45.10, 52.17, 52.87, 61.93, 65.47,
86.74, 122.53, 124.50, 125.38, 126.21, 128.45, 129.35, 129.68, 130.42,
131.06, 132.27, 132.92, 134.05, 137.90, 141.80, 153.15, 168.47, 169.18,
169.94, 173.84. Anal. Calcd for C34H32ClF3N4O6S: C, 56.94; H, 4.50;
N, 7.81. Found: C, 56.83; H, 4.71; N, 7.52.
4.1.6. N-[(2S,3S)-2-tert-Butylthio-5-oxo-tetrahydrofuran-3-yl]-2-
[(3R)-3-hydroxy methyl-2-oxo-5-phenyl-2,3-dihydro-benzo[e][1,4]
diazepin-1-yl]-acetamide (11b)
Compound 11b was obtained from 9b (110 mg, 0.18 mmol)
employing the procedure described for compound 11a. The
obtained residue was purified by flash column chromatography
4.1.9. (4-Chloro-2-trifluoromethyl-phenyl)-carbamic acid 1-
{[(2S,3S)-2-tert-butylthio-5-oxo-tetrahydrofuran-3-ylcarbamoyl]-
methyl}-(3R)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]
diazepin-3-ylmethyl ester (2b)
(CH2Cl2/MeOH 9:1). Yield: 72 mg, 80%. [
a
]
20 - 93.1 (c ¼ 1.20, CHCl3).
D
Compound 2b was prepared from 11b (72 mg, 0.15 mmol)
employing the procedure described for compound 2a. The crude
was purified by flash column chromatography (CH2Cl2/MeOH
Rf ¼ 0.63 (CH2Cl2/MeOH 9:1). 1H NMR (300 MHz, CDCl3): 1.32 (s,
9H), 2.63 (dd, 2H, J ¼ 17.3 Hz, J ¼ 8.2 Hz, J ¼ 8.5 Hz, Δ ¼ 37.6 Hz),
n
95:5). Yield: 70 mg, 67%. [
a
]
20 - 45.3 (c ¼ 0.25, CHCl3). Mp 96e98 ꢂC.
3.23 (bs, 1H), 3.85 (t, 1H, J ¼ 6.3 Hz), 4.11 (d, 1H, J ¼ 15.1 Hz),
4.20e4.29 (m, 1H), 4.38e4.49 (m, 1H), 4.78 (d, 1H, J ¼ 15.1 Hz),
4.91e4.99 (m, 1H), 5.96 (d, 1H, J ¼ 6.0 Hz), 7.23e7.73 (m, 10H). 13C
NMR (75 MHz, CDCl3): 31.32, 34.09, 44.94, 51.96, 52.44, 62.53,
63.75, 86.66, 122.47, 125.07, 128.29, 129.45, 129.58, 130.25, 130.59,
131.88, 138.33, 142.26, 168.48, 170.05, 170.98, 174.67.
D
Rf ¼ 0.28 (CH2Cl2/MeOH 95:5). 1H NMR (300 MHz, CDCl3): 1.25 (s,
9H), 2.62 (dd, 2H, J ¼ 18.2 Hz, J ¼ 8.2 Hz, J ¼ 8.5 Hz, Δ ¼ 43.5 Hz),
n
4.03 (t, 1H, J ¼ 6.0 Hz), 4.17 (d, 1H, J ¼ 16.2 Hz), 4.59e4.68 (m, 1H),
4.80 (d, 1H, J ¼ 16.2 Hz), 4.82e4.97 (m, 1H), 5.00e5.12 (m, 1H), 5.98
(d, 1H, J ¼ 5.5 Hz), 7.03 (bs, 1H), 7.09 (d, 1H, J ¼ 6.2 Hz), 7.30e7.71
(m, 10H), 8.07e8.19 (m, 2H). 13C NMR (75 MHz, CDCl3): 31.29, 34.28,
45.04, 51.99, 52.78, 62.02, 65.36, 86.70, 122.52, 124.55, 125.32,
126.23,128.33,129.45,129.56,130.24,130.64,131.98,132.25,132.93,
134.10, 137.99, 142.20, 152.97, 168.63, 169.05, 170.09, 174.31. Anal.
Calcd for C34H32ClF3N4O6S: C, 56.94; H, 4.50; N, 7.81. Found: C,
57.23; H, 4.32; N, 8.11.
4.1.7. N-[(2S,3S)-2-Benzyloxy-5-oxo-tetrahydrofuran-3-yl]-2-
[(3R)-3-hydroxymethyl-2-oxo-5-phenyl-2,3-dihydro-benzo[e][1,4]
diazepin-1-yl]-acetamide (12a) and N-[(2R,3S)-2-benzyloxy-5-oxo-
tetrahydrofuran-3-yl]-2-[(3R)-3-hydroxymethyl-2-oxo-5-phenyl-
2,3-dihydro-benzo[e][1,4]diazepin-1-yl]-acetamide (12b)
Compound 12a and 12b were prepared from 10 (176 mg,
0.28 mmol) employing the procedure described for compound 11a.
The obtained residue was purified by flash column chromatography
(CH2Cl2/MeOH 95:5). Yield: 0.22 mmol, 78%, of which 0.12 mmol,
55% of the trans isomer 12a and 0.10 mmol, 45% of the cis isomer
12b.12a: Rf ¼ 0.46 (CH2Cl2/MeOH 95:5); 1H NMR (300 MHz, CDCl3):
4.1.10. (4-Chloro-2-trifluoromethyl-phenyl)-carbamic acid 1-
{[(2S,3S)-2-benzyloxy-5-oxo-tetrahydrofuran-3-ylcarbamoyl]-
methyl}-(3R)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]
diazepin-3-ylmethyl ester (3a)
Compound 3a was prepared from 12a (62 mg, 0.12 mmol)
employing the procedure described for compound 2a. The crude
was purified by flash chromatography (using CH2Cl2/MeOH 95:5).
Yied: 56 mg, 63%. Mp 119e123 ꢂC. Rf ¼ 0.64 (CH2Cl2/MeOH 95:5);
1H NMR (300 MHz, CDCl3): 2.50 (dd, 2H, J ¼ 17.3 Hz, J ¼ 8.5 Hz,
2.56 (dd, 2H, J ¼ 17.3 Hz, J ¼ 8.6 Hz, J ¼ 10.3 Hz, Δ ¼ 51.1 Hz), 2.59
n
(bs, 1H), 3.78 (t, 1H, J ¼ 6.7 Hz), 4.32 (d, 1H, J ¼ 15.6 Hz), 4.17e4.42
(m, 2H), 4.55 (d,1H, J ¼ 11.7 Hz), 4.64 (d,1H, J ¼ 15.6 Hz), 4.73 (d,1H,
J ¼ 11.7 Hz), 4.68e4.81 (m, 1H), 5.49 (d, 1H, J ¼ 5.3 Hz), 6.86 (d, 1H,
J ¼ 7.5 Hz), 7.13e7.80 (m, 14H). 13C NMR (75 MHz, CDCl3): 32.60,
47.89, 52.16, 62.83, 63.80, 70.84, 99.15, 122.23, 125.18, 128.36,
128.40, 128.52, 128.68, 129.47, 129.66, 130.40, 130.95, 132.09,
135.35, 138.05, 141.94, 168.26, 169.89, 171.20, 172.97. 12b: Rf ¼ 0.40
(CH2Cl2/MeOH 95:5); 1H NMR (300 MHz, CDCl3): 2.78 (dd, 2H,
J ¼ 10.4 Hz, Δ ¼ 51.9 Hz), 3.96 (t, 1H, J ¼ 6.3 Hz), 4.39 (d, 1H,
n
J ¼ 15.7 Hz), 4.52 (d, 1H, J ¼ 11.5 Hz), 4.59 (d, 1H, J ¼ 15.7 Hz), 4.68
(d, 1H, J ¼ 11.5 Hz), 4.60e4.78 (m, 1H), 4.87e5.01 (m, 2H), 5.46 (d,
1H, J ¼ 5.2 Hz), 6.75 (d, 1H, J ¼ 8.0 Hz), 6.93 (bs, 1H), 7.26e7.64 (m,
16H), 8.08 (d, 1H, J ¼ 9.1 Hz). 13C NMR (75 MHz, CDCl3): 32.51, 47.92,
52.11, 61.84, 65.56, 71.01, 99.30, 122.42, 123.97, 125.29, 126.14,
126.22, 128.39, 128.44, 128.52, 128.67, 129.39, 129.71, 130.51, 131.04,
132.23, 132.95, 135.36, 137.96, 141.89, 152.93, 168.09, 169.02, 169.78,
172.90. Anal. Calcd for C37H30ClF3N4O7: C, 56.94; H, 4.50; N, 7.81.
Found: C, 56.71; H, 4.73; N, 7.60.
J ¼ 18.2 Hz, J ¼ 8.0 Hz, J ¼ 1.8 Hz, Δ ¼ 78.8 Hz), 3.24 (bs, 1H), 3.93 (t,
n
1H, J ¼ 6.2 Hz), 4.30 (d, 1H, J ¼ 15.7 Hz), 4.24e4.35 (m, 1H),
4.36e4.46 (m, 1H), 4.47e4.55 (m, 1H), 4.60 (d, 1H, J ¼ 15.7 Hz), 4.66
(d, 1H, J ¼ 11.4 Hz), 4.84 (d, 1H, J ¼ 11.4 Hz), 5.52 (bs, 1H), 7.23e7.75
(m, 15H). 13C NMR (75 MHz, CDCl3): 33.33, 52.31, 52.39, 62.62,
63.78, 71.19, 106.03, 122.44, 125.17, 128.14, 128.26, 128.40, 128.54,
129.37, 129.67, 130.29, 130.91, 132.15,135.90, 138.10, 142.03, 168.54,
170.18, 171.12, 174.74.
4.1.11. (4-Chloro-2-trifluoromethyl-phenyl)-carbamic acid 1-
{[(2R,3S)-2-benzyloxy-5-oxo-tetrahydrofuran-3-ylcarbamoyl]-
methyl}-(3R)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]
diazepin-3-ylmethyl ester (3b)
4.1.8. (4-Chloro-2-trifluoromethyl-phenyl)-carbamic acid 1-
{[(2R,3S)-2-tert-butylthio-5-oxo-tetrahydrofuran-3-ylcarbamoyl]-
methyl}-(3R)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]
diazepin-3-ylmethyl ester (2a)
To 0.1 M solution in dry CH2Cl2 of alcohol 11a (112 mg,
0.22 mmol) was added 4-Cl,2-CF3C6H3NCO (70 mL, 0.46 mmol) and
Compound 3b was prepared from 12b (50 mg, 0.10 mmol)
employing the procedure described for compound 2a. The crude
was purified by flash chromatography (CH2Cl2/MeOH 95:5). Yield:
47 mg, 65%. Mp 105e108 ꢂC. Rf ¼ 0.59 (CH2Cl2/MeOH 95:5); 1H
NMR (300 MHz, CDCl3): 2.66 (dd, 2H, J ¼ 18.1 Hz, J ¼ 8.0 Hz,
the mixture was stirred under N2 atmosphere for 12 h. After this
time, the mixture was washed with water and dried. The crude was
purified by flash chromatography (CH2Cl2/MeOH 95:5). Yield:
J ¼ 2.0 Hz, Δ ¼ 70.0 Hz), 4.03 (t, 1H, J ¼ 6.3 Hz), 4.27 (d, 1H,
n
J ¼ 15.7 Hz), 4.34e4.42 (m, 1H), 4.58 (d, 1H, J ¼ 11.3 Hz), 4.59 (d, 1H,
J ¼ 15.7 Hz), 4.76 (d, 1H, J ¼ 11.3 Hz), 4.87e5.04 (m, 2H), 5.48 (bs,
1H), 7.15 (d, 1H, J ¼ 6.3 Hz), 7.19 (bs,1H), 7.24e7.66 (m, 16H), 8.02 (d,
1H, J ¼ 8.8 Hz). 13C NMR (75 MHz, CDCl3): 33.16, 52.45, 52.48, 61.94,
65.30, 71.21, 106.05, 122.56, 124.87, 125.30, 126.14, 126.21, 128.06,
128.22, 128.41, 128.51, 128.65, 129.25, 129.31, 129.71, 130.43, 130.94,
132.29, 132.86, 134.14, 135.95, 138.02, 141.99, 153.25, 168.61, 169.24,
113 mg, 70%. [
a
]
20 - 23.1 (c ¼ 1.30, CHCl3). Mp 115e118 ꢂC. Rf ¼ 0.30
D
(CH2Cl2/MeOH 95:5). 1H NMR (300 MHz, CDCl3): 1.37 (s, 9H), 2.66
(dd, 2H, J ¼ 18.1 Hz, J ¼ 8.2 Hz, J ¼ 4.9 Hz, Δ ¼ 43.5 Hz), 4.09 (t, 1H,
n
J ¼ 6.3 Hz), 4.26e4.33 (m, 1H), 4.46 (d, 1H, J ¼ 15.9 Hz), 4.61 (d, 1H,
J ¼ 15.9 Hz), 4.96 (dd, 1H, J ¼ 11.3 Hz and J ¼ 6.3 Hz), 5.08 (dd, 1H,
J ¼ 11.3 Hz and J ¼ 6.3 Hz), 5.55 (d, 1H, J ¼ 4.7 Hz), 6.70 (d, 1H,

R. Ettari et al. / European Journal of Medicinal Chemistry 45 (2010) 3228e3233
3233
169.95, 174.69. Anal. Calcd for C37H30ClF3N4O7: C, 56.94; H, 4.50; N,
7.81. Found: C, 57.20; H, 4.41; N, 8.05.
compounds but without trypanosomes, respectively. After 24 h,
20 L of Alamar Blue were added to each well and the plates were
m
incubated again for a further 24 h. Absorbance were then measured
at 550 nm with a reference wavelength of 630 nm. IC50 values were
calculated by linear interpolation as described [33]. Experiments
were repeated twice.
4.2. Pharmacology
4.2.1. Enzyme assays
The preliminary screening was performed with 100, 30 and 2
m
M inhibitor concentrations using an equivalent amount of DMSO
as negative control. Product release from substrate hydrolysis (Cbz-
Phe-Arg-AMC, 40 M for FP-2 and 10 M for rhodesain) was
determined continuously over a period of 10 min. Compounds
showing at least 50% inhibition at 30 M were subjected to detailed
assays. These were performed in a 50 mM sodium acetate buffer, pH
5.5 containing 10 mM DTT with Cbz-Phe-Arg-AMC (40 or 10 M) as
substrate [24]. The Km values used to correct Kiapp values were
determined to 21.5 M (FP-2) [27] and 0.9 M (rhodesain) [26].
Inhibitor solutions were prepared from stocks in DMSO. Each assay
was performed twice in 96-well-plates in a total volume of 300 L.
A Varian Cary Eclipse spectrofluorometer Varian, Darmstadt,
Germany with a microplate reader (excitation 365 nm, emission
460 nm) was used. Product formation was monitored continuously
for 10e15 min at room temperature. The apparent Kiapp values were
obtained by a Dixon plot [28] using equation [E]0/[E]a ¼ 1 þ [I]/Kiapp
and correction to zero substrate concentration from Ki ¼ Kiapp/(1þ
[S] Kꢁm1) with [E]0 as enzyme activity in the absence, and [E]a as
residual enzyme activities in the presence of the inhibitor.
Acknowledgments
m
m
We would like to thank the DAAD Vigoni project (2009/10) for
partial support of this work. TS thanks the German Research Society
(DFG, SFB630) for financial support.
m
m
References
m
m
[1] http://www.who.int/neglected_diseases/en.
m
[2] World Malaria Report.http://malaria.who.int/wmr2008 (2008).
[3] E. Ashley, R. McGready, S. Proux, F. Nosten, Travel Med. Infect. Dis. 4 (2006)
159e173.
[4] M. Schlitzer, ChemMedChem 2 (2007) 944e986.
[5] WHO, Global AMDP Database-Afro. WHO, 2007.
[6] http://news.bbc.co.uk/2/hi/asia-pacific/8073118.stm.
[7] R. Brun, O. Balmer, Curr. Opin. Infect. Dis. 19 (2006) 415e420.
[8] http://www.who.int/mediacentre/factsheets/fs259/en.
[9] J.P. Mallari, A. Shelat, A. Kosinski, C.R. Caffrey, M. Connelly, F. Zhu, J.
H. McKerrow, R.K. Guya, Bioorg. Med. Chem. Lett. 18 (2008) 2883e2885.
[10] T.C. O’Brien, Z.B. Mackey, R.D. Fetter, Y. Choe, A.J. O’Donoghue, M. Zhou, C.
S. Craik, C.R. Caffrey, J.H. McKerrow, J. Biol. Chem. 283 (2008) 28934e28943.
[11] A. Lüscher, H.P. de Koning, P. Mäser, Curr. Pharm. Des. 13 (2007) 555e567.
[12] P.S. Sijwalai, P.J. Rosenthal, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 4384.
[13] M. Dua, P. Raphael, P.S. Sijwali, P.J. Rosenthal, M. Hanspal, Mol. Biochem.
Parasitol. 118 (2001) 95e99.
[14] C.R. Caffrey, E. Hansell, K.D. Lucas, L.S. Brinen, A.J. Alvarez Hernandez, J. Cheng,
S.L. Gwaltney II, W.R. Roush, M.Y. Stierhof, D. Bogyo, D. Steverding, J.
H. McKerrow, Mol. Biochem. Parasitol. 11 (2001) 861e873.
[15] N. Micale, A.P. Kozikowski, R. Ettari, S. Grasso, M. Zappalà, J. J-Jeong, A. Kumar,
M. Hanspal, A.H. Chishti, J. Med. Chem. 49 (2006) 3064.
[16] R. Ettari, E. Nizi, M.E. Di Francesco, M.A. Dude, G. Pradel, R. Vicik,
T. Schirmeister, N. Micale, S. Grasso, M. Zappalà, J. Med. Chem. 51 (2008)
988e996.
[17] R. Ettari, E. Nizi, M.E. Di Francesco, N. Micale, S. Grasso, M. Zappalà, R. Vicík,
T. Schirmeister, ChemMedChem 3 (2008) 1030e1033.
[18] R. Ettari, N. Micale, T. Schirmeister, R. Vicík, E. Nizi, M.E. Di Francesco,
S. Grasso, M. Zappalà, J. Med. Chem. 52 (2009) 2157e2160.
[19] R. Ettari, F. Bova, M. Zappalà, S. Grasso, N. Micale, Med. Res. Rev. 30 (2010)
136e167.
[20] S.D. Linton, Curr. Top. Med. Chem. 5 (2005) 1697e1716.
[21] N. Micale, R. Vairagoundar, A.G. Yakovlev, A.P. Kozikowski, J. Med, Chem 47
(2004) 6455e6458.
[22] G. Cainelli, E. Mezzina, M. Panunzio, Tetrahedron Lett. 31 (1990) 3481e3484.
[23] P.S. Sijwali, L.S. Brinen, P.J. Rosenthal, Protein Expr. Purif. 22 (2001) 128e134.
[24] K.C. Pandey, S.X. Wang, P.S. Sijwali, A.L. Lau, J.H. McKerrow, P.J. Rosenthal,
Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 9138e9143.
[25] A. Evers, S. Heppner, M. Leippe, C. Gelhaus, Biol. Chem. 389 (2008)
1523e1525.
4.2.2. Drug screening on P. falciparum cultures
The compounds were screened in quadruplicates against the
human malaria pathogen P. falciparum at concentrations between
100 and 0.0488 mM. P. falciparum (strain FCBR) was maintained in
continuous culture basically according to Trager and Jensen [29].
Parasites were cultured in human red blood cells (RBC) (blood
group ARh þ) in RPMI 1640 medium supplemented with 25 mM
HEPES (Molecular Probes, Invitrogen), 20 mM sodium bicarbonate,
and 0.5% AlbuMAX I (Molecular Probes, Invitrogen) at 2.5% (v/v)
hematocrit. Cultures were maintained at 37 ꢂC with a gaseous
phase of 94% N2, 1% O2, and 5% CO2. Synchronized ring stages of P.
falciparum strain FCBR were plated in 96-well-plates at a para-
sitemia of 1%, in the presence of the compounds (dissolved in
DMSO and diluted 10-fold in 50% ethanol before added to the cells).
Incubation of parasites with an equal concentration of 50% ethanol
alone was used as a negative control. To kill the plasmodia
completely for the positive control, the parasites were incubated in
ꢀ
ꢀ
presence of 2 mM chloroquine. The viability of the parasites was
screened subsequently using a previously published assay [25].
[26] R. Vicik, V. Hoerr, M. Glaser, M. Schultheis, E. Hansell, J.H. McKerrow,
U. Holzgrabe, C.R. Caffrey, A. Ponte-Sucre, H. Moll, A. Stich, T. Schirmeister,
Bioorg. Med. Chem. Lett. 16 (2006) 2753e2757.
4.2.3. Drug screening on T. brucei brucei cultures
Trypomastigote forms of T. b. brucei laboratory strain TC 221
were cultured in Baltz medium according to standard conditions
[30]. Trypanocidal activity was determined using the Alamar Blue
assay [31,32] in a 96-well plate format and a MR 700 Microplate
ELISA Reader. Trypanosomes were added to culture medium con-
taining various concentrations of test compound and 1% solvent to
give a cell concentration of 104 cells mLꢁ1 in a final volume of
[27] C. Gelhaus, R. Vicik, R. Hilgenfeld, C.L. Schmidt, M. Leippe, T. Schirmeister, Biol.
Chem. 385 (2004) 435e438.
[28] P.J.F. Henderson, Biochem. J. 127 (1972) 321e333.
[29] W. Trager, J.B. Jensen, Science 193 (1976) 673e675.
[30] T. Baltz, D. Baltz, C. Giroud, J. Crocket, EMBO J. 4 (1985) 1273e1277.
[31] B. Räz, M. Iten, Y. Grether-Bühler, R. Kaminsky, R. Brun, Acta Trop. 68 (1997)
139e147.
[32] K. Merschjohann, F. Sporer, D. Steverding, M. Wink, Planta Med. 67 (2001)
623e627.
200
mL. Positive and negative controls comprised wells containing
medium, 1% solvent and trypanosomes, and wells with test
[33] W. Huber, J.C. Koella, Acta Trop. 55 (1993) 257e261.