Synthesis and Molecular Modeling Studies of Derivatives of a Highly Potent Peptidomimetic Vinyl Ester as Falcipain-2 Inhibitors

Article abstract of DOI:10.1002/cmdc.201200274

Herein we report the synthesis of a set of constrained peptidomimetics endowed with an electrophilic vinyl ester warhead and structurally related to a previously identified lead compound, a potent and irreversible inhibitor of falcipain-2 (FP-2). FP-2 is the main hemoglobinase of the malaria parasite P.falciparum. The new compounds were evaluated for their inhibition against FP-2, and the results were rationalized on the basis of docking experiments. These studies underscore the pivotal role of both the ester function at the P1' site and the trifluoromethyl group of the P3 side chain in determining the correct orientation of the Michael acceptor warhead in the catalytic site, and as a consequence, the potency of the inhibitors as well as their reversible or irreversible mode of inhibition.

Full text of DOI:10.1002/cmdc.201200274

MED
DOI: 10.1002/cmdc.201200274
Synthesis and Molecular Modeling Studies of Derivatives
of a Highly Potent Peptidomimetic Vinyl Ester as Falcipain-
2 Inhibitors
Roberta Ettari,*[a] Nicola Micale,[b] Giovanni Grazioso,[a] Floriana Bova,[b] Tanja Schirmeister,[c]
Silvana Grasso,[b] and Maria Zappalꢀ[b]
Herein we report the synthesis of a set of constrained peptido-
mimetics endowed with an electrophilic vinyl ester warhead
and structurally related to a previously identified lead com-
pound, a potent and irreversible inhibitor of falcipain-2 (FP-2).
FP-2 is the main hemoglobinase of the malaria parasite P. falci-
parum. The new compounds were evaluated for their inhibi-
tion against FP-2, and the results were rationalized on the
basis of docking experiments. These studies underscore the
pivotal role of both the ester function at the P1’ site and the
trifluoromethyl group of the P3 side chain in determining the
correct orientation of the Michael acceptor warhead in the cat-
alytic site, and as a consequence, the potency of the inhibitors
as well as their reversible or irreversible mode of inhibition.
Introduction
Malaria is currently endemic in 106 countries, with an estimat-
ed 216 million clinical cases and nearly 655000 deaths in
2010.[1] Five parasite species of the Plasmodium genus are in-
fective for humans, with the most severe form of malaria
caused by P. falciparum.[2]
into a dipeptide framework, mimicking a specific sequence re-
quired for interaction with the target enzyme. Vinyl sulfones,[11]
phosphonates,[12] amides,[13] esters,[13] nitriles,[13] and ketones[14]
have been selected as P1-site electrophilic warheads that act
as Michael acceptors to covalently trap the active site thiol
function of the target enzyme. Among all synthesized peptido-
mimetics, vinyl ester 1 (Figure 1)[13] turned out to be the most
potent (k2 =3570000mÀ1 minÀ1) and selective FP-2 inhibitor. To
investigate the structure–activity relationship of the selected
lead compound, the 4-chloro-2-trifluoromethylphenyl ring
linked by a carbamoyl moiety to the side chain of the P3 site
serine residue was replaced with other aromatic rings or with
For the treatment of uncomplicated malaria from 2001, the
World Health Organization (WHO) recommended artemisinin-
based combination therapies (ACTs),[3] with the dihydroartemi-
sinin–piperaquine association being the most effective.[4] In
contrast, the treatment of severe malaria is usually based on
the administration of parenteral antimalarial drugs. The first-
choice treatment is intravenous artesunate; quinine is an ac-
ceptable alternative if artesunate is not available. Following ini-
tial parenteral treatment, it is necessary to continue and com-
plete the treatment with a full course of an effective ACT.[4] Un-
fortunately, evidence of resistance to artemisinin has been re-
ported recently.[5] For this reason, new antimalarial drugs that
target metabolic pathways of the parasite and that are able to
bypass resistance mechanisms enacted by the parasite are
needed.[6]
In this context, a new important target for antimalarial drug
design is falcipain-2 (FP-2), a hemoglobinase of the P. falcipa-
rum food vacuole. FP-2, in concert with other Plasmodium pro-
teases, is involved in hemoglobin degradation[7] in order to
ensure the availability of free amino acids for parasite survival.
Additionally, FP-2 orchestrates selective proteolytic events
during the release of parasites from human red blood cells
through its ability to cleave cytoskeletal proteins (i.e., ankyrin
and the band-4.1 protein) during the late trophozoite and
schizont stages.[8] For these reasons, FP-2 is considered a prom-
ising target for the discovery of novel antimalarial drugs.[9]
In this regard, our research group has been working for the
past few years on the development of novel peptidomimetics
containing a 1,4-benzodiazepine (BDZ) scaffold[10] introduced
Figure 1. Structures of vinyl esters 1–5.
[a] Dr. R. Ettari, Dr. G. Grazioso
Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”
Universitꢀ di Milano, Via Mangiagalli 25, 20133 Milano (Italy)
E-mail: roberta.ettari@unimi.it
[b] Dr. N. Micale, Dr. F. Bova, Prof. S. Grasso, Prof. M. Zappalꢀ
Dipartimento Farmaco Chimico
University of Messina, Viale Annunziata, 98168 Messina (Italy)
[c] Prof. Dr. T. Schirmeister
Institute of Pharmacy and Biochemistry
University of Mainz, Staudinger Weg 5, 55099 Mainz (Germany)
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(cyclo)alkyl groups in order to investigate the ability of the S3
pocket to accommodate groups of various size (Figure 1). Re-
placement of the above-mentioned group either with aromatic
rings (4-methoxyphenyl 2 or naphthyl 3) or (cyclo)alkyl groups
(n-butyl 4 or c-hexyl 5) led to reversible inhibition of the target
enzyme and a decrease in affinity.[15]
Table 1. Inhibition of FP-2 by compounds 1–16 and 25.
Compd
R
R’
KI [mm][a]
1[b,c]
2
3
4
Me
Me
Me
Me
Me
Et
iBu
Bn
Et
iBu
Et
iBu
Et
4-Cl-2-CF3C6H3
4-MeOC6H4
1-naphthyl
n-butyl
0.017Æ0.004
15.1Æ1.4
3.48Æ0.21
>100
Herein we report the synthesis and biological evaluation of
novel vinyl esters 6–16 obtained by replacing the methyl ester
group of parent inhibitors 1–5 with an ethyl, isobutyl, or
benzyl group to evaluate size and characteristics of the S1’ lip-
ophilic pocket of the target. Moreover, we carried out docking
studies to explain the varying degrees of inhibitory activity of
compounds 6–16 toward FP-2 as well as their different charac-
teristics in binding modes.
5
c-hexyl
89.0Æ1.6
3.7Æ1.3
6[b,d]
7[b,e]
8[b,f]
9
4-Cl-2-CF3C6H3
4-Cl-2-CF3C6H3
4-Cl-2-CF3C6H3
4-MeOC6H4
4-MeOC6H4
1-naphthyl
1-naphthyl
n-butyl
n-butyl
c-hexyl
c-hexyl
4-Cl-2-CF3C6H3
1.57Æ0.02
0.44Æ0.08
5.4Æ0.7
10
11
12
13
14
15
16
25
E-64
3.5Æ0.3
0.92Æ0.05
2.06Æ0.04
>100
iBu
Et
iBu
H
>100
>100
>100
>100
Results and Discussion
0.29Æ0.09
Synthesis
[a] Values represent the mean ÆSD of two independent measurements,
each performed in duplicate. [b] Time-dependent inhibition. [c] kinac
=
The designed inhibitors 6–16 were synthesized by starting
from P3-carbamate derivatives 17–21, in turn obtained as pre-
viously described.[13] The key step in the synthesis of vinyl
esters 6–16 (Scheme 1) is the olefin cross-metathesis (CM) re-
action, a rapid and efficient method selected to functionalize
the terminal olefin of 17–21. These intermediates were allowed
to react with the CM partners 22–24 by using the Hoveyda–
Grubbs second-generation catalyst to give 6–16 as E isomers.
CM reactions were performed under microwave irradiation to
promote the pharmacophore introduction over shorter times
(from 24 to 1 h) and in excellent yields, due to the excellent re-
activity of acrylates 22–24 in CM reactions.
0.06Æ0.01 minÀ1
,
k2 =3570000Æ400000mÀ1 minÀ1
.
[d] kinac =0.02Æ
0.01 minÀ1, k2 =5600Æ2200mÀ1 minÀ1. [e] kinac =0.008Æ0.001 minÀ1, k2 =
5100Æ100mÀ1 minÀ1
.
[f] kinac =0.010Æ0.001 minÀ1
,
k2 =25300Æ
3200mÀ1 minÀ1
.
was used as negative control, and the irreversible standard in-
hibitor of clan CA family C1 cysteine proteases (papain family),
namely E-64,[18] was used as positive control. For compounds
that passed the initial screen, continuous assays (progress
curve method)[19] were performed to determine the first-order
rate constants of inhibition kinac (minÀ1), dissociation constants
KI (mm), and second-order rate constants of inhibition k2
(MÀ1 minÀ1), with k2 =kinac/KI. For time-independent inhibition,
KI was determined by using a Dixon plot.[20] Among the new
synthesized compounds, only 13–16 did not pass the prelimi-
nary screen, in agreement with the inactivity of the parent
compounds 4 and 5.
As shown by data listed in Table 1, replacement of the
methyl group at the P1’ site of the lead compound 1 by ethyl
(i.e. 6), isobutyl (i.e. 7), or benzyl groups (i.e. 8) led to com-
pounds that maintain the irreversible inhibition mechanism
with a drastic decrease in both affinity and inhibitory potency
(e.g., KI =17 nm and k2 =3570000mÀ1 minÀ1 for 1 versus KI =
3.7 mm and k2 =5600mÀ1 minÀ1 for 6). However, compound 6,
with an ethyl group, showed the lowest enzyme affinity with
respect to analogues 7 and 8, bearing bulkier groups at P1’
site. In contrast, the presence of a bulkier substituent in the re-
versible inhibitors 9–12 slightly increased the enzyme affinity
relative to the parent methyl derivatives 2 and 3 (e.g., KI =
5.4 mm for 9 versus KI =15.1 mm for 2).
Scheme 1. Reagents and conditions: a) Hoveyda–Grubbs catalyst, CH2Cl2,
1008C, MW, 1 h.
Irreversible inhibitors 6–8 were also tested against the P. fal-
ciparum strain FCBR. Dose-dependent effects of compounds
on parasite development were quantified by using a previously
reported assay.[21] At variance with results of the enzymatic
assay, in which derivatives 6–8 were found to be less potent
than lead compound 1, the abilities of these derivatives to in-
hibit parasite development were similar to that of 1. Indeed,
Biological activity and molecular modeling studies
Compounds 6–16 (Table 1) were tested for their inhibitory ac-
tivity against recombinant FP-2[16] using Cbz-Phe-Arg-AMC as
fluorogenic substrate.[17] First, a preliminary screen with inhibi-
tors at 100 mm was performed. An equivalent volume of DMSO
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Peptidomimetic Vinyl Ester Derivatives as FP-2 Inhibitors
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compounds 6–8 possess IC50 values in the micromolar range
(19.1Æ3.7, 12.5Æ1.1, and 9.4Æ1.0 mm, respectively), similar to
that of parent compound 1 (IC50: 12.0Æ2.1 mm).[13] Such a differ-
ent outcome in the functional assay can be reasonably attrib-
uted to an increase in lipophilicity in compounds 6–8. The hy-
pothesis of the free carboxylic acid as the active form of these
esters was discarded by testing 25, readily obtained by basic
hydrolysis of 1, against FP-2. Compound 25 is inactive as an
FP-2 inhibitor (Table 1), and this result is consistent with the
decreased reactivity of acrylic acids versus acrylic esters as Mi-
chael acceptors.[22]
Compounds 6–8 could assume an alternate binding mode
through variation of the torsion angle between the vinyl and
the adjacent amide groups. In fact, gauche and anti geometries
were observed in the structures of compounds 1 and 6–8, re-
spectively. In comparison with 1, this diversity led to an in-
crease of ~2 ꢂ in the distance between Cb in 6–8 and the
sulfur atom of Cys42, which is responsible for Michael adduct
formation. As a consequence, we can assume that the homolo-
gation of 1 to give esters 6–8 had a detrimental effect on the
biological activity of 6–8 due to both the increased distance
and the suboptimal orientation of the vinyl ester moiety into
the catalytic site of the enzyme. Furthermore, the higher bind-
ing affinity observed on passing from 6 to 8 can be attributed
to the improved hydrophobic interactions between 8 and the
FP-2 binding cleft defined by residues Asn173, Val152, and
Trp206.
To explain the structure–activity relationships of the com-
pounds under evaluation, we carried out docking calculations
on a computational model of FP-2 built on the basis of its crys-
tal structure with the covalent inhibitor E-64. In this investiga-
tion, the low k2 values shown by compounds 6–8 (Table 1,
footnote) clearly indicate that the formation of covalent ad-
ducts is disfavored. Thus, we simulated the formation of com-
plexes between the inhibitors and FP-2, the first step of the
ligand–enzyme recognition process. With this goal, we applied
the recently developed PLP scoring function,[23] implemented
in the GOLD 5.1 algorithm, which should allow us to acquire
the optimal binding modes of compounds 1 and 6–8.
After a calculation in which 1000 poses for compounds
1 and 6–8 were computed, a common binding mode for the
BDZ moieties was found. Conversely, the vinyl ester warheads,
responsible for the kinetic profile of the molecules, adopted
a different binding mode. Although the carbonyl group of the
electrophilic moiety of 1 and 6–8 forms a hydrogen bond with
the side chain of Trp206, the P1’ substituent shows a different
orientation. In detail, we found that the methyl ester group of
1 interacts with a cavity of limited dimension delimited by
Gln36, Ser41, and Ser205, whereas the homologated chains of
compounds 6–8 reside on the surface of residues Asn173,
Val152, and Trp206 (Figure 2).
The same docking protocol was then applied in order to ex-
plain the inactivity of compound 25. As shown in Figure 2, the
orientation of the electrophilic warhead of 25 is similar to that
of 1, even if the carbonyl group forms a hydrogen bond with
the backbone of Ser41 and not with the side chain of Trp206.
The inactivity of compound 25 as an FP-2 inhibitor may be ex-
plained by the fact that acrylic acids are much less reactive as
Michael acceptors than the corresponding acrylate esters.[22a]
Acrylic acids are negatively charged at physiological pH, and
this unequivocally leads to inversion of the polarity of Cb,
which must be positively charged to react as a Michael accept-
or.
An observation of the results listed in Table 1 shows that
whereas 6–8 possess residual irreversible activity on the
enzyme, compound 2 is nearly ineffective compared with 1.
Indeed, replacement of chlorine with a methoxy group and re-
moval of the trifluoromethyl group from the distal aryl moiety
led to a derivative endowed with reversible activity and low af-
finity for FP-2. Docking calculations with 2 indicated that the
methoxy group is located in the same position previously
found for the chloro group of 1, while the common molecular
portion can engage with the catalytic site through the same
hydrogen bond interactions found earlier for reference com-
pound 1. Thus, the stability of the FP-2–1 and FP-2–2 com-
plexes was evaluated by molecular dynamics (MD) simulations
in which the RMSD values of the ligands versus simulation
time were monitored (Figure 3). The plot shows that the RMSD
value of compound 1 is moderately stable, whereas that of 2
increased gradually during the simulation time. Further analysis
of the trajectories confirm that the distal aryl moiety of 2
changed its binding mode, leading to an incorrect orientation
of the Michael warhead in the catalytic site, and this precluded
covalent inhibition of the enzyme. These results are in line
with the poor biological activity experimentally observed for
compounds 2–5 and clearly indicate that the trifluoromethyl
group of 1 is pivotal for the ligand–enzyme complementarities
and for correct orientation of the vinyl ester warhead on the
surface of the catalytic residues.
Figure 2. Surface representation of FP-2 together with an enlargement of
the catalytic site. The predicted binding modes of compounds 1, 6–8, and
25 are represented. Carbon atoms are colored in green, yellow, magenta,
cyan and blue, respectively.
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ruthenium] (10.5 mg, 0.0170 mmol). The resulting mixture was
heated under microwave irradiation at 1008C for 1 h. The solvent
was then removed under reduced pressure, and the residue was
purified by flash chromatography, eluting with light petroleum/
EtOAc 5:5 to afford the title compound 6 (56 mg, 99%). 1H NMR
(300 MHz, CDCl3): d=1.27 (t, J=7.1 Hz, 3H), 4.04–4.12 (m, 3H),
4.15 (q, J=7.1 Hz, 2H), 4.33 (d, J=15.0 Hz, 1H), 4.69 (d, J=15.0 Hz,
1H), 4.95–5.04 (m, 1H), 5.08–5.14 (m, 1H), 5.84 (d, J=15.6 Hz, 1H)
6.60 (bs, 1H), 6.83 (bt, 1H), 7.09 (bs, 1H), 7.30–7.80 (m, 11H),
8.09 ppm (d, J=9.1 Hz, 1H); 13C NMR (75 MHz, CDCl3): d=14.20,
40.90, 57.32, 61.43, 62.29, 65.16, 116.43, 120.27, 122.72, 123.45,
124.25, 124.57, 127.70, 128.15, 128.68, 129.43, 129.55, 129.76,
131.60, 133.55, 136.22, 140.18, 140.21, 142.90, 157.16, 166.58,
167.14, 169.15, 170.10 ppm; Anal. calcd for C32H28ClF3N4O6: C 58.50,
H 4.30, N 8.53, found: C 58.10, H 4.48, N 8.32.
(R,E)-Isobutyl 4-(2-(3-((4-chloro-2-(trifluoromethyl)phenylcarba-
moyloxy)methyl)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e]-
[1,4]diazepin-1-yl)acetamido)but-2-enoate (7): (R)-(1-(2-(allylami-
no)-2-oxoethyl)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e]-
Figure 3. History of RMSD values for compounds 1 and 2 in the MD simula-
tion (5 ns) starting from the docked conformations.
[1,4]diazepin-3-yl)methyl 4-chloro-2-(trifluoromethyl)phenylcarba-
mate 17 (50 mg, 0.085 mmol) was reacted with isobutyl acrylate
23 (109 mg, 0.85 mmol) according to the same procedure de-
Conclusions
1
scribed for 6 to give the title compound 7 (58 mg, 99%). H NMR
(300 MHz, CDCl3): d=0.95 (s, 3H), 0.97 (s, 3H), 1.92–1.99 (m, 1H),
3.90 (d, J=6.9 Hz, 2H), 4.04–4.17 (m, 3H), 4.34 (d, J=15.4 Hz, 1H),
4.69 (d, J=15.4 Hz, 1H), 4.74–5.01 (m, 1H), 5.04–5.13 (m, 1H), 5.85
(d, J=15.6 Hz, 1H), 6.63 (bs, 1H), 6.82 (bt, 1H), 7.10 (bs, 1H), 7.30–
7.79 (m, 11H), 8.08 ppm (d, J=8.8 Hz, 1H); 13C NMR (75 MHz,
CDCl3): d=19.30, 27.95, 40.49, 52.82, 62.19, 65.70, 70.92, 122.22,
122.92, 124.76, 125.51, 126.42, 128.65, 129.99, 130.74, 131.18,
132.61, 133.20, 138.36, 142.35, 143.32, 157.18, 166.56, 167.12,
169.12, 170.11 ppm; Anal. calcd for C34H32ClF3N4O6: C 59.61, H 4.71,
N 8.18, found: C 59.31, H 4.52, N 8.43.
With this work we clearly defined, through docking studies,
the structural requirements for efficacious interaction with FP-2
of a class of constrained peptidomimetic inhibitors endowed
with a C-terminal electrophilic warhead. In particular, the ho-
mologation of the ester moiety at the P1’ site of our lead com-
pound 1 turned out to be detrimental owing to an interaction
of the bulkier ester moieties with a different cavity of the cata-
lytic site of the target enzyme. Moreover, these studies indicate
that the trifluoromethyl group of the P3 side chain of 1 is fun-
damental in determining the correct orientation of the a,b-un-
saturated electrophilic warhead, and consequently, the binding
mode of this type of inhibitor.
(R,E)-Benzyl
4-(2-(3-((4-chloro-2-(trifluoromethyl)phenylcarba-
moyloxy)methyl)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e]-
[1,4]diazepin-1-yl)acetamido)but-2-enoate (8): (R)-(1-(2-(allylami-
no)-2-oxoethyl)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e]-
[1,4]diazepin-3-yl)methyl 4-chloro-2-(trifluoromethyl)phenylcarba-
mate 17 (50 mg, 0.085 mmol) was reacted with benzyl acrylate 24
(138 mg, 0.85 mmol) according to the same procedure described
for 6 to give the title compound 8 (61 mg, 99%). 1H NMR
(300 MHz, CDCl3): d=4.04–4.10 (m, 3H), 4.34 (d, J=15.1 Hz, 1H),
4.68 (d, J=15.1 Hz, 1H), 4.94–5.04 (m, 1H), 5.06–5.12 (m, 1H), 5.17
(s, 2H), 5.88 (d, J=15.6 Hz, 1H) 6.60 (bs, 1H), 6.86 (bt, 1H), 7.08
(bs, 1H), 7.27–7.78 (m, 16H), 8.07 ppm (d, J=8.8 Hz, 1H); 13C NMR
(75 MHz, CDCl3): d=40.49, 52.81, 62.20, 65.70, 66.61, 121.87,
122.97, 124.79, 125.57, 126.52, 128.53, 128.68, 128.82, 129.45,
129.98, 130.72, 131.19, 132.64, 133.20, 134.70, 136.07, 138.35,
142.32, 144.05, 153.40, 167.37, 169.50, 169.64, 170.25 ppm; Anal.
calcd for C37H30ClF3N4O6: C 61.80, H 4.20, N 7.79, found: C 61.65, H
4.32, N 7.56.
Experimental Section
All reagents and solvents were obtained from commercial suppliers
and were used without further purification. Reactions under micro-
wave irradiation were performed on a Biotage Emrys Optimizer ap-
paratus. Elemental analyses were carried out on a Carlo Erba
Model 1106 (elemental analyzer for C, H, and N), and the results
are within Æ0.4% of theoretical values. Merck silica gel 60 F254
plates were used for analytical TLC, flash column chromatography
was performed on Merck silica gel (200–400 mesh) or was conduct-
ed with pre-packed cartridges on a Bꢃchi MPLC system. 1H and
13C NMR spectra were recorded in CDCl3 on a Varian Gemini 300
1
spectrometer at 300 K and 300 and 75 MHz, respectively. H NMR
chemical shifts (d) are expressed in ppm relative to TMS as internal
standard, and coupling constants (J) in Hz. 13C NMR chemical shifts
are referenced to CDCl3 (central peak, d=77.0 ppm).
(R,E)-Ethyl
4-(2-(3-((4-methoxyphenylcarbamoyloxy)methyl)-2-
oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)acetami-
do)but-2-enoate (9): (R)-(1-(2-(allylamino)-2-oxoethyl)-2-oxo-5-
phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)methyl naphtha-
len-1-ylcarbamate 18 (50 mg, 0.097 mmol) was reacted with ethyl
acrylate 22 (98 mg, 0.97 mmol) according to the same procedure
described for 6 to give the title compound 9 (44 mg, 77%).
1H NMR (300 MHz, CDCl3): d=0.82–0.88 (m, 3H), 3.76 (s, 3H), 4.01
(m, 1H), 4.10–4.22 (m, 5H), 4.76 (d, J=15.6 Hz, 1H), 4.92–5.08 (m,
2H), 5.92 (d, J=15.6 Hz, 1H), 6.81 (d, J=9.1 Hz, 2H), 6.88–6.95 (m,
2H), 7.26–7.81 ppm (m, 11H); 13C NMR (75 MHz, CDCl3): d=14.10,
(R,E)-Ethyl 4-(2-(3-((4-chloro-2-(trifluoromethyl)phenylcarbamoyl-
oxy)methyl)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e]-
[1,4]diazepin-1-yl)acetamido)but-2-enoate (6): To a solution of
(R)-(1-(2-(allylamino)-2-oxoethyl)-2-oxo-5-phenyl-2,3-dihydro-1H-
benzo[e][1,4]diazepin-3-yl)methyl 4-chloro-2-(trifluoromethyl)phe-
nylcarbamate 17 (50 mg, 0.085 mmol) in dry CH2Cl2 (5 mL) was
added ethyl acrylate (22) (85 mg, 0.85 mmol) followed by Hovey-
da–Grubbs second-generation catalyst [(1,3-bis-(2,4,6-trimethylphe-
nyl)-2-imidazolidinylidene)dichloro-(o-isopropoxyphenylmethylene)
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40.40, 53.56, 55.76, 61.45, 62.75, 64.49, 114.41, 114.53, 122.84,
123.11, 125.23, 128.20, 128.53, 129.61, 129.98, 130.56, 130.95,
132.35, 138.51, 142.53, 142.47, 144.04, 154.25, 166.57, 169.17,
169.82, 170.2 ppm; Anal. calcd for C32H32N4O7: C 65.74, H 5.52, N
9.58, found: C 65.98, H 5.41, N 9.89.
give the title compound 13 (40 mg, 69%). 1H NMR (300 MHz,
CDCl3): d=1.03 (t, J=7.1 Hz, 3H), 1.37–1.59 (m, 4H), 3.27 (q, J=
6.6 Hz, 2H), 4.09 (m, 1H) 4.22–4.37 (m, 5H), 4.86 (d, J=15.1 Hz,
1H), 4.96–5.10 (m, 2H), 5.42 (bt, 1H), 5.97 (d, J=15.9 Hz, 1H), 6.93–
6.98 (m, 2H), 7.37–7.76 (m, 8H), 7.90 ppm (d, J=8.5 Hz, 1H);
13C NMR (75 MHz, CDCl3): d=10.68, 15.06, 19.47, 26.36, 40.41,
47.25, 53.18, 60.84, 62.78, 64.21, 111.53, 125.45, 128.62, 129.40,
130.00, 130.27, 130.69, 131.06, 132.53, 138.46, 142.56, 143.56,
149.49, 160.81, 170.13, 171.14, 172.14 ppm; Anal. calcd for
C29H34N4O6: C 65.15, H 6.41, N 10.48, found: C 64.90, H 6.58, N
10.25.
(R,E)-Isobutyl 4-(2-(3-((4-methoxyphenylcarbamoyloxy)methyl)-2-
oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)acetami-
do)but-2-enoate (10): (R)-(1-(2-(allylamino)-2-oxoethyl)-2-oxo-5-
phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)methyl naphtha-
len-1-ylcarbamate 18 (50 mg, 0.097 mmol) was reacted with isobu-
tyl acrylate 23 (124 mg, 0.97 mmol) according to the same proce-
dure described for 6 to give the title compound 10 (40 mg, 67%).
1H NMR (300 MHz, CDCl3): d=0.86 (d, J=6.6 Hz, 3H), 0.93 (d, J=
6.6 Hz, 3H), 2.26 (m, 1H), 3.76 (s, 3H), 3.91 (d, J=6.6 Hz, 2H), 4.01
(m, 1H), 4.11–4.23 (m, 3H), 4.77 (d, J=15.2 Hz, 1H), 4.95–5.04 (m,
2H), 5.88 (d, J=15.9 Hz, 1H), 6.81 (d, J=8.5 Hz, 2H), 6.86–6.99 (m,
3H), 7.26–7.62 (m, 10H), 7.84 ppm (d, J=8.8 Hz, 1H); 13C NMR
(75 MHz, CDCl3): d=19.73, 19.89, 47.59, 53.56, 55.76, 62.71, 64.49,
70.11, 114.34, 114.53, 122.84, 123.67, 125.23, 128.60, 129.26, 129.98,
130.46, 130.62, 131.57, 132.27, 138.41, 142.53, 142.47, 144.04,
158.13, 166.57, 169.17, 169.82, 170.2 ppm; Anal. calcd for
C34H36N4O7: C 66.65, H 5.92, N 9.14, found: C 66.56, H 5.61, N 9.35.
(R,E)-Isobutyl
phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)acetamido)-
but-2-enoate (14): (R)-(1-(2-(allylamino)-2-oxoethyl)-2-oxo-5-
4-(2-(3-((butylcarbamoyloxy)methyl)-2-oxo-5-
phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)methyl butylcar-
bamate 20 (50 mg, 0.108 mmol) was reacted with isobutyl acrylate
23 (138 mg, 1.08 mmol) according to the same procedure de-
scribed for 6 to give the title compound 14 (38 mg, 62.5%).
1H NMR (300 MHz, CDCl3): d=0.85–1.34 (m, 13H), 1.87 (m, 1H),
3.13 (q, J=6.8 Hz, 2H), 3.90 (d, J=6.6 Hz, 2H), 3.94 (m, 1H) 4.14–
4.23 (m, 2H), 4.21 (d, J=14.8 Hz, 1H), 4.73 (d, J=14.8 Hz, 1H),
4.82–4.97 (m, 2H), 5.27 (bt, 1H), 5.84 (d, J=15.9 Hz, 1H), 6.79–6.88
(m, 2H), 7.20–7.63 (m, 8H), 7.76 ppm (d, J=8.2 Hz, 1H); 13C NMR
(75 MHz, CDCl3): d=12.48, 19.33, 20.15, 29.94, 32.64, 40.55, 41.10,
53.23, 62.83, 64.23, 71.00, 112.49, 125.36, 128.53, 129.37, 130.01,
130.76, 130.95, 131.10, 132.53, 138.44, 142.47, 143.55, 149.50,
160.85, 170.11, 171.15, 172.12 ppm; Anal. calcd for C31H38N4O6: C
66.17, H 6.81, N 9.96. found: C 66.02, H 6.98, N 9.80.
(R,E)-Ethyl 4-(2-(3-((naphthalen-1-ylcarbamoyloxy)methyl)-2-oxo-
5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)acetamido)-
but-2-enoate (11): (R)-(1-(2-(allylamino)-2-oxoethyl)-2-oxo-5-phenyl-
2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)methyl naphthalen-1-yl-
carbamate 19 (50 mg, 0.093 mmol) was reacted with ethyl acrylate
22 (93 mg, 0.93 mmol) according to the same procedure described
for 6 to give the title compound 11 (42 mg, 74%). 1H NMR
(300 MHz, CDCl3): d=1.17 (t, J=7.1 Hz, 3H), 3.94 (m, 1H), 4.01–
4.13 (m, 4H), 4.23 (d, J=15.1 Hz, 1H), 4.72 (d, J=15.1 Hz, 1H),
4.95–5.11 (m, 2H), 5.86 (d, J=15.6 Hz, 1H), 6.81–6.87 (m, 2H), 7.23–
7.91 ppm (m, 16H); 13C NMR (75 MHz, CDCl3): d=15.10, 40.46,
53.19, 62.66, 63.18, 65.00, 109.42, 119.88, 121.29, 121.58, 123.14,
124.70, 125.56, 126.01, 126.31, 128.67, 128.89, 129.38, 130.07,
130.55, 131.75, 132.60, 133.90, 134.34, 139.41, 142.51, 144.20,
153.90, 154.70, 166.53, 170.13, 171.14, 171.25 ppm; Anal. calcd for
C35H32N4O6: C 69.52, H 5.33, N 9.27, found: C 69.73, H 5.09, N 9.45.
(R,E)-Ethyl
phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)acetamido)-
but-2-enoate (15): (R)-(1-(2-(allylamino)-2-oxoethyl)-2-oxo-5-
4-(2-(3-((cyclohexylcarbamoyloxy)methyl)-2-oxo-5-
phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)methyl cyclohexyl-
carbamate 21 (50 mg, 0.102 mmol) was reacted with ethyl acrylate
22 (102 mg, 1.02 mmol) according to the same procedure de-
1
scribed for 6 to give the title compound 15 (40 mg, 70%). H NMR
(300 MHz, CDCl3): d=1.05–1.37 (m, 9H), 1.55–1.68 (m, 2H), 1.87–
1.90 (m, 2H), 3.43 (m, 1H), 3.88–3.95 (m, 2H), 4.08–4.26 (m, 3H),
4.70 (d, J=15.1 Hz, 1H), 4.81–4.96 (m, 2H), 5.82 (d, J=15.9 Hz, 1H),
6.80–6.87 (m, 2H), 7.24–7.61 (m, 8H), 7.74 ppm (d, J=8.2 Hz, 1H);
13C NMR (75 MHz, CDCl3): d=14.77, 24.78, 29.91, 33.51, 40.41,
49.10, 53.05, 60.81, 62.81, 64.15, 122.14, 122.97, 125.53, 128.64,
128.64, 128.92, 130.07, 131.11, 132.55, 138.46, 142.62, 143.65,
166.50, 168.60, 171.10, 171.27 ppm; Anal. calcd for C31H36N4O6: C
66.41, H 6.47, N 9.99, found: C 66.75, H 6.56, N 9.86.
(R,E)-Isobutyl 4-(2-(3-((naphthalen-1-ylcarbamoyloxy)methyl)-2-
oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)acetami-
do)but-2-enoate (12): (R)-(1-(2-(allylamino)-2-oxoethyl)-2-oxo-5-
phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)methyl naphtha-
len-1-ylcarbamate 19 (50 mg, 0.093 mmol) was reacted with isobu-
tyl acrylate 23 (120 mg, 0.93 mmol) according to the same proce-
dure described for 6 to give the title compound 12 (43 mg,
(R,E)-Isobutyl 4-(2-(3-((cyclohexylcarbamoyloxy)methyl)-2-oxo-5-
phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)acetamido)-
1
72.7%). H NMR (300 MHz, CDCl3): d=0.83–0.85 (d, J=6.6 Hz, 6H),
1.84 (m, 1H), 3.75 (d, J=6.5 Hz, 2H), 3.91 (m, 1H), 4.01–4.13 (m,
2H), 4.23 (d, J=15.1 Hz, 1H), 4.72 (d, J=15.1 Hz, 1H), 4.98–5.14 (m,
2H), 5.87 (d, J=15.9 Hz, 1H), 6.81 (dt, J=15.9, 4.7 Hz, 1H), 6.87 (bs,
1H), 7.24–7.92 ppm (m, 16H); 13C NMR (75 MHz, CDCl3): d=19.23,
27.86, 40.43, 53.13, 62.64, 64.95, 71.00, 111.60, 119.96, 121.40,
121.98, 123.14, 125.49, 126.00, 126.21, 128.66, 129.38, 130.08,
130.69, 131.14, 132.57, 132.95, 134.34, 138.43, 142.52, 143.64,
154.74, 166.44, 168.90, 170.12, 171.17, 171.30 ppm; Anal. calcd for
C37H36N4O6: C 70.24, H 5.74, N 8.85, found: C 70.11, H 5.97, N 8.70.
but-2-enoate
(16):
(R)-(1-(2-(allylamino)-2-oxoethyl)-2-oxo-5-
phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)methyl cyclohexyl-
carbamate 21 (50 mg, 0.102 mmol) was reacted with isobutyl acry-
late 23 (130 mg, 1.02 mmol) according to the same procedure de-
scribed for 6 to give the title compound 16 (37 mg, 61.6%).
1H NMR (300 MHz, CDCl3): d=0.93 (d, J=6.8 Hz, 6H), 1.09–1.25 (m,
6H), 1.50–1.61 (m, 2H), 1.81–1.90 (m, 2H), 2.26 (m, 1H), 3.46 (m,
1H), 3.89–3.95 (m, 5H), 4.24 (d, J=15.1 Hz, 1H), 4.71 (d, J=15.1 Hz,
1H), 4.81–4.92 (m, 2H), 5.83 (d, J=15.6 Hz, 1H), 6.80–6.87 (m, 2H),
7.22–7.61 (m, 8H), 7.75 ppm (d, J=7.8 Hz, 1H); 13C NMR (75 MHz,
CDCl3): d=19.55, 25.36, 29.00, 29.98, 33.52, 40.43, 50.22, 53.10,
62.81, 64.14, 70.98, 121.99, 123.14, 125.44, 128.65, 129.41, 130.07,
131.04, 131.52, 135.90, 138.44, 142.44, 143.50, 166.28, 169.51,
171.15, 171.30 ppm; Anal. calcd for C33H40N4O6: C 67.33, H 6.85, N
9.52, found: C 67.44, H 6.59, N 9.70.
(R,E)-Ethyl 4-(2-(3-((butylcarbamoyloxy)methyl)-2-oxo-5-phenyl-
2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)acetamido)but-2-
enoate (13): (R)-(1-(2-(allylamino)-2-oxoethyl)-2-oxo-5-phenyl-2,3-di-
hydro-1H-benzo[e][1,4]diazepin-3-yl)methyl butylcarbamate 20
(50 mg, 0.108 mmol) was reacted with ethyl acrylate 22 (108 mg,
1.08 mmol) according to the same procedure described for 6 to
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(R,E)-4-{2-[3-(4-Chloro-2-trifluoromethylphenylcarbamoyloxy-
methyl)-2-oxo-5-phenyl-2,3-dihydrobenzo[e][1,4]diazepin-1-yl]-
acetoxy}but-2-enoic acid (25): To a solution of compound 1 syn-
thesized according to a previously described procedure[13] (50 mg,
0.077 mmol) in a mixture of MeOH/H2O (10 mL, 1:1) was added
LiOH (5.5 mg, 0.23 mmol) at 08C and stirred at room temperature
until the disappearance of starting material (TLC monitoring). The
solvent was concentrated under reduced pressure. A 10% aqueous
solution of citric acid was added to the resulting residue, the acid
was extracted with EtOAc, and the organic phase was dried
(Na2SO4) and concentrated to give acid 25, which was purified by
flash column chromatography using CHCl3/MeOH (9:1) (43.6 mg,
chain sulfur atom of residue Cys42. The PLP fitness function and
distribution of torsion angles were chosen to evaluate the quality
of the docking results;[27] van der Waals and hydrogen bonding
radii were set at 4.0 and 3.0 ꢂ, respectively, and genetic algorithm
parameters were kept at the default values. The FP-2–1 and FP-2–2
complexes were then submitted to minimization, equilibration,
and MD simulation by adopting the protocol previously applied
for the FP-2 model setup. The plot shown in Figure 3 was obtained
by VMD[30] and GNUPLOT.[31] The image in Figure 2 was acquired by
PyMOL software (PyMOL Molecular Graphics System).
1
90%). H NMR (300 MHz, CD3OD): d=3.96–4.07 (m, 2H), 4.07 (t, J=
Acknowledgements
6.5 Hz, 1H), 4.30 (d, J=15.2 Hz, 1H), 4.63 (d, J=15.2 Hz, 1H), 4.97
(m, 1H), 5.10 (m, 1H), 5.83 (d, J=15.7 Hz, 1H), 6.58 (bs, 1H), 6.83
(bt, 1H), 7.07 (bs, 1H), 7.22–7.73 (m,11H), 8.08 (d, J=8.8 Hz, 1H),
9.41 ppm (bs, 1H); 13C NMR (75 MHz, CD3OD): d=40.51, 52.84,
62.25, 65.79, 121.61, 121.93, 122.78, 122.99, 124.82, 125.60, 126.57,
128.30, 128.57, 128.65, 128.81, 129.47, 129.96, 130.70, 131.20,
132.63, 136.09, 142.30, 144.07, 153.42, 168.57, 169.52, 169.66,
170.10 ppm; Anal. calcd for C30H24ClF3N4O6: C 57.29, H 3.85, N 8.91,
found: C 57.40, H 3.70, N 9.21.
We thank Prof. Dr. M. Leippe and Dr. C. Gelhaus (Zoologisches In-
stitut, Universitꢁt Kiel) for assays on P. falciparum. We also thank
the Deutscher Akademisher Austausch Dienst (DAAD) (Vigoni
project 2011) for partial support of this work. R.E. acknowledges
the support of her postdoctoral fellowship from “Dote Ricerca”:
FSE, Regione Lombardia. T.S. thanks the Deutsche Forschungsge-
meinschaft (DFG) (SFB630) for financial support.
Enzyme assays: The preliminary screen for FP-2 inhibition was per-
formed with inhibitor concentrations of 100 mm using an equiva-
lent amount of DMSO as negative control. Product release from
substrate hydrolysis (Cbz-Phe-Arg-AMC, 40 mm) was determined
continuously over a period of 10 min. Compounds showing at
least 50% inhibition 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 mm) as substrate.[18]
Inhibitor solutions were prepared from stocks in DMSO. Each assay
was performed twice in 96-well plates in a total volume of 300 mL.
A Varian Cary Eclipse spectrofluorimeter (Varian, Darmstadt, Germa-
ny) with a microplate reader (lex: 365 nm, lem: 460 nm) was used.
Standard units of FP-2 were added to the reaction mixture contain-
ing the fluorogenic substrate and increasing compound concentra-
tions (from 0 to 100 mm). To determine first-order inactivation rate
constants (kobs) for time-dependent inhibition, progress curves
(fluorescence (F) versus time) were analyzed by nonlinear regres-
sion analysis using the equation F=A(1Àexp(Àkobs t))+B.[19] Prod-
uct formation was monitored continuously for 15 min at room
temperature. Pseudo-first-order rate constants kobs were fit to the
Keywords: cysteine proteases
inhibitors · peptidomimetics
· docking · falcipain-2 ·
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inhibitor concentrations using the hyperbolic equation: kobs
=
kinac[I]/(KIapp+[I]), yielding the first-order rate constant kinac and the
apparent inhibition constant KIapp. Inhibition constants KI were ob-
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Received: May 30, 2012
Published online on && &&, 0000
ChemMedChem 0000, 00, 1 – 8
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
&
7
&
ÞÞ
These are not the final page numbers!

FULL PAPERS
R. Ettari,* N. Micale, G. Grazioso, F. Bova,
T. Schirmeister, S. Grasso, M. Zappalꢀ
Constrained peptidomimetics 6–16 are
equipped with an electrophilic vinyl
ester warhead and are structurally relat-
ed to a previously identified potent and
irreversible inhibitor of falcipain-2 (FP-2),
the main hemoglobinase of P. falcipa-
rum. The new compounds were evaluat-
ed as inhibitors of FP-2, and docking ex-
periments were performed to rationalize
the results.
&& – &&
Synthesis and Molecular Modeling
Studies of Derivatives of a Highly
Potent Peptidomimetic Vinyl Ester as
Falcipain-2 Inhibitors
&8
&
www.chemmedchem.org
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 8
ÝÝ
These are not the final page numbers!