Peptidomimetics containing a vinyl ketone warhead as falcipain-2 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.02.058

The design, chemical synthesis, and enzymatic activity evaluation of a set of falcipain-2 inhibitors are reported. These compounds contain a proven peptidomimetic recognition motif based on a benzo[1,4]diazepin-2-one (1,4-BDZ) framework built on a dipepti

Full text of DOI:10.1016/j.ejmech.2011.02.058

European Journal of Medicinal Chemistry 46 (2011) 2058e2065
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Peptidomimetics containing a vinyl ketone warhead as falcipain-2 inhibitors
,
a
a
b
a
Roberta Ettari a , Maria Zappalà , Nicola Micale , Giovanni Grazioso , Salvatore Giofrè ,
*
Tanja Schirmeister c, Silvana Grasso a
a Dipartimento Farmaco-Chimico, University of Messina, Viale Annunziata, 98168 Messina, Italy
b Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy
c Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, chemical synthesis, and enzymatic activity evaluation of a set of falcipain-2 inhibitors are
reported. These compounds contain a proven peptidomimetic recognition motif based on a benzo[1,4]
diazepin-2-one (1,4-BDZ) framework built on a dipeptide sequence, and a Michael acceptor terminal
moiety capable of deactivating the cysteine protease active site. Our goal is to modify the P3 site of this
motif in order to identify the structural requirements for the interaction with the target.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 17 June 2010
Received in revised form
21 February 2011
Accepted 23 February 2011
Available online 3 March 2011
Keywords:
Peptidomimetics
Falcipain-2 inhibitors
Cysteine proteases
Docking studies
1. Introduction
processes of host erythrocyte rupture, erythrocyte invasion, and
hemoglobin degradation. Falcipain-2 (FP-2) of P. falciparum is
Half of the world’s population is at risk of malaria, and an esti-
mated 247 million cases led to nearly 881 000 deaths in 2006. The
World malaria report 2008 describes the global distribution of
cases and deaths, how WHO-recommended control strategies have
been adopted and implemented in endemic countries, sources of
funding for malaria control, and recent evidence that prevention
and treatment can alleviate the burden of the disease [1]. Five
species of a parasite belonging to the genus Plasmodium can infect
humans; the most serious forms of the disease are caused by
Plasmodium falciparum. Malaria infection caused by Plasmodium
vivax, Plasmodium ovale and Plasmodium malariae instead, brings
on a milder form of the disease in humans that is not generally fatal.
A fifth species of Plasmodium, named Plasmodium knowlesi, has
macaques as natural vertebrate host but recently it has been
regnognized infective also for humans [2].
a papain-family (clan CA, family C1) cysteine protease and is likely
the major hemoglobinase in the food vacuole of erythrocytic
parasites [5]. FP-2 is also able to promote host cell rupture cleaving
erythrocyte membrane skeletal proteins [6]. Therefore, the inhibi-
tion of FP-2 represents a promising strategy for discovery of novel
anti-malarial drugs.
Our research group has actively been involved on the synthesis
of novel peptidomimetic FP-2 inhibitors containing a 1,4-benzodi-
azepine (1,4-BDZ) scaffold [7e10], introduced internally to
a peptide sequence which mimics the dipeptide
D-Ser-Gly, and
different electrophilic warheads able to interact with the thiol
group of the cysteine active site by forming a reversible or irre-
versible covalent bond. In this context, we decided to synthesize
new derivatives in which the
a, b unsaturated electrophilic
warhead and the benzo[1,4]diazepin-2-one (1,4-BDZ) framework
were left unmodified, while the P3 site underwent profound
modifications, with the aim of gaining a better insight about the
characteristics of the pocket that could accommodate different
functional groups positioned at this site. Vinyl ketone 1 [10] (Fig. 1)
was selected as lead compound owing a good inhibitory activity
against the target enzyme (k2nd ¼ 4400 Mꢀ1 sꢀ1) and the strongest
The increasing resistance of malaria parasites to anti-malarial
drugs, the lack of widely available vaccines that provide a high level
of protection for a sustained period, and the inadequate control of
mosquito vectors demand new approaches to drug development
[3,4]. One promising strategy to develop new drugs has been to
target proteases of malaria parasites which play pivotal roles in the
potency against cultured P. falciparum (IC50 ¼ 9.3
mM), additionally
the ketone group possesses a major stability to metabolic reactions
in comparison to other functions, such as the ester moiety already
employed by our group [10].
* Corresponding author. Tel.: þ39 090 6766466; fax: þ39 090 6766402.
E-mail address: rettari@pharma.unime.it (R. Ettari).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.058

R. Ettari et al. / European Journal of Medicinal Chemistry 46 (2011) 2058e2065
2059
commercially available allylamine to afford the coupling products
14e16. The introduction of the required vinyl ketone warhead was
realized by olefin cross-metathesis (CM) reaction, using Hovey-
daeGrubbs 2nd generation catalyst, a phosphine-free N-heterocy-
clic carbene ruthenium complex which actually represents the
catalyst of choice for these reactions [13]. Thus, CM reactions
between the tripeptides 14e16 and methyl vinyl ketone has been
carried out under microwave irradiation in an efficient and rapid
manner to afford compounds 2e4. The introduction of an electron-
withdrawing substituent onto the olefin usually requires a rela-
tively large amount of catalyst and a prolonged reaction time and/
or results in a low conversion and yield of the expected CM product.
These results are normally attributed to the fact that a relatively
high temperature, used for several hours, can cause a gradual
decomposition of the catalyst. This problem has been overcome by
exposure of the reaction mixture to microwave heating at a high
temperature (100 ꢁC) for a short time [14].
The synthesis of the methyl derivative 5 was realized starting
from the carboxylic acid 17 [7], (Scheme 2), which was converted
into the corresponding mixed anhydride by reaction with i-BuO-
COCl and condensed in situ with 2-aminoacetophenone to afford
compound 18. According to our previously reported procedure [7],
this intermediate was refluxed in acidic conditions to afford the 1,4-
BDZ 19 substituted at position 3. Then, the hydroxyl group was
protected and the intermediate 20 was reacted with ethyl bro-
moacetate to introduce the side chain at the N-1 of the 1,4-BDZ. The
ester moiety was hydrolyzed to afford the corresponding acid 21
which was then coupled to the allylamine. The coupling product 22
was then deprotected to the serine hydroxyl group and treated with
the required isocyanate to provide the carbamate 23. The terminal
olefin was then easily functionalized via CM to afford compound 5.
Fig. 1. Structure of vinyl ketones 1e5.
Specifically, the N-(4-chloro-2-trifluoromethylphenyl)-carba-
moyloxy moiety of 1 linked at C-3 of the BDZ nucleus by means of
a methylene group has been replaced with an alicyclic, i.e. mor-
pholine (2) and piperidine (3), according to previously reported
studies on FP-2 inhibitors [11], or aliphatic amine, i.e. i-butylamine
(4), and the phenyl ring at C-5 of the 1,4-BDZ core was substituted
with a less bulky group, i.e. methyl (5).
Herein, we report the synthesis of vinyl ketones 2e5 (Fig. 1),
their biological evaluation against the FP-2 enzyme and molecular
modeling studies. Additionally, selectivity against the target
enzyme was estimated by testing the new compounds against
some human cysteine proteases of the papain-family such as
cathepsins B and L.
2. Results and discussion
2.1. Chemistry
2.2. Biological activity
Synthesis of target compounds 2e4 was accomplished as
depicted in Scheme 1. Desilylation of 1,4-BDZ derivative 6,
synthesized according to a previously reported procedure [7],
provided the free alcohol 7 which was easily converted to alicyclic
or aliphatic amine via Mitsunobu reaction [12], by using the
appropriate amine as a nucleophile in the presence of diethyl
azodicarboxylate (DEAD) and triphenylphosphine(TPP). The Mit-
sunobu reaction [12], firstly performed in standard conditions
(room temperature, 12 h) was not successful and the desired
products 8e10 were obtained under microwaves irradiation at
100 ꢁC for 2 h. The ester functionality in 8e10 was then hydrolyzed
and the corresponding carboxylic acids 11e13 were coupled to the
Compounds 2e5 were tested against FP-2 in standard fluores-
cence assays as published previously [15]. The hydrolysis of Cbz-
Phe-Arg-AMC (aminomethyl coumarin) in the absence or presence
of the respective inhibitor was measured by following the fluo-
rescence increase due to release of AMC. The well-known cysteine
protease inhibitor E-64 [16] and DMSO were used as a positive and
negative control respectively. First, initial screenings with 100 and
30
mM inhibitor concentration were performed. For inhibitors
which displayed considerable activity in this screening, continuous
assays (progress curve method, Fig. 2) [17] were performed to
determine the kinetic parameters (Table 1). In cases where kobs vs.
Scheme 1. Reagents and conditions: (a) TBAF, THF, rt, 5 h; (b) morpholine, piperidine or i-butylamine, TPP, DEAD, THF, MW, 100 ꢁC, 2 h; (c) LiOH, MeOH, 0 ꢁC-rt, 6 h; (d) allylamine,
HATU, CH2Cl2, rt, 12 h; (e) methyl vinyl ketone, Hoveyda Catalyst, CH2Cl2, 100 ꢁC, MW, 1 h.

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R. Ettari et al. / European Journal of Medicinal Chemistry 46 (2011) 2058e2065
Scheme 2. Reagents and conditions: (a) i-BuOCOCl, NMM, CH2Cl2, 0 ꢁC-rt, 30 min, then 2-aminoacetophenone, reflux, 20 min, rt, 12 h; (b) HCl/MeOH, reflux, 5 h, then NaHCO3,
MeOH, rt, 12 h; (c) TBS-Cl, imidazole, CH2Cl2, 0 ꢁC-rt, 12 h; (d) BrCH2COOEt, NaH, 0 ꢁC-rt, 5 h; (e) LiOH, MeOH, 0 ꢁC-rt, 6 h; (f) allylamine, HATU, CH2Cl2, rt, 12 h; (g) TBAF, THF, rt, 5 h;
(h) 4-Cl, 2-CF3C6H3NCO, rt, 12 h; (i) methyl vinyl ketone, Hoveyda Catalyst, CH2Cl2, 100 ꢁC, MW, 1 h.
[I] plots showed to be hyperbolic, both constants, kinac and KI, were
determined (Fig. 3). In cases where kobs vs. [I] plots were restricted
to the linear range, only the second-order rate constants of inhi-
in agreement with the expected mechanism of inhibition, we
carried out docking calculations taking for granted the formation of
the Michael adducts between the b-carbon of the a, b-unsaturated-
carbonyl ligands and the side chain of Cys42, present in the cata-
lytic site of the protein.
bition k2nd (Mꢀ1
s
ꢀ1) was determined (Fig. 4). The irreversible
inhibition of this class of compounds has been confirmed in our
previous studies by means of dialysis experiments [8], and can be
deduced from the observed time-dependent inhibition (Fig. 2).
In order to rationalize the activity profile of compounds 1e5, we
performed molecular modeling calculations on a computational
model of the enzyme derived from the recently published X-ray
structure of the FP-2 co-crystallized with E-64 [18]. Subsequently,
The results put in evidence that inhibitor 1 occupies entirely the
catalytic crevice of the enzyme creating a remarkable H-bond
network with various FP-2 residues (Fig. 5A). In particular, while
the three carbonyl groups of 1 behave as H-bond acceptors with the
side chain of Trp206 and the backbone of Gly83 and Ile85, the NH
group of the carbamoyl moiety acts as an H-bond donor with the
side chain of Asp234. Moreover, several hydrophobic contacts
further stabilize the FP-2/1 complex, i.e. the accommodation of the
aryl moiety in a cleft shaped by the side chains of Phe236, Ile85,
Leu84 and Asn86. On the contrary, the lack of some of these
contacts is detrimental on the potency of compound 5 since the
5-phenyl ring of derivative 1 is replaced by a methyl group.
The replacement of the arylcarbamoyl moiety of 1 by an amine
(e.g. compounds 2e4) reduces the potency of the ligands due to the
lack of the above-mentioned H-bonds and hydrophobic contacts
300
200
100
0
Table 1
Inhibition of FP-2 by 1e5a.
Compd k2nd (Mꢀ1 sꢀ1
)
kinac (minꢀ1
)
KI (mM)
c
c
1b
2
3
4400 ꢂ 500
200 ꢂ 33
c
c
80% inhib. at 100 mM, n.i. at 30 mM
4
5
>100
c
c
0
2
4
6
8
10
12
14
217 ꢂ 23
E-64d
(26.2 ꢂ 6.6) ꢃ 103 0.46 ꢂ 0.07 0.29 ꢂ 0.09
time [min]
a
All results are the average of at least two independent measurements, each
performed in duplicate. measurement time: 15 min.
Fig. 2. Inhibition of cathepsin L by cpd. 1. Progress curves at increasing inhibitor
concentrations (0, 5, 10, 20, 25, 30, 40, 50 M) show time-dependent inhibition,
b
m
Data reported in ref. 10.
c
indicating an irreversible inactivation of the enzyme. The progress curves were fit to
the equation: F ¼ A(1ꢀexp(ꢀkobst)) þ B to yield the kobs values (F ¼ fluorescence units,
B ¼ background fluorescence, t ¼ time).
Since the kobs vs. [I] diagrams were restricted to the linear range, only the
second-order rate constant could be determined as kobs/[I], for an example see Fig. 4.
d
Data reported in Ref. [8].

R. Ettari et al. / European Journal of Medicinal Chemistry 46 (2011) 2058e2065
2061
0,1
0,08
0,06
0,04
0,02
0
-1
= 0.11 min ; app = 8.6 µM
kinac
KI
app
KI KI
Km
=
k2nd kinac KI
/ (1 + [S]/ ) = 4.9 µM
=
/
= 374 M-1 s-1
0
20
40
60
I[µM]
Fig. 3. kobs vs. [I] plot of inhibition of cathepsin L by cpd. 1. Non-linear regression
analysis to the hyperbolic equation kobs ¼ kinac[I]/(KIapp þ [I]) yields kinac and the
apparent KI (KIapp). The inhibition constant KI was obtained by correction to zero-
substrate concentration using the ChengePrusoff equation KI ¼ KIapp/(1 þ [S]/Km). The
second-order rate constant was obtained by: k2nd ¼ kinac/KI.
created by the arylcarbamoyl moiety. Moreover, two steric clashes
could be created among the cyclic amine of 2 (Fig. 5B) and 3 and the
wall of the binding cleft formed by the side chain of Asp234 and
Ile85. Apparently, the steric hindrance of the isobutyl chain of
compound 4 is lower than the six-membered ring of derivatives 2
or 3 but, after a conformational minimization of the complex, we
noted a repulsive interaction between the positively charged
nitrogen of the amine and the nitrogen atom of Gly83. This
outcome could account for the inactivity of derivative 4.
Fig. 5. Surface representation of the FP-2 together with an enlargement of the catalytic
site. The predicted binding mode of 1 (A) and 2 (B) is represented. Carbon atoms are
colored in green and cyan, respectively. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article).
Compounds 3 and 4, which do not inhibit FP-2, were also inactive
toward cathepsins B and L.
In conclusion, our findings on this class of constrained pepti-
domimetics, supported by docking experiments, clearly indicate
that the phenylcarbamoyl portion appended to the P3 serine
For the most active of the synthesized inhibitors, i.e. compound
5, an additional assay against the P. falciparum strain FCBR was
performed, according to a previously published procedure [19]:
compound 5 turned out to be inactive (IC50 > 100 mM), in agree-
hydroxyl group remains
a structural requirement of utmost
ment with the low enzyme inhibition.
importance in the interaction with the FP-2 enzyme and that
a simplification at C-5 of the 1,4-BDZ scaffold does not fit with an
enhancement of the inhibitory potency. All this information will be
kept in mind for the progress of the research of new compounds
with improved FP-2 inhibitory properties.
The unsaturated ketones 2e5 were also tested against the
papain-family human cysteine proteases cathepsins B and L
(Table 2). Compounds 2 and 5, weak inhibitors of FP-2, proved to
generally possess k2nd values toward cathepsins B and L 1e2 orders
of magnitude lower than those reported for FP-2 inhibition.
3. Experimental protocols
kobs [I]-1 = 1300M-1 s-1
3.1. Chemistry
k2nd = kobs [I]-1 (1 + [S] Km-1) = 3720M-1 s-1
0.04
All reagents and solvents were obtained from commercial
suppliers and were used without further purification. Elemental
analyses were carried out on a C. Erba Model 1106 (Elemental
Table 2
Inhibition of Cathepsins B and L by 1e5a.
0.02
0
Cathepsin B
Cathepsin L
M) k2nd ) KI (mM)
kinac (minꢀ1
Compd k2nd
kinac
KI (m
(M-1 s-1
)
(minꢀ1
)
(Mꢀ1 s1)
1
2
3
4
5
50 ꢂ 12 0.02 ꢂ 0.01 6.0 ꢂ 0.3 383 ꢂ 2 0.12 ꢂ 0.02 5.9 ꢂ 3.4
b
b
>100
>100
9.6 ꢂ 0.5
>100
>140
0
10
20
30
>100
b
b
15.5 ꢂ 0.2
23.3 ꢂ 9.2 0.12 ꢂ 0.01 82 ꢂ 16
I[µM]
a
All results are the average of at least two independent measurements, each
Fig. 4. kobs vs. [I] plot of inhibition of falcipain-2 by cpd. 1. Since the values are
restricted to the linear range, the k2nd value is calculated from the slope of ꢀth1e curve
performed in duplicate.
Since the kobs vs. [I] diagrams were restricted to the linear range, only the
second-order rate constant could be determined as kobs/[I], for an example see Fig. 4.
b
and correction to zero-substrate concentration: k2nd z kobs [I]ꢀ1 (1 þ [S]Km
)

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R. Ettari et al. / European Journal of Medicinal Chemistry 46 (2011) 2058e2065
Analyzer for C, H and N) and the results are within ꢂ0.4% 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 conducted using pre-
packed cartridges on an MP-LC BUCHI system. 1H and 13C NMR
spectra were recorded in CDCl3 on a Varian Gemini 300 spec-
0.29 mmol), isobutylamine (42 mg, 0.58 mmol), triphenylphos-
phine (228 mg, 0.87 mmol), and diethyl azodicarboxylate (151 mg,
0.87 mmol). The title compound was obtained after purification by
flash column chromatography (light petroleum/EtOAc 6:4).
Rf ¼ 0.29 (light petroleum/EtOAc 6:4). Yield 95%. 1H NMR (300 MHz,
CDCl3): 1.18 (t, 3H, J ¼ 7.1 Hz), 1.23 (s, 6H), 2.00 (m, 1H), 2.94e3.02
(m, 2H), 3.88 (t, 1H, J ¼ 5.7 Hz), 4.08e4.27 (m, 3H), 4.36 (d, 1H,
J ¼ 15.5 Hz), 4.45 (m, 1H), 4.63 (d, 1H, J ¼ 15.5 Hz), 7.24e7.71 (m,
9H). 13C NMR (75 MHz, CDCl3): 14.16, 14.90, 20.62, 51.57, 61.01,
62.03, 62.35, 64.10, 121.78, 124.52, 127.34, 128.97, 129.25, 129.43,
131.17, 131.38, 139.09, 140.75, 168.63, 169.66, 170.02.
trometer. 1H chemical shifts are expressed in
TMS as internal standard and coupling constants (J) in hertz. 13C
chemical shifts are referenced to CDCl3 (central peak,
¼ 77.0 ppm).
d (ppm) relative to
d
3.1.1. (3-Hydroxymethyl-2-oxo-5-phenyl-2,3-dihydro-benzo[e][1,4]
diazepin-1-yl)-acetic acid ethyl ester (7)
To a solution of 6 (419 mg, 0.9 mmol) in dry THF (20 mL) was
added TBAF (1.35 mL, 1 M in THF). The mixture was stirred until the
disappearance of the starting material (TLC monitoring). After
removing of THF, the mixture was diluted with EtOAc (200 mL) and
washed with water (2 ꢃ 200 mL). The organic layer was dried,
filtered and evaporated under reduced pressure to give the
deprotected product 7 which was used for the next step without
any further purification. Rf ¼ 0.19 (light petroleum/EtOAc 6:4). Yield
95%. 1H NMR (300 MHz, CDCl3): 1.18 (t, 3H, J ¼ 7.1 Hz,), 2.79 (bs, 1H),
3.88 (t, 1H, J ¼ 5.7 Hz,), 4.08e4.27 (m, 3H), 4.41 (m, 1H), 4.49
(d, J ¼ 14.8 Hz, 1H), 4.58 (d, 1H,J ¼ 14.8 Hz), 7.20e7.65 (m, 9H). 13C
NMR (75 MHz, CDCl3): 14.30, 49.96, 61.92, 63.25, 64.04, 121.75,
124.93,128.50, 128.68, 129.94, 130.70,130.75, 130.80, 131.97, 138.82,
142.38, 170.12, 170.76.
3.1.5. (3-Morpholin-4-ylmethyl-2-oxo-5-phenyl-2,3-dihydro-benzo
[e][1,4]diazepin-1-yl)-acetic acid (11)
To a solution of ester 8 (113 mg, 0.27 mmol) in a mixture
methanol/water 1:1 (20 mL) was added LiOH (19 mg, 0.81 mmol)
keeping the temperature at 0 ꢁC and stirred at room temperature
until the disappearance of the starting material (TLC monitoring).
The solvent was concentrated under reduced pressure and the
mixture was treated with 10% citric acid and extracted with EtOAc
(200 mL), dried and concentrated to give the acid 11. Yield 99%. 1H
NMR (300 MHz, CDCl3): 2.77e2.93 (m, 4H), 3.76e3.85 (m, 2H), 3.91
(t, 1H, J ¼ 5.6 Hz), 4.12e4.25 (m, 3H), 4.40 (m, 1H), 4.47 (d, 1H,
J ¼ 17.3), 4.56 (d, 1H, J ¼ 17.3), 7.20e7.62 (m, 9H). 13C NMR (75 MHz,
CDCl3): 49.96, 53.01, 63.25, 64.04, 66.80, 121.75, 124.94, 128.50,
128.68, 129.95, 130.71, 130.81, 131.97, 138.82, 168.60, 170.01, 173.25.
3.1.2. (3-Morpholin-4-ylmethyl-2-oxo-5-phenyl-2,3-dihydro-benzo
[e][1,4] diazepin-1-yl)-acetic acid ethyl ester (8)
3.1.6. (2-Oxo-5-phenyl-3-piperidin-1-ylmethyl-2,3-dihydro-benzo
[e][1,4] diazepin-1-yl)-acetic acid (12)
To a mixture of the alcohol 7 (100 mg, 0.29 mmol), morpholine
(50 mg, 0.58 mmol), and triphenylphosphine (228 mg, 0.87 mmol)
in THF (5 mL) was added diethyl azodicarboxylate (151 mg,
0.87 mmol). The reaction mixture was stirred at 100 ꢁC for 2 h
under microwaves irradiation. After this time the solvent was
removed under reduced pressure. Diethyl ether was added to the
residue to precipitate triphenylphosphine oxide and diethyl azo-
dicarboxylate which were filtered off. The filtrate was evaporated
and the crude was purified by flash column chromatography (light
petroleum/EtOAc 6:4). Rf ¼ 0.33 (light petroleum/EtOAc 6:4). Yield
93%. 1H NMR (300 MHz, CDCl3): 1.18 (t, 3H, J ¼ 7.1 Hz,), 2.78e2.94
(m, 4H), 3.77e3.88 (m, 2H), 3.90 (t, 1H, J ¼ 5.5 Hz,), 4.14e4.27 (m,
5H), 4.41 (m, 1H), 4.48 (d, 1H, J ¼ 17.3), 4.58 (d, 1H, J ¼ 17.3),
7.21e7.65 (m, 9H). 13C NMR (75 MHz, CDCl3): 14.30, 49.96, 53.01,
61.92, 63.25, 64.04, 66.73, 121.75, 124.94, 128.50, 128.68, 129.95,
130.71, 130.81, 131.97, 138.82, 168.60, 170.01, 171.23.
The reaction has been carried out according to the same
procedure described for compound 11, by using ester 9 (104 mg,
0.25 mmol) and LiOH (18 mg, 0.75 mmol). Yield 99%. 1H NMR
(300 MHz, CDCl3): 1.24e1.30 (m, 6H), 2.30e2.35 (m, 4H), 3.88 (t,1H,
J ¼ 5.5 Hz), 4.28e4.36 (m, 2H), 4.58 (d, 1H, J ¼ 17.3), 4.80 (d, 1H,
J ¼ 17.3), 7.31e7.70 (m, 9H). 13C NMR (75 MHz, CDCl3): 28.11, 49.76,
53.11, 63.45, 64.14, 121.76, 124.84, 128.70, 128.68, 129.85, 130.72,
130.71, 131.87, 138.92, 168.26, 170.11, 173.05.
3.1.7. [3-(Isobutylamino-methyl)-2-oxo-5-phenyl-2,3-dihydro-
benzo[e][1,4] diazepin-1-yl]-acetic acid (13)
The reaction has been carried out according to the same proce-
dure described for compound 11, by using ester 10 (112 mg,
0.27 mmol) and LiOH (855 mg, 35.7 mmol). Yield 99%. 1H NMR
(300 MHz, CDCl3): 1.22 (s, 6H), 2.02 (m,1H), 2.96e3.04 (m, 2H), 3.88
(t,1H, J ¼ 5.7 Hz), 4.12 (m, 1H), 4.37 (d,1H, J ¼ 15.5 Hz), 4.44 (m, 1H),
4.62 (d,1H, J ¼ 15.5 Hz), 7.25e7.70 (m, 9H).13C NMR (75 MHz, CDCl3):
14.18, 20.82, 51.97, 61.10, 62.32, 64.05, 121.78, 124.42, 127.24, 128.67,
129.15, 129.33, 131.27, 131.35, 139.19, 140.65, 168.53, 169.86, 171.25.
3.1.3. (2-Oxo-5-phenyl-3-piperidin-1-ylmethyl-2,3-dihydro-benzo
[e][1,4] diazepin-1-yl)-acetic acid ethyl ester (9)
The reaction has been carried out according to the same proce-
dure described for compound 8, using alcohol
7
(100 mg,
3.1.8. N-Allyl-2-(3-morpholin-4-ylmethyl-2-oxo-5-phenyl-2,3-
dihydro-benzo[e][1,4]diazepin-1-yl)-acetamide (14)
0.29 mmol), piperidine (49 mg, 0.58 mmol), triphenylphosphine
(228 mg, 0.87 mmol), and diethyl azodicarboxylate (151 mg,
0.87 mmol). The title compound was obtained after purification by
flash column chromatography (light petroleum/EtOAc6:4). Rf ¼ 0.36
(light petroleum/EtOAc 6:4). Yield 87%. 1H NMR (300 MHz, CDCl3):
1.18 (t, 3H, J ¼ 7.1 Hz), 1.23e1.29 (m, 6H), 2.31e2.36 (m, 4H), 3.88 (t,
1H, J ¼ 5.5 Hz), 4.16e4.23 (m, 3H), 4.40 (m,1H), 4.48 (d,1H, J ¼ 17.3),
4.58 (d, 1H, J ¼ 17.3), 7.23e7.65 (m, 9H). 13C NMR (75 MHz, CDCl3):
14.26, 28.65, 49.95, 53.11, 61.90, 63.25, 64.06, 121.65, 124.93, 128.50,
128.78, 129.85, 130.51, 130.71, 131.87, 138.72, 168.56, 170.11, 171.13.
To a solution of 11 (105 mg, 0.27 mmol) in DMF (10 mL) at 0 ꢁC
was added HATU (154 mg, 0.40 mmol). After 5 min, the ice bath was
removed and allylamine (31 mg, 0.54 mmol) was added. The
mixture was stirred at room temperature for 12 h. After this time
the mixture was diluted with EtOAc and washed with water. The
organic layer was dried, filtered and evaporated under reduced
pressure and the crude was purified by flash chromatography
(CHCl3/MeOH 9:1). Rf ¼ 0.58 (CHCl3/MeOH 9:1). Yield 98%. 1H NMR
(300 MHz, CDCl3): 2.80e2.95 (m, 4H), 3.82e3.89 (m, 3H), 4.16e4.27
(m, 5H), 4.32 (d, 1H, J ¼ 15.4 Hz), 4.41 (m, 1H), 4.59 (d, 1H,
J ¼ 15.4 Hz), 5.02e5.11 (m, 2H), 5.75 (m, 1H), 6.39 (bs, 1H), 7.11e7.69
(m, 9H). 13C NMR (75 MHz, CDCl3): 42.27, 52.49, 52.63, 62.48, 63.13,
64.22, 116.82, 122.76, 125.28, 128.58, 129.78, 129.96, 130.54, 131.03,
132.35, 133.71, 138.54, 157.01, 168.27, 170.29, 171.32.
3.1.4. [3-(Isobutylamino-methyl)-2-oxo-5-phenyl-2,3-dihydro-
benzo[e][1,4] diazepin-1-yl]-acetic acid ethyl ester (10)
The reaction has been carried out according to the same
procedure described for compound 8, using alcohol 7 (100 mg,

R. Ettari et al. / European Journal of Medicinal Chemistry 46 (2011) 2058e2065
2063
3.1.9. N-Allyl-2-(2-oxo-5-phenyl-3-piperidin-1-ylmethyl-2,3-
dihydro-benzo[e][1,4]diazepin-1-yl)-acetamide (15)
(m, 1H, NeCHb.), 4.60 (d, 1H, J ¼ 15.4, NCHbCO), 6.02 (d, 1H,
J ¼ 15.8 Hz, NCH2CH]CHe), 6.51 (bs, 1H, NH), 6.63 (bt, 1H,
NCH2CH]CHe), 7.26e7.71 (m, 9H, CH Ar). 13C NMR (75 MHz,
CDCl3): 28.79 (piper (CH2)2), 29.84 (COCH3), 40.76 (NCH2CH]
CHe), 52.64 (piper (CH2)2), 52.90 (NCH2CO), 63.13 (NeCH2), 64.22
(CH-3), 122.72 (CH Ar), 125.23 (CH Ar), 128.50 (CH Ar), 128.61 (CH
Ar), 129.79 (CH Ar), 129.83 (CH Ar), 130.62 (CH Ar), 131.08 (CH Ar),
131.18 (NCH2CH]CHe), 138.74 (C Ar), 140.10 (C Ar), 142.10
(NCH2CH]CHe), 168.17 (C Ar), 170.11 (CO), 171.26 (CO), 195.96
(COCH3). Anal. Calcd for C28H32N4O3: C, 71.16; H, 6.83; N, 11.86;
found: C, 71.01; H, 7.09; N, 11.90.
The reaction has been carried out according to the same
procedure described for compound 14, by using acid 12 (98 mg,
0.25 mmol), HATU (142 mg, 0.37 mmol) and allylamine (28 mg,
0.50 mmol). The title compound was obtained after purification by
flash column chromatography (CHCl3/MeOH 9:1). Rf ¼ 0.61 (CHCl3/
MeOH 9:1). Yield 95%. 1H NMR (300 MHz, CDCl3): 1.25e1.31
(m, 6H), 2.31e2.36 (m, 4H), 3.82e3.89 (m, 3H), 4.25 (m,1H), 4.32 (d,
1H, J ¼ 15.4), 4.41 (m, 1H), 4.60 (d, 1H, J ¼ 15.4), 5.03e5.07(m, 2H),
5.71 (m, 1H), 6.26 (m, 1H), 7.23e7.70 (m, 9H). 13C NMR (75 MHz,
CDCl3): 28.68, 42.37, 52.19, 52.53, 62.28, 63.03, 116.82, 122.26,
125.48, 128.88, 129.79, 129.94, 130.52, 131.13, 132.35, 133.70, 138.53,
157.01, 168.37, 170.19, 171.22.
3.1.13. 2-[3-(Isobutylamino-methyl)-2-oxo-5-phenyl-2,3-dihydro-
benzo[e][1,4]diazepin-1-yl]-N-(4-oxo-pent-2-enyl)-acetamide (4)
Compound 16 (109 mg, 0.26 mmol) was reacted with methyl
vinyl ketone (182 mg, 2.6 mmol) according to the same procedure
described for 2. The title compound was obtained after purification
by flash column chromatography (CHCl3/MeOH 9:1). Rf ¼ 0.35
3.1.10. N-Allyl-2-[3-(isobutylamino-methyl)-2-oxo-5-phenyl-2,3-
dihydro-benzo[e][1,4]diazepin-1-yl]-acetamide (16)
The reaction has been carried out according to the same
procedure described for compound 14, by using acid 13 (102 mg,
0.27 mmol), HATU (154 mg, 0.40 mmol) and allylamine (31 mg,
0.54 mmol). The title compound was obtained after purification by
flash column chromatography (CHCl3/MeOH 9:1) Rf ¼ 0.54 (CHCl3/
MeOH 9:1). Yield 97%. 1H NMR (300 MHz, CDCl3): 1.24 (s, 6H), 2.01
(m, 1H), 2.94e3.03 (m, 2H), 3.89 (t, 1H, J ¼ 5.7 Hz), 4.07e4.15
(m, 2H), 4.24 (m, 1H), 4.37 (d, 1H, J ¼ 15.5 Hz), 4.46 (m, 1H), 4.65 (d,
1H, J ¼ 15.5 Hz), 5.03e5.12 (m, 2H), 5.73 (m, 1H), 6.53 (bs, 1H), 6.77
(bs, 1H), 7.22e7.69 (m, 9H). 13C NMR (75 MHz, CDCl3): 14.67, 20.81,
42.27, 51.27, 62.49, 63.09, 64.25, 116.81, 122.73, 122.77, 128.56,
128.61, 129.94, 129.98, 130.57, 131.05, 132.37, 138.52, 142.44, 168.60,
171.10, 171.20.
(CHCl3/MeOH9:1). Yield 87%. [
a
]
D
20: ꢀ34.3 (c ¼ 0.16, CHCl3).1H NMR
(300 MHz, CDCl3): 1.30 (s, 6H, (CH3)2CHe), 2.22 (m,1H, (CH3)2CHe),
2.26 (s, 3H, COCH3), 2.84e2.92 (m, 2H, (CH3)2CHeCH2e), 3.90 (t,1H,
J ¼ 5.7 Hz, CH-3), 4.08e4.16 (m, 2H, NCH2CH]CHe), 4.26 (m, 1H,
CHaeNei-Bu), 4.38 (d, 1H, J ¼ 15.5 Hz, NCHaCO), 4.48 (m, 1H,
CHbeNei-Bu), 4.64 (d, 1H, J ¼ 15.5 Hz, NCHbCO), 6.16 (d, 1H,
J ¼ 15.0 Hz, NCH2CH]CHe), 6.60e6.75 (m, 2H, NCH2CH]CHe and
NH), 7.31e7.73 (m, 9H, CH Ar). 13C NMR (75 MHz, CDCl3): 14.57
((CH3)2CH), 20.51 ((CH3)2CHe), 29.97 (COCH3), 40.59 (NCH2CH]
CHe), 52.69 ((CH3)2CHeCH2), 63.04 (NCH2CO), 64.31 (CH2eNei-
Bu), 65.41 (CH-3), 122.80 (CH Ar), 125.43 (CH Ar), 128.63 (CH Ar),
129.91 (CH Ar), 130.59 (CH Ar), 131.12 (CH Ar), 132.43 (NCH2CH]
CHe), 138.48 (C Ar), 142.14 (C Ar), 142.36 (NCH2CH]CHe), 168.60
(C Ar), 170.33 (CO), 171.41 (CO), 197.89 (COCH3). Anal. Calcd for
C27H32N4O3: C, 70.41; H, 7.00; N, 12.16; found C, 70.36; H, 7.32; N,
12.03.
3.1.11. N-(4-oxo-pent-2-enyl)-2-(2-oxo-5-phenyl-3-morpholin-1-
ylmethyl-2,3-dihydro-benzo[e][1,4]diazepin-1-yl)-acetamide (2)
To a solution of 14 (115 mg, 0.27 mmol) in dry CH2Cl2 (5 mL) was
added methyl vinyl ketone (189 mg, 2.7 mmol) followed by Hovey-
daeGrubbs2ndgenerationcatalyst[(1,3-bis-(2,4,6-trimethylphenyl)-
2-imidazolidinylidene)-dichloro-(o-isopropoxy phenyl methylene)
ruthenium] (16.8 mg, 0.027 mmol). The resulting mixture was heated
under microwave irradiationat 100 ꢁC for 2 h. The titlecompoundwas
obtained after purification by flash column chromatography (CHCl3/
MeOH9:1). Rf ¼ 0.38 (CHCl3/MeOH9:1). Yield85%.1H NMR(300 MHz,
CDCl3): 2.21 (s, 3H, COCH3), 2.80e2.97 (m, 4H, morph (CH2)2),
3.83e3.95 (m, 3H, NCH2CH]CH and CH-3), 4.16e4.23 (m, 5H, morph
(CH2)2 and NeCHa), 4.31 (d, 1H, J ¼ 16.5 Hz, NCHaCO), 4.41 (m, 1H,
NeCHb), 4.58 (d, 1H, J ¼ 16.5 Hz, NCHbCO), 6.06 (d, 1H, J ¼ 15.9 Hz,
NCH2CH]CHe), 6.61 (bt, 1H, NCH2CH]CHe), 7.26e7.71 (m, 9H, CH
Ar).13C NMR (75 MHz, CDCl3): 29.94 (COCH3), 40.62 (NCH2CH]CHe),
52.74 (morph (CH2)2), 52.80 (NCH2CO), 63.03 (NeCH2), 64.32 (CH-3),
65.90 (morph (CH2)2), 122.82 (CH Ar), 125.43 (CH Ar), 128.60 (CH Ar),
128.65 (CH Ar), 129.89 (CH Ar), 129.93 (CH Ar), 130.62 (CH Ar), 131.08
(CH Ar), 131.17 (NCH2CH]CHe), 138.54 (C Ar), 140.01 (C Ar), 142.12
(NCH2CH]CHe), 168.27 (C Ar), 170.31 (CO), 171.36 (CO), 195.85
(COCH3). Anal. Calcd for C27H30N4O4: C, 68.34; H, 6.37; N,11.81; found
C, 68.59; H, 6.27; N, 12.11.
3.1.14. (R)-4-(2-Benzoyl-methylcarbamoyl)-2,2-dimethyl-
oxazolidine-3-carboxylic acid tert-butyl ester (18)
To a solution of acid 17 (4.7 g, 19 mmol) in dry CH2Cl2 (80 mL) at
0 ꢁC was added N-methyl morpholine (2.31 mL, 21 mmol) followed
by isobutyl chloroformate (2.72 mL, 21 mmol). After 30 min,
a solution of 2-aminoacetophenone (2.56 g, 19 mmol) in CH2Cl2
(10 mL) was added to refluxing reaction mixture dropwise over
20 min. After stirring for 12 h at room temperature, the reaction
mixture was washed with 2 N HCl (100 mL), aqueous NaHCO3
(100 mL), and water (100 mL). The organic layer was dried and
concentrated to a residue purified by flash chromatography (light
petroleum/EtOAc 8:2). Rf ¼ 0.55 (light petroleum/EtOAc 8:2) to give
the product 18. Yield 75%. [
a
]
20: þ90.67 (c ¼ 1.65, CHCl3). 1H NMR
D
(300 MHz, CDCl3): 1.23e1.86 (m, 15H), 2.63 (s, 3H), 4.25e4.27 (m,
2H), 4.47 (m,1H), 7.15 (m,1H), 7.56 (m,1H), 7.89 (m,1H), 8.80 (d,1H,
J ¼ 8.5 Hz), 12.18 (s, 1H). 13C NMR (75 MHz, CDCl3): 28.52, 62.30,
67.22, 80.50, 97.30, 121.03, 122.90, 123.05, 131.70, 135.08, 140.37,
152.07, 171.02, 201.09.
3.1.15. (R)-3-Hydroxymethyl-5-methyl-1,3-dihydrobenzo[e][1,4]
diazepin-2-one (19)
3.1.12. N-(4-oxo-pent-2-enyl)-2-(2-oxo-5-phenyl-3-piperidin-1-
ylmethyl-2,3-dihydro-benzo[e][1,4] diazepin-1-yl)-acetamide (3)
Compound 15 (102 mg, 0.24 mmol) was reacted with methyl
vinyl ketone (168 mg, 2.4 mmol) according to the same procedure
described for 2. The title compound was obtained after purification
by flash column chromatography (CHCl3/MeOH 9:1). Rf ¼ 0.42
(CHCl3/MeOH 9:1). Yield 83%. 1H NMR (300 MHz, CDCl3): 1.25e1.31
(m, 6H, piper (CH2)3), 2.21 (s, 3H, COCH3), 2.30e2.35 (m, 4H, piper
(CH2)2), 3.89 (t, 1H, J ¼ 5.6 Hz, CH-3), 4.03e4.08 (m, 2H, NCH2CH]
CHe), 4.25 (m, 1H, NeCHa), 4.32 (d, 1H, J ¼ 15.4, NCHaCO), 4.41
To a solution of 18 (5.07 g,14 mmol) in MeOH (60 mL) was added
6 N HCl (11.6 mL). The mixture was refluxed for 5 h, after this time
the solvent was concentrated under reduced pressure and the
crude was extracted with EtOAc (200 mL) and washed with
NaHCO3 (2 ꢃ 200 mL). The organic phase was dried and again
concentrated. The mixture was solved in MeOH (60 mL) and stirred
until complete conversion of the starting material into the desired
product (12 h). The crude was purified by flash chromatography
(light petroleum/EtOAc 8:2). Rf ¼ 0.25 (light petroleum/EtOAc 8:2).

2064
R. Ettari et al. / European Journal of Medicinal Chemistry 46 (2011) 2058e2065
Yield 92%. [
a
]
20: ꢀ1.95 (c ¼ 1.95, CHCl3). 1H NMR (300 MHz,
3.1.19. (4-Chloro-2-trifluoromethyl-phenyl)-carbamic acid 1-allyl
carbamoyl methyl-5-methyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]
diazepin-3-ylmethyl ester (23)
D
CDCl3): 2.50 (s, 3H), 3.61 (t, 1H, J ¼ 6.3 Hz), 4.16 (m, 1H), 4.28 (m,
1H), 7.11 (d, 1H, J ¼ 7.9 Hz), 7.24 (m, 1H), 7.49 (m, 1H), 7.58 (d, 1H,
J ¼ 9.0 Hz,), 9.21 (bs, 1H). 13C NMR (75 MHz, CDCl3): 26.25, 62.83,
63.31, 121.57, 124.60, 128.73, 129.28, 131.84, 136.29, 171.02.
Step 1. To a solution of 22 (4.7 g, 11.4 mmol) in dry THF (50 mL)
was added TBAF (17.1 mL, 1 M in THF). The mixture was stirred until
the disappearance of the starting material (TLC monitoring), then
diluted with EtOAc and washed with water. The organic layer was
dried, filtered and evaporated under reduced pressure to give the
deprotected product which was used for the next step without
further purification. Rf ¼ 0.25 (CHCl3/MeOH 99:1). Yield 99%. 1H
NMR (300 MHz, CDCl3): 2.51 (s, 3H), 3.68 (t, 1H, J ¼ 5.7 Hz),
3.88e3.96 (m, 2H), 4.08 (d, 1H, J ¼ 15.1 Hz), 4.16e4.32 (m, 2H), 4.60
(d, 1H, J ¼ 15.1 Hz), 5.10e5.21 (m, 2H), 5.82 (m, 1H), 6.48 (bs, 1H),
7.31 (m,1H), 7.48e7.65 (m, 3H).
Step 2. To a solution of deprotected alcohol (3.4 g, 11.3 mmol) in
dry CH2Cl2 (50 mL) was added 4-Cl, 2-CF3C6H3NCO (3.4 mL,
22.6 mmol) and the mixture was stirred under N2 atmosphere for
12 h. After this time, the solvent was concentrated under reduced
pressure extracted with EtOAc and washed. The crude was purified
by flash chromatography (CHCl3/MeOH 99:1). Rf ¼ 0.52 (CHCl3/
MeOH 99:1). Yield 80%. 1H NMR (300 MHz, CDCl3): 2.50 (s, 3H), 3.87
(t,1H, J ¼ 6.6 Hz), 4.10 (d, 1H, J ¼ 14.5 Hz), 4.11e4.15 (m, 2H), 4.68 (d,
1H, J ¼ 14.5 Hz), 4.77 (m, 1H), 4.97 (m, 1H), 5.11e5.20 (m, 2H), 5.85
(m, 1H), 7.03 (bs, 1H), 7.31e7.67 (m, 6H), 8.02 (d, 1H, J ¼ 8.8 Hz). 13C
NMR (75 MHz, CDCl3): 25.15, 40.50, 53.71, 61.48, 65.22, 114.36,
116.70, 121.32, 123.12, 124.63, 126.42, 126.82, 127.65, 132.56, 133.16,
134.23, 140.21, 153.69, 164.21, 170.01, 171.12.
3.1.16. (R)-3-(tert-Butyl-dimethyl-silanyloxymethyl)-5-methyl-1,3-
dihydro-benzo[e][1,4]diazepin-2-one (20)
To a solution of alcohol 19 (2.6 g, 12.8 mmol) in CH2Cl2 (70 mL)
were added imidazole (1.9 g, 28 mmol) and TBS-Cl (4.8 g, 32 mmol)
at 0 ꢁC. After stirring 12 h at room temperature the mixture was
washed with water and dried. The crude was purified by flash
chromatography to afford the clean product (light petroleum/EtOAc
8:2) Rf ¼ 0.37 (light petroleum/EtOAc 8:2). Yield 99%. [
a]
20: ꢀ1.95
D
(c ¼ 1.95, CHCl3). 1H NMR (300 MHz, CDCl3): 0.23 (s, 3H), 0.24 (s,
3H), 1.01 (s, 9H), 2.57 (s, 3H), 3.79 (t, 1H, J ¼ 6.3 Hz), 4.37 (m, 1H),
4.69 (m, 1H), 7.12e7.68 (m, 4H), 10.27 (bs, 1H). 13C NMR (75 MHz,
CDCl3): ꢀ4.94, 18.07, 25.94, 26.27, 63.62, 63.70, 121.89, 124.10,
128.45, 129.30, 131.44, 136.93, 164.51, 171.01.
3.1.17. (R)-[3-(tert-Butyl-dimethyl-silanyloxymethyl)-2-oxo-5-
methyl-2,3-dihydrobenzo[e][1,4] diazepin-1-yl]-acetic acid (21)
Step 1. To a suspension of NaH (368 mg, 15.3 mmol) in dry DMF
(10 mL) was added a solution 0.1 M of 20 (4.07 g,12.8 mmol) in DMF
(20 mL) at 0 ꢁC under nitrogen and then slowly reaction mixture
brought into room temperature. After stirring 1 h at room temper-
ature, ethyl bromoacetate (2.13 mL,19.2 mmol, d ¼ 1.506) was added
via syringe and further stirred for 12 h. The reaction mixture was
quenched with NH4Cl and extracted with EtOAc (100 mL). The
organic layer was washed with water (2 ꢃ 100 mL), dried, concen-
trated and the resulting residue was purified by flash chromatog-
raphy (light petroleum/EtOAc 8:2) to afford the clean product.
3.1.20. (4-Chloro-2-trifluoromethyl-phenyl)-carbamic acid
5-methyl-2-oxo-1-[(4-oxo-pent-2-enylcarbamoyl)-methyl]-
2,3-dihydro-1H-benzo[e][1,4] diazepin-3-lmethyl ester (5)
Compound 23 (100 mg, 0.19 mmol) was reacted with methyl
vinyl ketone (133 mg, 1.9 mmol) according to the same procedure
described for 2. The title compound was obtained after purification
by flash column chromatography (CHCl3/MeOH 99:1). Rf ¼ 0.33
(CHCl3/MeOH 99:1). Yield 80%. 1H NMR (300 MHz, CDCl3): 2.25
(s, 3H, COCH3), 2.52 (s, 3H, CH3-5), 3.87 (t, 1H, J ¼ 6.6 Hz, CH-3), 4.10
(d,1H, J ¼ 14.5 Hz, NCHaCO), 4.11e4.15 (m, 2H, NCH2CH]CHe), 4.68
(d, 1H, J ¼ 14.5 Hz, NCHbCO), 4.77 (m, 1H, CHaOCONHAr), 4.97 (m,
1H, CHbOCONHAr), 6.09 (d, 1H, J ¼ 16.0 Hz, NCH2CH]CHe), 6.70
(bt, 1H, NCH2CH]CHe), 7.03 (bs, 1H, NH), 7.31e7.67 (m, 6H, CH Ar),
8.02 (d,1H, J ¼ 8.8 Hz, CH Ar). 13C NMR (75 MHz, CDCl3): 25.18 (CH3-
5), 29.96 (COCH3), 40.60 (NCH2CH]CHe), 53.21 (NCH2CO), 61.58
(CH-3), 65.73 (CH2OCONHAr), 114.60 (C Ar), 121.65 (C Ar), 123.07
(CH Ar),124.62 (CH Ar), 126.11 (CH Ar),126.42 (CH Ar),127.85 (C Ar),
131.06 (C Ar), 132.30 (CH Ar), 133.16 (NCH2CH]CHe), 140.10 (C Ar),
142.25 (NCH2CH]CHe), 153.90 (CO), 164.10, (C Ar), 170.10 (CO),
171.02 (CO), 195.95 (COCH3). Anal. Calcd for C30H25F3N4O5: C, 62.28;
H, 4.36; N, 9.68; found C, 62.16; H, 4.45, N, 9.43.
Rf ¼ 0.51 (light petroleum/EtOAc 8:2). Yield 99%. [
a]
20: ꢀ7.42
D
(c ¼ 0.35, CHCl3).1H NMR (300 MHz, CDCl3): 0.08 (s, 3H), 0.09 (s, 3H),
0.87 (s, 9H),1.22 (t, 3H, J ¼ 4.4 Hz), 2.49 (s, 3H), 3.63 (t,1H, J ¼ 7.1 Hz),
4.09e4.20 (m, 4H), 4.48e4.68 (m, 2H), 7.24e7.57 (m, 4H). 13C NMR
(75 MHz, CDCl3): ꢀ5.02,14.27,18.01, 25.46, 26.15, 49.80, 61.57, 63.99,
64.72, 121.98, 125.23, 127.58, 127.62, 131.38, 140.80, 168.95.169.02.
Step 2. To solution of ester (5.1 g, 12.7 mmol) in a mixture
methanol/water 1:1 (60 mL) was added LiOH (1.8 g, 76.2 mmol)
keeping the temperature at 0 ꢁC and stirred at room temperature
until the disappearance of the starting material (TLC monitoring).
The solvent was concentrated under reduced pressure and the
mixture was treated with 10% citric acid and extracted with EtOAc,
dried and concentrated to give the acid 21. Yield 99%. [
a
]
20: ꢀ3.75
D
(c ¼ 0.08, CHCl3). 1H NMR (300 MHz, CDCl3): 0.07 (s, 3H), 0.08 (s,
3H), 0.88 (s, 9H), 2.50 (s, 3H), 3.60 (t, 1H, J ¼ 7.1 Hz), 4.10e4.21 (m,
2H), 4.48e4.69 (m, 2H), 7.25e7.58 (m, 4H). 13C NMR (75 MHz,
CDCl3): ꢀ5.01,18.02, 25.36, 26.05, 49.70, 61.37, 64.32,121.78,125.13,
127.38, 127.32, 131.68, 140, 80, 168.75, 173.12.
3.2. Pharmacology
3.1.18. N-Allyl-2-[3-(tert-butyl-dimethyl-silanyloxymethyl)-5-methyl-
2-oxo-2,3-dihydro-benzo[e][1,4] diazepin-1-yl]-acetamide (22)
3.2.1. Enzyme assays
To a solution of 21 (4.7 g, 12.6 mmol) in CH2Cl2 (60 mL) at 0 ꢁ
C
The preliminary screening for FP-2 inhibition was performed
was added HATU (9.5 g, 25.2 mmol). After 5 min, the ice bath was
removed and allylamine (1.8 mL, 25.2 mmol, d ¼ 0.761) was added.
The mixture was stirred at room temperature for 12 h. After this
time the mixture was washed with water. The organic layer was
dried, filtered and evaporated under reduced pressure and the
crude was purified by flash chromatography (CHCl3/MeOH 99:1).
Rf ¼ 0.37 (CHCl3/MeOH 99:1). Yield 90%. 1H NMR (300 MHz, CDCl3):
0.12 (s, 3H), 0.15 (s, 3H), 0.90 (s, 9H), 2.50 (s, 3H), 3.67 (t, 1H,
J ¼ 5.7 Hz), 3.87e3.95 (m, 2H), 4.07 (d, 1H, J ¼ 15.1 Hz), 4.15e4.33
(m, 2H), 4.60 (d, 1H, J ¼ 15.1 Hz), 5.09e5.20 (m, 2H), 5.81 (m, 1H),
6.46 (bs, 1H), 7.30 (m, 1H), 7.48e7.66 (m, 3H).
with 100
amount of DMSO as negative control. Product release from substrate
hydrolysis (Cbz-Phe-Arg-AMC, 40 M) was determined continu-
ously 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 [16].
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
mM and 30 mM inhibitor concentrations using an equivalent
m
m
m
m
VarianCary Eclipse spectrofluorometerVarian, Darmstadt, Germany
with a microplate reader (excitation 365 nm, emission 460 nm) was

R. Ettari et al. / European Journal of Medicinal Chemistry 46 (2011) 2058e2065
2065
used. Standard units of FP-2 were added to the reaction mixture
containing the fluorogenic substrate and increasing from 0 to
Damavandi for her contribution in the early phase of the molecular
modeling studies. T.S. thanks the German Research Society (DFG,
SFB630) for financial support.
100
mM concentrations of compounds. To determine first-order
inactivation rate constants (kobs) for the time-dependent inhibition,
progress curves (fluorescence (F) vs. time) were analyzed by non-
linear regression analysis using the equation F ¼ A(1 ꢀ exp
(ꢀkobst)) þ B [17]. Product formation was monitored continuously
for 15 min at room temperature. Pseudo-first order rate constants
kobs were fit to the inhibitor concentrations using the hyperbolic
equation: kobs ¼ kinac[I]/(KIapp þ [I]) yielding the first-order rate
constant kinac and the apparent inhibition constant KIapp. Inhibition
constants KI were obtained by correction to zero-substrate
concentration using the ChengePrusoff equation KI ¼ KIapp/(1 þ [S]/
Km). Second-order rate constants were obtained by: k2nd ¼ kinac/KI. In
cases where the plots of the kobs values against the inhibitor
concentrations [I] were restricted to the linear range it was possible
to calculate, by means of the program GraFit [20], only k2nd values by
using the equation k2nd z kobs[I]ꢀ1 (1 þ [S]Kmꢀ1). The Km value was
References
[1] World Malaria Report 2008 http://malaria.who.int/wmr2008.
[2] J. Cox-Singh, T.M.E. Davis, K.-S. Lee, S.S.G. Shamsul, A. Matusop, S. Ratnam,
H.A. Rahman, D.J. Conway, B. Singh, Clin. Infect. Dis. 46 (2008) 165e171.
[3] A. Mital, Curr. Med. Chem. 14 (2007) 759e773.
[4] S. Jana, J. Paliwal, Int. J. Antimicrob. Agents 30 (2007) 4e10.
[5] A. Singh, P.J. Rosenthal, J. Biol. Chem. 34 (2004) 35236e35241.
[6] M. Hanspal, M. Dua, Y. Takakuwa, A.H. Chishti, A. Mizuno, Blood 100 (2002)
1048e1054.
[7] 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) 3064e3067.
[8] 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.
ꢀ
[9] R. Ettari, E. Nizi, M.E. Di Francesco, N. Micale, S. Grasso, M. Zappalà, R. Vicík,
T. Schirmeister, ChemMedChem 3 (2008) 1030e1033.
[10] R. Ettari, N. Micale, T. Schirmeister, R. Vicík, E. Nizi, M.E. Di Francesco,
determined to 21.5
m
M (FP-2) [21]. Assays with cathepsins B and L
ꢀ
were performed as described previously [22]. Cbz-Phe-Arg-AMC
was used as substrate (80 mM for cathepsin B, 5 mM for cathepsin L).
S. Grasso, M. Zappalà, J. Med. Chem. 52 (2009) 2157e2160.
[11] B.R. Shenai, B.J. Lee, A. Alvarez-Hernandez, P.Y. Chong, C.D. Emal, R.J. Neitz,
W.R. Roush, P.J. Rosenthal, Antimicrob. Agents Chemother. 47 (2003)
154e160.
[12] T.Y.S. But, P.H. Toy, Chem. Asian J. 2 (2007) 1340e1355.
[13] R. Ettari, N. Micale, J. Organomet. Chem. 692 (2007) 3574e3576.
[14] Y. Coquerel, J. Rodriguez, Eur. J. Org. Chem. (2008) 1125e1132.
[15] F. Schulz, C. Gelhaus, B. Degel, R. Vicik, S. Heppner, A. Breuning, M. Leippe,
J. Gut, P.J. Rosenthal, T. Schirmeister, ChemMedChem 2 (2007) 1214e1224.
[16] 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.
[17] W.X. Tian, C.L. Tsou, Biochemistry 21 (1982) 1028e1032.
[18] I.D. Kerr, J.H. Lee, K.C. Pandey, A. Harrison, M. Sajid, P.J. Rosenthal, L.S. Brinen,
J. Med. Chem. 52 (2009) 852e857.
[19] A. Evers, S. Heppner, M. Leippe, C. Gelhaus, Biol. Chem. 389 (2008)
1523e1525.
3.3. Molecular modeling
The structure of the FP-2, retrieved from the Protein Data Bank
(code 3BPF), was geometrically refined following the molecular
modeling standard procedures: minimization, equilibration and
molecular dynamics simulation [23]. Ligands were built by Sybyl 8.0
[24] and preliminarily minimized by Gaussian 03 [25] at DFT/HF/6-
31g(d) level. The amino group of ligands 2, 3 and 4 were considered
in the ionized form to better simulate the physiological conditions.
Docking calculations were then performed with the GOLD 4.1
program [26] into the binding cleft depicted by the presence of E-64
molecule. The cavity was detected with an active site radius of 20.0 Å
from the side chain sulfur atom of residue Cys42 and, accordingly to
the hypothesized mechanism of reaction [8], the formation of the
Michael adducts by Cys42 sulfur atom and carbon in beta position of
the ligands, were simulated. The goldscore fitness function and the
distribution of torsion angles were chosen to evaluate the quality of
the docking results. Van der Waals and hydrogen bonding radii were
set at 4.0 and 3.0 Å, respectively; genetic algorithm parameters were
kept at the default value. The complexes obtained by the ligand best
docked poses within FP-2 were further geometry optimized by
means of molecular mechanics method (Tripos force field) imple-
memented in Sybyl 8.0. Figures were acquired by the PyMOL soft-
ware (The PyMOL Molecular Graphics System, Version 0.99).
[20] GraFit, Version 3.0. Erithacus Software Ltd., London, 1992.
[21] C. Gelhaus, R. Vicik, R. Hilgenfeld, C.L. Schmidt, M. Leippe, T. Schirmeister, Biol.
Chem. 385 (2004) 435e438.
[22] R. Vicik, M. Busemann, C. Gelhaus, N. Stiefl, J. Scheiber, W. Schmitz, F. Schulz,
M. Mladenovic, B. Engels, M. Leippe, K. Baumann, T. Schirmeister, Chem-
MedChem 1 (2006) 1126e1141.
[23] G. Grazioso, A. Cavalli, M. De Amici, M. Recanatini, C. De Micheli, J. Comp.
Chem. 29 (2008) 2593e2602.
[24] Tripos Inc., 1699 South Hanley Rd., St. Louis, MO 63144.
[25] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb,
J.R. Cheeseman, J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant,
J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi,
G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara,
K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,
H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian, J.B. Cross, V. Bakken,
C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin,
R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth,
P. Salvador, J.J. Dannenberg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels,
M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman,
J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu,
A. Liashenko, P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-
Laham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson,
W. Chen, M.W. Wong, C. Gonzalez, J.A. Pople, Gaussian 03, Revision C.02.
Gaussian, Inc., Wallingford CT, 2004.
Acknowledgments
We would like to thank the DAAD Vigoni project (2009/10) for
partial support of this work. G.G. thanks Dr. Pardis Tabee
[26] Gold V. 4.1, Cambridge Crystallographic Data Centre: Cambridge, UK.