Constrained peptidomimetics as antiplasmodial falcipain-2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.06.010

Herein we report the synthesis of a series of novel constrained peptidomimetics 2-10 endowed with a dipeptide backbone (d-Ser-Gly) and a vinyl ester warhead, structurally related to a previously identified lead compound 1, an irreversible inhibitor of fal

Full text of DOI:10.1016/j.bmc.2010.06.010

Bioorganic & Medicinal Chemistry 18 (2010) 4928–4938
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Constrained peptidomimetics as antiplasmodial falcipain-2 inhibitors
a
a
b
Floriana Bova a, Roberta Ettari a, , Nicola Micale , Caterina Carnovale , Tanja Schirmeister ,
*
Christoph Gelhaus c, Matthias Leippe c, Silvana Grasso a, Maria Zappalà a
a Dipartimento Farmaco-Chimico, Università di Messina, Viale Annunziata, 98168 Messina, Italy
b Institute of Pharmacy and Food Chemistry, University of Würzburg Am Hubland, 97074 Würzburg, Germany
c Zoological Institute, University of Kiel, Olshausenstrasse 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:
Herein we report the synthesis of a series of novel constrained peptidomimetics 2–10 endowed with a
Received 7 April 2010
Revised 28 May 2010
Accepted 4 June 2010
Available online 10 June 2010
dipeptide backbone (D-Ser-Gly) and a vinyl ester warhead, structurally related to a previously identified
lead compound 1, an irreversible inhibitor of falcipain-2, the main haemoglobinase of lethal malaria par-
asite Plasmodium falciparum. The new compounds were evaluated for their inhibition against falcipain-2,
as well as against cultured P. falciparum. The inhibitory activity of the synthesized compounds was also
evaluated against another protozoal cysteine protease, namely rhodesain of Trypanosoma brucei
rhodesiense.
Keywords:
Peptidomimetics
Falcipain-2 inhibitors
Rhodesain inhibitors
Cysteine proteases
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
a,b-unsaturated electrophilic functions such as vinyl-sulfones, -
phosphonates, -ketones, -nitriles, -amides, -esters,6–8 that react
as classical ‘Michael acceptors’, leading to an alkylated enzyme
by irreversible addition of the thiol group of the active site cys-
teine. Among all the synthesized inhibitors, a surprising inhibitory
potency was observed for the vinyl ester 1 (Fig. 1) which showed
an impressive value of second-order rate constant of inhibition
(k2nd = 3,570,000 Mꢀ1 minꢀ1), a potency two times higher than that
of the standard inhibitor E-64 (k2nd = 1,586,000 Mꢀ1 minꢀ1) and
the highest enzymatic affinity (Ki = 17 nM). Furthermore, lead
compound 1 displayed an excellent inner-class selectivity towards
the target enzyme with respect to human cysteine proteases
cathepsins B and L. Compound 1 was also tested against cultured
Malaria, a disease caused by several species of protozoa belong-
ing to the genus Plasmodium, remains to be one of the most deadly
parasitic diseases affecting about 250 million people worldwide
and leading to more than 1 million deaths each year.1 Taking into
consideration that current antimalarial therapeutics become
increasingly ineffective because of parasitic resistance, there exists
an urgent need to develop and pursue new therapeutic strategies.2
Many drug targets can be related to the functions of distinct
organellar structures. Of particular interest is the lysosomal food
vacuole of P. falciparum parasite, the site of extensive hemoglobin
degradation. One of the main proteases involved in this metabolic
process is named falcipain-2 (FP-2). Its primary functions include
degradation of host hemoglobin, required for sustaining the meta-
bolic needs of rapidly growing parasite,3 together with the cleav-
age of host cytoskeletal proteins4 to facilitate the invasion and
rupture of erythrocytes.
P. falciparum strain FCBR, showing an IC50 value of 12 l
M.8
In view of these consistent results, we designed novel pepti-
domimetic analogues of compound 1, whereas the electrophilic
warhead was left unmodified and recognition elements were
added along the constrained dipeptide framework, in order to
In view of its key role in P. falciparum life cycle, in the last dec-
ades a lot of FP-2 inhibitors have been developed5 and in this re-
gard our research group has been actively involved in the design
and synthesis of novel peptidomimetic FP-2 inhibitors. In this
context, these FP-2 inhibitors are based on a 1,4-benzodiazepine
scaffold, as b-turn mimetic, introduced internally to a peptide
O
CF3
N
H
N
N
O
O
N
H
sequence with mimics the fragment
D-Ser-Gly, and various
O
O
O
Cl
1
* Corresponding author. Tel.: +39 090 6766466; fax: +39 090 6766402.
E-mail address: rettari@pharma.unime.it (R. Ettari).
Figure 1. Structure of FP-2 irreversible inhibitor 1.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.010

F. Bova et al. / Bioorg. Med. Chem. 18 (2010) 4928–4938
4929
identify key interactions between inhibitor and the binding sites of
the enzyme.
condensed in situ with the appropriate 2-aminobenzophenone to
afford compounds 12–14. Then, 12–14 underwent hydrolysis in
acidic conditions, and the resultant 1,4-benzodiazepine derivatives
15–17 were O-protected with TBS (18–20) and functionalized at N-
1 with ethyl bromoacetate followed by hydrolysis of the ester moi-
ety to give 21–23.
The synthons 21–23 were coupled with commercially available
allylamine 25 by means of HATU to afford intermediates 27–29,
whereas the unsubstituted synthon 24 was coupled with 3-
methyl-1-vinyl-butylamine 26 (synthesized according to Albeck’s
work),10 to give the intermediate 30. Then, fluoride promoted desi-
lylation of 27–30, followed by treatment with the 4-chloro-2-tri-
fluoromethyl isocyanate, afforded carbamates 31–34.
Specifically, at the P1 site the amino acid glycine was switched
with leucine (i.e., 2), useful to evaluate the relevance of the amino-
acid chain in the process of recognition of the ligand by the en-
zyme. The P3 site was modified by introducing substituents with
different electronic properties (i.e., Br, 4 or OCH3, 5) at C-40 of the
phenyl substituent on the benzodiazepine scaffold, and by intro-
ducing a chlorine atom (i.e., 3) on the fused benzene ring. More-
over, the 4-chloro-2-trifluoromethylphenyl group appended to
the side chain of serine at the P3 site by means of a carbamoyl
moiety, was replaced by other aromatic rings (i.e., naphthyl, 7 or
4-methoxyphenyl, 6) selected on the basis of our previous findings
on related reversible inhibitors,9 and (cyclo)alkyl groups (i.e., butyl
8, cyclohexyl, 9 or adamantyl, 10) in order to find out the capacity
of this pocket in accommodating groups with different steric
hindrance.
The introduction of the required methyl ester warhead has been
realized by cross-metathesis reaction using Hoveyda–Grubbs
second generation catalyst,11
carbene–ruthenium complex.
a phosphine-free N-heterocyclic
All compounds were tested on recombinant FP-2 and on cul-
tured plasmodia in order to establish a structure–activity relation-
ship for these recognition elements built on the lead structure 1.
Selectivity against the target enzymes was also estimated, testing
inhibitors against papain-family human cysteine proteases such
as cathepsins B and L. The most interesting compounds were also
tested for their activity against rhodesain, another cysteine prote-
ase of papain-like family isolated from T. brucei rhodesiense, the
causing agent of the sleeping sickness, spotting it as an alternative
target.
Thus, cross-metathesis reactions between the tripeptides 31–
34 and methyl acrylate proceeded under microwave irradiation12
in an efficient and rapid manner to afford compounds 2–5
(Scheme 2).
By using the same synthetic procedure as for compounds 2–5,
compound 24 was coupled with allylamine and deprotected to give
356 which was in sequence reacted with the suitable isocyanate,
and conjugated through cross-metathesis to methyl acrylate to af-
ford compounds 6–10 (Scheme 3).
2.2. Pharmacology
2. Results and discussion
2.1. Chemistry
Compounds 2–10 (Table 1) were tested for their inhibitory
activity against recombinant FP-213 using Cbz-Phe-Arg-AMC as
fluorogenic substrate.14 First, a preliminary screening with inhibi-
tor concentrations of 100 lM was performed. An equivalent vol-
The constrained dipeptide framework substituted at the aro-
matic rings 21–23 (Scheme 1) were prepared according to a previ-
ously reported method for the unsubstituted analogue 24.9
The oxazolidine derivative 119 was converted into the
corresponding mixed anhydride with i-butyl chloroformate and
ume of DMSO was used as negative control, and the irreversible
standard inhibitor of clan CA, family C1 cysteine proteases (pa-
pain-family), namely E-64,15 was used as positive control. For com-
pounds which passed the initial screening, continuous assays
(progress curve method)16 were performed to determine the
first-order rate constants of inhibition kinac (minꢀ1), the dissocia-
tion constants Ki (lM), and the second-order rate constants of inhi-
R'
R
R'
R
bition k2nd (Mꢀ1 minꢀ1), with k2nd = kinac/Ki. In the case of time
independent inhibition, Ki was determined using a Dixon plot.17
Among the novel synthesized compounds, only 5 and 8 didn’t
pass the preliminary screening. When a Leu residue was intro-
duced at the P1 site (2) instead of the Gly residue of lead com-
pound 1, a strong decrease both in term of affinity and inhibitory
potency was noticed (Ki = 17 nM and k2nd = 3,570,000 Mꢀ1 minꢀ1
Boc
O
N
a
b
O
NH
N
NH
COOH
O
O
Boc
HO
N
O
11
for 1 vs Ki = 2.22 l
M and k2nd = 18,000 Mꢀ1 minꢀ1 for 2). This up-
15, R= Cl, R'= H
16, R= H, R'= Br
, R= H, R'= OCH3
12, R= Cl, R'= H
13
, R= H, R'= Br
shot is in disagreement to what is observed with other papain-like
cysteine protease inhibitors, which usually accommodate bulky
side chains closely associated with the electrophilic moiety.18 Still,
we observed a similar trend in our previous work, when we
switched the Gly residue at the P1 site of 1 with a homoPhe resi-
due.6 So, we can assess for this class of FP-2 inhibitors endowed
with a vinyl ester warhead, that the insertion of side chains at
the P1 site is not a favorable design to construct a suitable recog-
nition motif.
17
14, R= H, R'= OCH3
R'
R
R'
R
c
d-e
COOH
N
N
NH
N
TBSO
TBSO
O
O
A comparable drop both in term of affinity and inhibitory po-
tency can be also observed in compounds with substituents at
the aromatic rings of the benzodiazepine scaffold, that is, 3–5, sug-
gesting that these aromatic portions of the skeleton are very sensi-
tive to structural changes.
With compound 1 as a reference point and with these addi-
tional binding motif clues, we began an exploration of the group
appended to the terminal serine hydroxyl by means of a carbamoyl
moiety. The substitution of the 4-chloro-2-trifluoromethylphenyl
21, R= Cl, R'= H
22, R= H, R'= Br
18
, R= Cl, R'= H
19
, R= H, R'= Br
20, R= H, R'= OCH3
23
, R= H, R'= OCH3
Scheme 1. Reagents and conditions: (a) i-BuOCOCl, N-methylmorpholine, CH2Cl2,
0 °C to room temperature, 30 min, then appropriate 2-aminobenzophenone, reflux,
20 min, room temperature, 12 h; (b) HCl/MeOH, reflux, 5 h, NaHCO3, then MeOH,
room temperature, 12 h; (c) TBS-Cl, imidazole, CH2Cl2, 0 °C to room temperature,
12 h; (d) NaH, BrCH2COOEt, 0 °C to room temperature, 5 h; (e) LiOH, MeOH, 0 °C to
room temperature, 6 h.

4930
F. Bova et al. / Bioorg. Med. Chem. 18 (2010) 4928–4938
R'
R'
R''
R''
R
R
O
a
N
COOH
+
H2N
N
N
O
N
O
N
H
25, R= H
TBSO
TBSO
26
, R= CH2CH(CH3)2
21, R'= Cl, R''= H
22, R'= H, R''= Br
27, R= R''= H, R'= Cl
28
, R= R'= H, R''= Br
23
, R'= H, R''= OCH3
29, R= R'= H, R''= OCH3
30, R= CH2CH(CH3)2,
R'= R''= H
24, R'= R''=
H
R'
R''
R
O
31, R= R''= H, R'= Cl
32
, R= R'= H, R''= Br
O N
O
N
H
N
F3C
33, R= R'= H, R''= OCH3
34, R= CH2CH(CH3)2,
R'= R''= H
N
H
b, c
O
Cl
R'
R''
R
O
2, R= CH2CH(CH3)2,
R'= R''= H
O
O
O
N
N
H
N
F3C
3
N
H
, R= R''= H, R'= Cl
d
4, R= R'= H, R''= Br
O
5
, R= R'= H, R''= OCH3
O
Cl
Scheme 2. Reagents and conditions: (a) HATU, CH2Cl2, room temperature, 12 h; (b) TBAF, THF, room temperature, 5 h; (c) 4-Cl-CF3–C6H3NCO, CH2Cl2, room temperature,
12 h; (d) methyl acrylate, Hoveyda catalyst, CH2Cl2, 100 °C, MW, 2 h.
After determining the inhibition potencies of these compounds,
the best candidates (2, 3, 4, 6, 7, and 10) were tested against P. fal-
ciparum strain FCBR (Table 1). The dose-dependent effects of the
O
a, b
compounds on parasite development were quantified using a pre-
24
N
N
viously published assay.19
N
H
HO
As shown in Table 1, all tested compounds displayed similar ex-
tent (micromolar range) of antiplasmodial activity against the test
parasites, comparable to that of the reference compound 1. In view
of these data, a SAR discussion turns out to be a difficult task. How-
ever compounds 4 and 10 were found to exhibit the better antiplas-
modial profile with IC50 values of 6.33 and 10.7 lM, respectively.
This may be due to inhibition of the related protease falcipain-3
which also is involved in hemoglobin degradation.
This set of compounds which showed significant enzymatic
inhibition and antiplasmodial activity (2, 3, 4, 6, 7, and 10), as well
as lead compound 1, were considered for an assay against another
papain-like protozoan cysteine protease, that is, rhodesain from T.
brucei rhodesiense (Table 2).
O
35
O
c, d
O
O
O
N
N
H
N
N
H
O
R
O
6
, R= 4-MeO-C6H4
7, R= 1-naphthyl
8
, R= n-C4H9
9, R= c-C6H11
10, R= 1-adamantyl
Kinetic measurements against rhodesain showed the same
trend of reaction mechanism and inhibitory activity of that ob-
served for FP-2 for all compounds except 1. Most importantly, all
new compounds showed a relevant affinity toward rhodesain with
Ki values three to fivefold lower than that of FP-2 (e.g.. 10,
Ki = 2.6 nM for rhodesain vs Ki = 14 nM for FP-2). The inhibitory
potencies of new compounds toward rhodesain also resulted high-
er comparing to that observed toward FP-2.
The most representative compound 10 showed a remarkable
value of second-order rate constant of inhibition (k2nd = 4,620,000
Mꢀ1 minꢀ1). These results indicate that the adamantyl group an-
chors to a hollow pocket of rhodesain by means of extended hydro-
phobic interactions and ensures proper alignment of the peptide
framework to the binding sites of the enzyme.
Scheme 3. Reagents and conditions: (a) allylamine, HATU, CH2Cl2, room temper-
ature, 12 h; (b) TBAF, THF, rt, 5 h; (c) RNCO, CH2Cl2, room temperature, 12 h; (d)
methyl acrylate, Hoveyda catalyst, CH2Cl2, 100 °C, MW, 2 h.
ring of 1 with other aromatic rings, that is, 6 and 7, or with (cy-
clo)alkyl groups, that is, 8 and 9, reduces the affinity for the target
enzyme, in particular for the (cyclo)alkyl derivatives. In addition,
the inhibition is reversible, and this can be probably due to a not
proper orientation of the electrophilic warhead, preventing an irre-
versible alkylation of the thiol group of the target enzyme.
Surprisingly, the introduction of a bulkier hydrophobic group at
the same site (i.e., compound 10) gave an impressive irreversible
inhibition with the same affinity and potency degree of lead com-
pound 1.

F. Bova et al. / Bioorg. Med. Chem. 18 (2010) 4928–4938
4931
Table 1
Inhibition of FP-2 and antiplasmodial activity of compounds 2–10a
R'
R''
R
O
O
O
O
N
N
O
H
N
N
H
O
R'"
Compd
R
R0
R00
R000
k2nd (Mꢀ1 minꢀ1
)
kinac (minꢀ1
)
Ki (lM)
P. falciparum IC50 (lM)
2
3
4
5
6
7
8
9
i-C4H9
H
Cl
H
H
H
H
H
H
H
H
H
H
Br
OCH3
H
H
H
H
H
4-Cl-2-CF3C6H4
4-Cl-2-CF3C6H4
4-Cl-2-CF3C6H4
4-Cl-2-CF3C6H4
4-MeO-C6H4
1-Naphthyl
n-C4H9
c-C6H11
1-Adamantyl
4-Cl-2-CF3C6H4
18,000 2000
76,000 26,000
24,500 900
0.040 0.001
0.030 0.002
0.038 0.002
2.22 0.37
0.46 0.18
1.56 0.15
>100
15.1 1.4
3.48 0.21
>100
89.0 1.6
0.014 0.007
0.017 0.004
0.29 0.09
26.9
28.9
6.33
H
H
H
H
H
H
H
H
H
b
b
b
b
33.1
17.6
b
b
10
1
E-64
1,160,000 570,000
3,570,000 400,000
1,586,000 400,000
0.013 0.001
0.06 0.01
0.46 0.07
10.7
12.0
H
a
All results include standard deviations from two independent measurements, each performed in duplicate.
Time independent inhibition.
b
Table 2
3. Conclusion
Inhibition of rhodesain of compounds 2–10a
In conclusion, the structural modifications realized on lead
compound 1 led to a drop in FP-2 inhibitory activity, with the
exception of compound 10 which, besides a good FP-2 inhibitory
properties, proved to inhibit rhodesain with an impressive potency
and, when tested against human cysteine proteases, to possess a
good selectivity towards the target enzymes. In view of the ob-
tained results, compound 10 can be considered as a promising try-
panocidal agent.
Compd
k2nd (Mꢀ1 minꢀ1
)
kinac (minꢀ1
)
Ki (lM)
2
3
4
6
7
36,000 1000
220,000 8000
46,000 4000
0.031 0.001
0.019 0.001
0.87 0.05
0.087 0.003
0.47 0.03
4.3 0.4
0.93 0.02
0.0026 0.0003
0.81 0.02
0.022 0.001
b
b
b
b
10
1
4,620,000 530,000
34,600 200
0.012 0.001
0.028 0.001
a
All results include standard deviations from two independent measurements,
each performed in duplicate.
b
4. Experimental
4.1. Chemistry
Time independent inhibition.
Selectivity against the target enzymes (protozoan cysteine pro-
teases) was also estimated, testing inhibitors against papain-family
human cysteine proteases such as cathepsins B and L (Table 3).
In most cases, the compounds inhibit the enzymes in a time
independent manner, showing Ki values one to two orders of mag-
nitude higher than that for FP-2 inhibition, except for compounds
5, 8, and 9 that inhibit cathepsins B and L with Ki values lower com-
paring to that observed toward FP-2. The most representative com-
pound 10 displayed an excellent selectivity toward FP-2 and
rhodesain with respect to the inhibition of cathepsins B and L.
4.1.1. Instruments and analyses
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 chromatogra-
phy was performed using Merck Silica Gel (200–400 mesh) on a
Table 3
Inhibition of human cathepsins B and L of compounds 2–10a
Compd
Cathepsin B
Cathepsin L
k2nd (Mꢀ1 minꢀ1
)
kinac (minꢀ1
)
Ki (l
M)
k2nd (Mꢀ1 minꢀ1
)
kinac (minꢀ1
)
Ki (lM)
b
b
2
3
4
5
6
7
8
9
10
1
39.1 2.7
6.1 0.5
>150
10.9 0.3
2.23 0.01
>150
29.8 3.4
>150
59.9 2.7
12.0 1.5
12.2 3.4
2.7 0.2
76.5 11.8
65.0 8.6
8.4 0.8
b
b
b
b
b
b
b
b
b
b
12,500 3300
6400 1400
0.15 0.01
2000 500
0.037 0.001
25
9
0.079 0.005
b
b
20.4 0.4
12.4 0.1
13.7 2.5
7.3 3.2
9400 500
0.025 0.001
b
b
b
b
b
b
550 50
0.036 0.001
b
b
6000 1000
0.044 0.004
a
All results include standard deviations from two independent measurements, each performed in duplicate.
Time independent inhibition.
b

4932
F. Bova et al. / Bioorg. Med. Chem. 18 (2010) 4928–4938
MP-LC BUCHI system. Preparative HPLC was performed on a Varian
PrepStar SD-1 instrument equipped with a ProStar 325 UV–vis
detector. The stationary phase was XTerra C18 column (5 lm,
19 ꢁ 150 mm); the mobile phase was a binary mixture of MeCN
(containing 0.1% TFA) and water (containing 0.1% TFA) 55:45 at
the flow rate of 5 mL minꢀ1. 1H and 13C NMR spectra were recorded
in CDCl3 on a Varian Gemini 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 = 77.0 ppm).
4.1.1.4. (R)-7-Chloro-3-(hydroxymethyl)-5-phenyl-1H-benzo[e]
[1,4]diazepin-2(3H)-one (15). To a solution of 12 (4.27 g, 9.3 mmol)
in MeOH (10 mL) was added 6 N HCl (4 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 and
washed with NaHCO3. The organic phase was dried and again
concentrated. The mixture was dissolved in MeOH and stirred
until complete conversion of the starting material into the desired
product (12 h). The crude product was purified by flash chro-
matography (petroleum ether/EtOAc 4:6) (1.96 g, 70%). Rf = 0.32
(petroleum ether/EtOAc 4:6). 1H NMR (300 MHz, CDCl3): 3.05 (br s,
1H, CH2OH), 3.72 (dd, 1H, J = 6.3 and J = 6.0 Hz, CHCH2), 4.24 (dd,
1H, J = 9.9 and J = 6.0 Hz, CHAOH), 4.43 (dd, 1H, J = 9.9 and
J = 6.3 Hz, CHBOH), 7.14–7.46 (m, 8H, Ar), 10.30 (s, 1H, NH). 13C
NMR (75 MHz, CDCl3): 62.97 (CH2OH), 64.18 (CHCH2), 123.22 (Ar),
128.58 (Ar), 128.91 (Ar), 129.17 (Ar), 129.98 (Ar), 130.72
(Ar), 131.93 (Ar), 137.45 (Ar), 138.91 (Ar), 168.72 (C@N), 171.41
(CO). Anal. Calcd for C16H13ClN2O2: C, 63.90; H, 4.36; N, 11.79.
Found: C, 64.09; H, 4.05 N, 12.13.
4.1.1.1. (R)-tert-Butyl 4-(2-benzoyl-4-chlorophenylcarbamoyl)-
2,2-dimethyloxazolidine-3-carboxylate (12). To a solution of acid
119 (3.0 g, 12.2 mmol) in 40 mL of dry CH2Cl2 at 0 °C N-methyl
morpholine (1.48 mL, 13.45 mmol) was added followed by i-butyl
chloroformate (1.75 mL, 13.45 mmol). After 30 min., a solution of
2-amino-5-chloro-benzophenone (2.83 g, 12.2 mmol) in CH2Cl2
was added dropwise to refluxing reaction mixture over 20 min.
After being stirred for 12 h at room temperature, the reaction mix-
ture was washed with dilute HCl, aqueous NaHCO3, and water. The
organic layer was dried and concentrated to a residue and, after
purification by column chromatography (petroleum ether/EtOAc
9:1) gave the product 12 (4.27 g, 76%). Rf = 0.31 (petroleum
ether/EtOAc 9:1). 1H NMR (300 MHz, CDCl3): 1.23–1.85 (m, 15H,
C(CH3)3 and C(CH3)2), 4.10–4.24 (m, 2H, CH2CH), 4.41–4.53 (m,
1H, CH2CH), 7.46–7.72 (m, 7H, Ar), 8.69 (m, 1H, Ar), 11.16 (br s,
1H, NH). 13C NMR (75 MHz, CHCl3): 24.42 (C(CH3)2), 28.51
(C(CH3)2), 62.07 (CH2CH), 67.03 (CH2CH), 76.23 (C(CH3)3), 106.31
(C(CH3)2), 123.17 (Ar), 123.27 (Ar), 125.62 (Ar), 127.96 (Ar),
128.69 (Ar), 130.21 (Ar), 132.62 (Ar), 133.18 (Ar), 133.82 (Ar),
147.91 (CO), 155.62 (CO), 171.08 (CO). Anal. Calcd for
4.1.1.5. (R)-5-(4-Bromophenyl)-3-(hydroxymethyl)-1H-benzo[e]
[1,4]diazepin-2(3H)-one (16). In analogy to 15, compound 16 was
obtained from 13 (5.46 g, 11.0 mmol) by acidic hydrolysis in meth-
anol (3.46 g, 90.4%). Rf = 0.34 (petroleum ether/EtOAc 4:6). 1H NMR
(300 MHz, CDCl3): 3.07 (br s, 1H, CH2OH), 3.78 (dd, J = 7.1 and
J = 5.5 Hz, 1H, CHCH2OH), 4.24 (dd, 1H, J = 11.5 and J = 5.5 Hz,
CHAOH), 4.42 (dd, 1H, J = 11.5 and J = 7.1 Hz, CHBOH), 7.15–7.626
(m, 8H, Ar), 9.42 (s, 1H, NH). 13C NMR (75 MHz, CDCl3): 62.97
(CH2OH), 64.18 (CHCH2OH), 121.71 (Ar), 124.02 (Ar), 125.46 (Ar),
127.39 (Ar), 131.21 (Ar), 131.62 (Ar), 132.23 (Ar), 132.29 (Ar),
138.07 (Ar), 138.31 (Ar), 168. 30 (C@N), 170.20 (CO). Anal. Calcd
for C16H13BrN2O2: C, 55.67; H, 3.80; N, 8.12. Found: C, 55.39; H,
4.07 N, 7.81.
C24H27ClN2O5: C, 68.31; H, 5.93; N, 6.10. Found: C, 67.93; H,
6.28; N, 6.33.
4.1.1.2. (R)-tert-Butyl 4-(2-(4-bromobenzoyl)phenylcarbamoyl)-
2,2-dimethyloxazolidine-3-carboxylate (13). With the same pro-
cedure employed for synthesis of 12, intermediate 13 was obtained
using 2-amino-40-bromo-benzophenone (3.34 g, 12.2 mmol).
Yield: 5.46 g (90%). Rf = 0.34 (petroleum ether/EtOAc 9:1). 1H
NMR (300 MHz, CDCl3): 1.22–1.86 (m, 15H, C(CH3)3 and C(CH3)2),
4.26–4.43 (m, 2H, CH2CH), 4.50 (m, 1H, CH2CH), 7.12 (m, 1H, Ar),
7.42–7.70 (m, 6H, Ar), 8.72 (d, 1H, J = 7.9 Hz, Ar), 11.25 (br s, 1H,
NH). 13C NMR (75 MHz, CHCl3): 22.98 (C(CH3)2), 23.98 (C(CH3)2),
28.18 (C(CH3)3), 66.73 (CH2CH), 67.00 (CH2CH), 77.37 (C(CH3)3),
108.32 (C(CH3)2), 121.41 (Ar), 122.46 (Ar), 124.33 (Ar), 128.16
(Ar), 129.95 (Ar), 131.67 (Ar), 132.35 (Ar), 133.17 (Ar), 133.88
(Ar), 151.36 (CO), 173.18 (CO), 198.51 (CO). Anal. Calcd for
4.1.1.6. (R)-3-(Hydroxymethyl)-5-(4-methoxyphenyl)-1H-ben-
zo[e] [1,4]diazepin-2(3H)-one (17). In analogy to 15, compound
17 was obtained from 14 (3.77 g, 8.3 mmol) by acidic hydrolysis
in methanol (1.82 g, 74%). Rf = 0.40 (petroleum ether/EtOAc 3:7)
1H NMR (300 MHz, CDCl3): 3.08 (br s, 1H, CH2OH), 3.78 (dd, 1H,
J = 7.1 and J = 5.5 Hz, CHCH2), 3.81 (s, 3H, OCH3), 4.24 (dd, 1H,
J = 11.5 Hz and J = 5.5 Hz, CHAOH), 4.41 (dd, 1H, J = 11.5 Hz and
J = 7.1 Hz, CHBOH), 6.86 (d, 2H, J = 8.5 Hz, Ar), 7.12–7.27 (m, 2H,
Ar), 7.34 (d, 1H, J = 7.7 Hz, Ar), 7.46–7.49 (m, 3H, Ar), 9.55 (br s,
1H, NH). 13C NMR (75 MHz, CDCl3): 55.80 (OCH3), 62.97 (CHCH2),
64.18 (CHCH2), 121.71 (Ar), 124.02 (Ar), 125.46 (Ar), 127.39 (Ar),
131.21 (Ar), 131.62 (Ar), 132.23 (Ar), 132.29 (Ar), 138.07 (Ar),
138.31 (Ar), 168.11 (C@N), 170.20 (CO). Anal. Calcd for
C24H27BrN2O5: C, 57.26; H, 5.41; N, 5.56. Found: C, 57.53; H,
C17H16N2O2: C, 68.91; H, 5.44; N, 9.45. Found: C, 69.07; H, 5.18;
5.20; N, 5.19.
N, 9.21.
4.1.1.3. (R)-tert-Butyl 4-(2-(4-methoxybenzoyl)phenyl-carbam-
oyl)-2,2-dimethyloxazolidine-3-carboxylate (14). With the same
procedure employed for synthesis of 12, intermediate 14 was ob-
tained using 2-amino-40-methoxy-benzophenone (2.76 g, 12.2
mmol). Yield: 3.77 g (63%). In this case 2-amino-40-methoxy-benzo-
phenone was synthesized according to Frye et al.20 Rf = 0.25 (petro-
leum ether/EtOAc 8:2). 1H NMR (300 MHz, CDCl3): 1.32–1.56 (m,
15H, C(CH3)3 and C(CH3)2), 3.83 (s, 3H, OCH3), 3.87–4.13 (m, 2H,
CH2CH), 4.57 (m, 1H, CH2CH), 7.10 (m, 2H, Ar), 7.29–7.76 (m, 5H,
Ar), 8.52 (d, 1H, J = 7.9 Hz, Ar), 11.25 (br s, 1H, NH). 13C NMR
(75 MHz, CDCl3): 24.98 (C(CH3)2), 28.98 (C(CH3)3), 55.96 (OCH3),
63.91 (CH2CH), 69.18 (CH2CH), 78.73 (C(CH3)3), 106.10 (C(CH3)2),
111.41 (Ar), 119.46 (Ar), 124.16 (Ar), 125.95 (Ar), 127.35 (Ar),
130.17 (Ar), 131.88 (Ar), 132.64 (Ar), 133.50 (Ar), 164.36 (CO),
172.75 (CO), 198.51 (CO). Anal. Calcd for C25H30N2O6: C, 66.06; H,
5.41; N, 5.56. Found: C, 66.37; H, 5.61; N, 5.23.
4.1.1.7. (R)-3-((tert-Butyldimethylsilyloxy)methyl)-7-chloro-5-
phenyl-1H-benzo[e][1,4]diazepin-2-(3H)-one (18). To a solution
of 15 (1.96 g, 6.5 mmol) in CH2Cl2 (40 mL) were added imidazole
(0.66 g, 9.75 mmol) and TBS-Cl (1.47 g, 9.75 mmol) at 0 °C. After
stirring 12 h at room temperature the mixture was washed with
water and dried. The crude product was purified by flash chroma-
tography to afford the title compound (petroleum ether/EtOAc 8:2)
(2.43 g, 89.8%). Rf = 0.35 (petroleum ether/EtOAc 8:2). 1H NMR
(300 MHz, CDCl3): 0.14 (s, 6H, Si(CH3)3), 0.92 (s, 9H, C(CH3)3),
3.69 (dd, 1H, J = 6.3 and J = 6.0 Hz, CHCH2), 4.25 (dd, 1H, J = 10.0
and J = 6.0 Hz, CHCHA), 4.56 (dd, 1H, J = 10.0 and J = 6.3 Hz, CHCHB),
7.14–7.46 (m, 8H, Ar), 10.30 (br s, 1H, NH). 13C NMR (75 MHz,
CDCl3): ꢀ4.83 (Si(CH3)2), 17.20 (C(CH3)3), 26.34 (C(CH3)3), 63.92
(CHCH2), 65.69 (CHCH2), 123.22 (Ar), 128.58 (Ar), 128.91 (Ar),
129.17 (Ar), 129.98 (Ar), 130.72 (Ar), 131.93 (Ar), 137.45 (Ar),
138.91 (Ar), 168.72 (C@N), 171.41 (CO). Anal. Calcd for

F. Bova et al. / Bioorg. Med. Chem. 18 (2010) 4928–4938
4933
C22H27ClN2O2Si: C, 63.67; H, 6.56; N, 8.54. Found: C, 64.01; H, 6.87;
N, 8.19.
mixture was treated with 10% citric acid and extracted with ethyl
acetate (3 ꢁ 30 mL), dried and concentrated to give the acid 21
(1.78 g, 70%). 1H NMR (300 MHz, CDCl3): 0.02 (s, 3H, Si(CH3)2),
0.03 (s, 3H, Si(CH3)2), 0.78 (s, 9H, C(CH3)3), 3.69 (dd, 1H, J = 6.3
and J = 6.0 Hz, CHCH2O), 4.31 (dd, 1H, J = 10.0 and J = 6.3 Hz,
CHCHA), 4.16–4.30 (m, 3H, CHCHB and NCH2), 7.11–7.33 (m, 7H,
Ar), 7.49 (d, 1H, J = 7.7 Hz, Ar). 13C NMR (75 MHz, CDCl3): ꢀ4.95
(Si(CH3)2), 23.22 (C(CH3)3), 26.90 (C(CH3)3), 49.54 (NCH2), 64.15
(CHCH2), 65.48 (CHCH2), 123.56 (Ar), 128.49 (Ar), 129.77 (Ar),
130.02 (Ar), 130.75 (Ar), 131.59 (Ar), 131.77 (Ar), 132.87 (Ar),
138.44 (Ar), 141.20 (Ar), 168.56 (C@N), 168.73 (CO), 170.92 (CO).
Anal. Calcd for C24H29ClN2O4Si: C, 60.94; H, 6.18; N, 5.92. Found:
C, 61.28; H, 5.87; N, 5.63.
4.1.1.8. (R)-5-(4-Bromophenyl)-3-((tert-butyldimethylsilyloxy)
methyl)-1H-benzo[e][1,4]diazepin-2-(3H)-one (19). With a simi-
lar procedure, 19 was prepared from 16 (3.46 g, 10.0 mmol), imid-
azole (1.0 g, 14.8 mmol), and TBS-Cl (2.26 g, 14.8 mmol). Yield:
3.92 g (84.7%). Rf = 0.32 (petroleum ether/EtOAc 8:2). 1H NMR
(300 MHz, CDCl3): 0.09 (s, 3H, Si(CH3)2), 0.10 (s, 3H, Si(CH3)2),
0.87 (s, 9H, C(CH3)3), 3.64 (dd, 1H, J = 6.3 and J = 6.0 Hz, CHCH2),
4.20 (dd, 1H, J = 10.1 and J = 6.3 Hz, CHCH2), 4.52 (dd, 1H, J = 10.1
and J = 6.0 Hz, CHCH2), 7.03 (t, 1H, J = 7.4 Hz, Ar), 7.17 (d, 2H,
J = 7.9 Hz, Ar), 7.29 (d, 2H, J = 8.5 Hz, Ar), 7.35–7.42 (m, 3H, Ar),
10.26 (br s, 1H, NH). 13C NMR (75 MHz, CDCl3): ꢀ4.83 (Si(CH3)3),
26.28 (C(CH3)3), 43.34 (C(CH3)3), 63.99 (CHCH2), 65.65 (CHCH2),
121.95 (Ar), 123.55 (Ar), 125.17 (Ar), 127.39 (Ar), 131.01 (Ar),
131.61 (Ar), 132.0 (Ar), 138.37 (Ar), 138.90 (Ar), 168.88 (Ar),
171.50 (CO). Anal. Calcd for C22H27BrN2O2Si: C, 57.51; H, 5.92; N,
6.10. Found: C, 57.90; H, 5.61; N, 6.44.
4.1.1.11. (R)-2-(5-(4-Bromophenyl)-3-((tert-butyldimethyl-silyl-
oxy)methyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)
acetic acid (22). Compound 22 was obtained from 19 (3.92 g,
8.5 mmol) employing the procedure described for compound 21.
The ethyl ester intermediate was obtained in 90.1% yield (4.16 g),
after purification by flash chromatography (petroleum ether/EtOAc
8:2). Rf = 0.54 (petroleum ether/EtOAc 8:2). 1H NMR (300 MHz,
CDCl3): 0.00 (s, 3H, Si(CH3)2), 0.02 (s, 3H, Si(CH3)2), 0.78 (s, 9H,
C(CH3)3), 1.03 (t, 3H, J = 6.8 Hz, OCH2CH3), 3.70 (dd, 1H, J = 6.3
and J = 5.8 Hz, CHCH2), 3.90–4.08 (m, 3H, OCH2CH3 and CHCHA),
4.10–4.21 (m, 1H, CHCHB), 4.36–4.52 (m, 3H, NCH2), 7.05–7.42
(m, 8H, Ar). 13C NMR (75 MHz, CDCl3): ꢀ4.90 (Si(CH3)2), 14.33
(OCH2CH3), 21.73 (C(CH3)3), 25.57 (C(CH3)3), 49.66 (NCH2), 61.67
(OCH2CH3), 64.19 (CHCH2), 65.46 (CHCH2), 122.06 (Ar), 124.82
(Ar), 125.16 (Ar), 128.52 (Ar), 129.79 (Ar), 130.22 (Ar), 131.46
(Ar), 131.58 (Ar), 131.99 (Ar), 137.93 (Ar) 142.61 (Ar), 168.28
(C@N), 168.99 (CO), 170.92 (CO). Compound 22 was obtained in
65.6% yield (2.61 g,). 1H NMR (300 MHz, CDCl3): 0.05 (s, 3H,
Si(CH3)2), 0.03 (s, 3H, Si(CH3)2), 0.78 (s, 9H, C(CH3)3), 3.70 (dd,
1H, J = 6.3 and J = 5.8 Hz, CHCH2), 4.16–4.22 (m, 1H, CHCHA),
4.38–4.54 (m, 3H, CHCHB and NCH2), 6.70 (br s, 1H, COOH), 7.09–
7.46 (m, 8H, Ar). 13C NMR (75 MHz, CDCl3): ꢀ4.95 (Si(CH3)2),
23.22 (C(CH3)3), 26.90 (C(CH3)3), 49.54 (NCH2), 64.15 (CHCH2),
65.48 (CHCH2), 123.56 (Ar), 128.49 (Ar), 129.77 (Ar), 130.02 (Ar),
130.75 (Ar), 131.59 (Ar), 131.77 (Ar), 132.87 (Ar), 138.44 (Ar),
141.20 (Ar), 168.56 (C@N), 168.73 (CO), 170.92 (CO). Anal. Calcd
for C24H29BrN2O4Si: C, 55.70; H, 5.65; N, 5.41. Found: C, 55.45; H,
5.99; N, 5.06.
4.1.1.9. (R)-3-((tert-Butyldimethylsilyloxy)methyl)-5-(4-meth-
oxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one (20). With a sim-
ilar procedure, 20 was prepared from 17 (1.82 g, 6.14 mmol),
imidazole (0.62 g, 9.2 mmol), and TBS-Cl (1.39 g, 9.2 mmol). Yield:
1.97 g (78.5%). Rf = 0.75 (petroleum ether/EtOAc 4:6). 1H NMR
(300 MHz, CDCl3): 0.09 (s, 3H, Si(CH3)2), 0.10 (s, 3H, Si(CH3)2), 0.87
(s, 9H, C(CH3)3), 3.10 (s, 3H, OCH3), 3.66 (dd, 1H, J = 6.3 and
J = 6.0 Hz, CHCH2), 4.24 (dd, 1H, J = 10.1 and J = 6.3 Hz, CHCH2),
4.41 (dd, 1H, J = 10.1 and J = 6.0 Hz, CHCH2), 6.86 (d, 2H, J = 8.5 Hz,
Ar), 7.12–7.27 (m, 2H, Ar), 7.34 (d, 1H, J = 7.7 Hz, Ar), 7.46–7.49
(m, 3H, Ar), 9.55 (br s, 1H, NH). 13C NMR (75 MHz, CDCl3): ꢀ4.83
(Si(CH3)3), 26.28 (C(CH3)3), 43.34 (C(CH3)3), 62.97 (CHCH2), 64.18
(CHCH2), 121.71 (Ar), 124.02 (Ar), 125.46 (Ar), 127.39 (Ar), 131.21
(Ar), 131.62 (Ar), 132.23 (Ar), 138.07 (Ar), 138.31 (Ar), 170.23
(CO). Anal. Calcd for C23H30N2O3Si: C, 67.28; H, 7.36; N, 6.82. Found:
C, 67.50; H, 7.59; N, 6.51.
4.1.1.10. (R)-2-(3-((tert-Butyldimethylsilyloxy)methyl)-7-chloro-
2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)acetic
acid (21). To a suspension of NaH (153 mg, 6.38 mmol) in dry DMF
(10 mL) was added a solution of 18 (2.43 g, 5.85 mmol) in DMF
(10 mL) at 0 °C under nitrogen and then the reaction mixture
was slowly brought to room temperature. After stirring 30 min.
at room temperature, ethyl bromoacetate (0.8 mL, 7.3 mmol) was
added via syringe and further stirred for 5 h. The reaction mixture
was quenched with satd aqueous NH4Cl (30 mL) and extracted
with ethyl acetate (2 ꢁ 50 mL). The combined organic layers were
washed with water, dried, concentrated, and the resulting residue
was purified on silica gel column chromatography (petroleum
ether/EtOAc 8:2) to obtain the ester derivative as a yellowish oil
(2.64 g, 90.3%). Rf = 0.51 (petroleum ether/EtOAc 8:2). 1H NMR
(300 MHz, CDCl3): 0.02 (s, 3H, Si(CH3)2), 0.03 (s, 3H, Si(CH3)2),
0.78 (s, 9H, C(CH3)3), 1.11 (t, 3H, J = 6.8 Hz, OCH2CH3), 3.69 (dd,
1H, J = 6.3 J = 6.0 Hz, CHCH2), 3.89–4.06 (m, 3H, OCH2CH3 and
CHCHA), 4.16–4.21 (m, 1H, CHCHB), 4.31–4.48 (m, 3H, NCH2),
7.11–7.33 (m, 7H, Ar), 7.49 (d, 1H, J = 7.7 Hz, Ar). 13C NMR
(75 MHz, CDCl3): ꢀ4.95 (Si(CH3)2), 14.22 (OCH2CH3), 23.22
(C(CH3)3), 26.90 (C(CH3)3), 49.54 (NCH2), 61.65 (OCH2CH3), 64.15
(CHCH2), 65.48 (CHCH2), 123.56 (Ar), 128.49 (Ar), 129.77 (Ar),
130.02 (Ar), 130.75 (Ar), 131.59 (Ar), 131.77 (Ar), 132.87 (Ar),
138.44 (Ar), 141.20 (Ar), 168.56 (C@N), 168.73 (CO), 170.92 (CO).
To a solution of the ester (2.64 g, 5.3 mmol) in a mixture of meth-
anol/water 1:1 (20 mL) was added LiOH (251 mg, 10.6 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
4.1.1.12. (R)-2-(3-((tert-butyldimethylsilyloxy)methyl)-5-(4-met
hoxyphenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)
acetic acid (23). Compound 23 was obtained from 20 (1.97 g,
4.8 mmol) employing the procedure described for compound 21.
The ethyl ester intermediate was obtained in 97.1% yield (2.24 g),
after purification by flash chromatography (petroleum ether/EtOAc
7:3). Rf = 0.63 (petroleum ether/EtOAc 7:3). 1H NMR (300 MHz,
CDCl3): 0.07 (s, 3H, Si(CH3)2), 0.08 (s, 3H, Si(CH3)2), 0.84 (s, 9H,
C(CH3)3), 1.10 (t, 3H, J = 7.1 Hz, OCH2CH3), 3.76 (s, 3H, OCH3),
3.73–3.87 (m, 1H, CHCH2), 4.01–4.26 (m, 2H, OCH2CH3 and
NCH2), 4.42–4.61 (m, 2H, CHCH2). 6.82 (d, 2H, J = 8.8 Hz, Ar), 7.14
(t, 1 H, J = 7.1 Hz, Ar), 7.23–7.28 (m, 2H, Ar), 7.46 (t, 1 H,
J = 7.1 Hz, Ar), 7.53 (d, 2H, J = 8.8 Hz, Ar). 13C NMR (75 MHz, CDCl3):
ꢀ4.90 (Si(CH3)2), 14.33 (OCH2CH3), 21.73 (C(CH3)3), 25.57
(C(CH3)3), 49.66 (NCH2), 55.81 (OCH3), 61.67 (OCH2CH3), 64.19
(CHCH2), 65.46 (CHCH2), 122.06 (Ar), 124.82 (Ar), 125.44 (Ar),
128.52 (Ar), 129.79 (Ar), 130.22 (Ar), 131.46 (Ar), 131.58 (Ar),
132.99 (Ar), 137.93 (Ar) 142.61 (Ar), 168.28 (C@N), 168.99 (CO),
170.92 (CO). Compound 23 was obtained in 84% yield (1.75 g,).
1H NMR (300 MHz, D2O): ꢀ0.03 (s, 3H, Si(CH3)2), 0.00 (s, 3H,
Si(CH3)2), 0.76 (s, 9H, C(CH3)3), 3.68 (s, 3H, OCH3), 3.72 (dd, 1H,
J = 6.3 and J = 5.8 Hz, CHCH2), 4.16–4.22 (m, 1H, CHCHA), 4.24–
4.46 (m, 3H, CHCHB and NCH2), 7.13–7.53 (m, 8H, Ar). 13C NMR

4934
F. Bova et al. / Bioorg. Med. Chem. 18 (2010) 4928–4938
(75 MHz, D2O): ꢀ4.97 (Si(CH3)2), 23.09 (C(CH3)3), 26.82 (C(CH3)3),
50.71 (NCH2), 55.54 (OCH3), 64.15 (CHCH2), 65.79 (CHCH2),
120.61 (Ar), 124.54 (Ar), 129.76 (Ar), 130.05 (Ar), 130.90 (Ar),
131.62 (Ar), 131.81 (Ar), 131.94 (Ar), 138.44 (Ar), 148.52 (Ar),
168.56 (C@N), 168.73 (CO), 170.92 (CO). Anal. Calcd for
and CHCHA), 4.41–4.63 (m, 2H, CHCHB and NCHB), 4.85–4.95 (m,
2H, CH@CH2), 5.51–5.61 (m, 1H, CH@CH2), 6.38 (br t, 1H, NH),
6.78 (d, 2H, J = 8.8 Hz, Ar), 7.15–7.52 (m, 6H, Ar). 13C NMR
(75 MHz, CDCl3): ꢀ5.23 (Si(CH3)2), 25.92 (C(CH3)3), 30.61
(C(CH3)3), 43.61 (NHCH2), 55.88 (OCH3), 56.10 (NCH2), 65.71
(CHCH2), 67.32 (CHCH2), 117.41 (CH@CH2), 121.00 (Ar), 123.20
(Ar), 124.70 (Ar), 128.22 (Ar), 129.64 (Ar), 130.20 (Ar), 131.20
(Ar), 131.46 (Ar), 132.74 (Ar), 134.26 (CH@CH2), 137.58 (Ar),
168.42 (N@C), 170.09 (CO), 171.20 (CO). Anal. Calcd for
C25H32N2O5Si: C, 64.08; H, 6.88; N, 5.98. Found: C, 64.40; H, 6.57;
N, 6.20.
4.1.1.13. (R)-N-Allyl-2-(3-((tert-butyldimethylsilyloxy)methyl)-
7-chloro-2-oxo-5-phenyl-2,3-dihydro-1H -benzo[e][1,4]diaze-
pin-1-yl)acetamide (27). To a solution 21 (300 mg, 0.63 mmol) in
CH2Cl2 (20 mL) at 0 °C was added HATU (361 mg, 0.95 mmol). After
5 min, the ice bath was removed and a solution of amine 25
(72 mg, 1.26 mmol) in CH2Cl2 (5 mL) was added. The resulting mix-
ture was stirred at room temperature for 12 h and washed with
water (2 ꢁ 50 mL). The organic layer was separated, dried
(Na2SO4), filtered and concentrated under reduced pressure. The
residue was purified by flash chromatography eluting with petro-
leum ether/EtOAc (5:5) to give title compound 27 (300 mg,
92.4%) as a yellow oil. Rf = 0.31 (petroleum ether/EtOAc 5:5). 1H
NMR (300 MHz, CDCl3): 0.02 (s, 3H, Si(CH3)2), 0.04 (s, 3H, Si(CH3)2),
0.80 (s, 9H, C(CH3)3), 3.67–3.72 (m, 3H, NHCH2 and CHCH2), 4.07 (d,
1H, J = 15.4 Hz, NCHA), 4.16 (dd, 1H, J = 10.2 and 6.3 Hz, CHCHA),
4.47 (dd, 1H, J = 10.2 and 6.3 Hz, CHCHB), 4.54 (d, 1H, J = 15.4 Hz,
NCHB), 4.91–4.95 (m, 2H, CH@CH2), 5.56–5.65 (m, 1H, CH@CH2),
6.38 (br t, 1H, NH), 7.15–7.60 (m, 8H, Ar). 13C NMR (75 MHz,
CDCl3): ꢀ5.75 (Si(CH3)2), 25.84 (C(CH3)3), 30.62 (C(CH3)3), 41.76
(NHCH2), 54.06 (NCH2), 62.97 (CHCH2), 68.19 (CHCH2), 116.24
(CH@CH2), 121.77 (Ar), 124.59 (Ar), 127.32 (Ar), 128.93 (Ar),
130.50 (Ar), 131.02 (Ar), 131.17 (Ar), 133.24 (CH@CH2), 138.06
(Ar), 168.34 (C@N), 171.17 (CO), 171.21 (CO). Anal. Calcd for
C
28H37N3O4Si: C, 66.24; H, 7.35; N, 8.28. Found: C, 66.00; H, 7.66;
N, 7.93.
4.1.1.16. 2-((R)-3-((tert-Butyldimethylsilyloxy)methyl)-2-oxo-5-
phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)-N-(5-meth-
ylhex-1-en-3-yl)acetamide (30). Compound 30 was obtained from
24 (200 mg, 0.60 mmol) and amine 26 (0.14 g, 1.2 mmol) employ-
ing the procedure described for compound 27. The residue was
purified by flash chromatography, eluting with petroleum ether/
EtOAc (8:2), to afford compound 30 (0.31 g, 80%). Rf = 0.35 (petro-
leum ether/EtOAc 5:5). 1H NMR (300 MHz, CDCl3): 0.07 (s, 3H,
Si(CH3)2), 0.08 (s, 3H, Si(CH3)2), 0.73–0.82 (m, 6H, CH(CH3)2), 0.89
(s, 9H, C(CH3)3), 1.27–1.52 (m, 3H, CH2CH and CH(CH3)2), 4.17–
4.30 (m, 3H, CHCHA, CHCH2 and NHCH), 4.38–4.43 (m, 2H, CHCHB
and NCHA), 4.57 (d, 1H, J = 15.9 Hz, NCHB), 4.85–5.22 (m, 2H,
CH@CH2), 5.51–5.60 (m, 1H, CH@CH2), 6.55 (br s, 1H, NH), 7.11–
7.51 (m, 9H, Ar). 13C NMR (75 MHz, CDCl3): ꢀ5.23 (Si(CH3)2),
20.70 (CH(CH3)2), 21.15 (C(CH3)3), 24.83 (CH(CH3)2), 25.99
(CH(CH3)2), 31.45 (CHCH2), 46.80 (CHCH2), 57.30 (NCH2), 62.34
(CHCH2), 63.87 (CHCH2), 114.90 (CH@CH2), 122.68 (Ar), 125.15
(Ar), 128.55 (Ar), 129.65 (Ar), 129.94 (Ar), 130.45 (Ar), 130.97
(Ar), 132.36 (CH@CH2), 138.44 (Ar), 138.56 (Ar), 142.34 (Ar),
167.82 (C@N), 170.15 (CO), 171.39 (CO). Anal. Calcd for
C27H34ClN3O3Si: C, 63.62; H, 6.69; N, 8.20. Found: C, 63.40; H,
7.01; N, 8.03.
C31H43N3O3Si: C, 69.75; H, 8.12; N, 7.87. Found: C, 69.99; H, 7.92;
N, 7.54.
4.1.1.14. (R)-N-Allyl-2-(5-(4-bromophenyl)-3-((tert-butyldimeth
ylsilyloxy)methyl)-2-oxo-2,3-dihydro-1H -benzo[e][1,4]diazepin-
1-yl)acetamide (28). Compound 28 was obtained from 22 (250 mg,
0.48 mmol) and amine 25 (55 mg, 0.96 mmol) employing the
procedure described for compound 27 The residue was purified by
flash chromatography, eluting with petroleum ether/EtOAc (5:5),
to afford compound 28 (240 mg, 89.3%). Rf = 0.33 (petroleum
ether/EtOAc 5:5). 1H NMR (300 MHz, CDCl3): 0.00 (s, 3H, Si(CH3)2),
0.02 (s, 3H, Si(CH3)2), 0.78 (s, 9H, C(CH3)3), 3.60–3.69 (m, 3H, NHCH2
and CHCH2), 4.13 (d, 1H, J = 15.1 Hz, NCHA), 4.10–4.15 (m, 1H,
CHCHA), 4.46 (dd, 1H, J = 10.1 and 7.4 Hz, CHCHB), 4.55 (d, 1H, J =
15.3 Hz, NCHB), 4.89–4.98 (m, 2H, CH@CH2), 5.52–5.63 (m, 1H,
CH@CH2), 6.38 (br t, 1H, NH), 7.09–7.18 (m, 2H, Ar), 7.34–7.57
(m, 6H, Ar). 13C NMR (75 MHz, CDCl3): ꢀ6.31 (Si(CH3)2), 26.22
(C(CH3)3), 28.70 (C(CH3)3), 42.21 (NHCH2), 64.05 (NCH2), 65.71
(CHCH2), 52.72 (CHCH2), 116.77 (CH@CH2), 123.00 (Ar), 125.19
(Ar), 125.53 (Ar), 1289.19 (Ar), 130.06 (Ar), 130.20 (Ar), 131.46
(Ar), 131.76 (Ar), 132.43 (Ar), 133.71 (CH@CH2), 137.58 (Ar),
167.42 (C@N), 170.09 (CO), 171.20 (CO). Anal. Calcd for C27H34
BrN3O3Si: C, 58.27; H, 6.16; N, 7.55. Found: C, 57.60; H, 6.47;
N, 7.86.
4.1.1.17. (R)-(1-(2-Allylamino)-2-oxoethyl)-7-chloro-2-oxo-5-
phenyl-2,3-dihydro-1H -benzo[e][1,4]diazepin-3-yl)methyl 4-
chloro-2-(trifluoromethyl)phenylcarbamate (31). To a solution
of 27 (300 mg, 0.58 mmol) in dry THF (10 mL) was added TBAF
(1 M solution in THF, 0.87 mL, 0.87 mmol) dropwise. The mixture
was stirred at room temperature until disappearance of the start-
ing material (TLC monitoring), and then it was diluted with EtOAc
(20 mL) and washed with water (3 ꢁ 20 mL). The organic layer was
separated, dried (Na2SO4), filtered, and concentrated under re-
duced pressure to give the deprotected compound, which was
purified by flash chromatography eluting with petroleum ether/
EtOAc 2:8 (150 mg, 64.4%). 1H NMR (300 MHz, CDCl3): 3.95–3.99
(m, 3H, NHCH2 and CHCH2), 4.34–4.39 (m, 2H, NCHA and CHCHA),
4.51 (m, 1H, CHCHB), 4.72 (d, 1H, J = 15.4 Hz, NCHB), 5.18–5.33
(m, 2H, CH@CH2), 5.88 (m, 1H, CH@CH2), 6.62 (br t, 1H, NH),
7.40–7.96 (m, 8H, Ar). To a solution of N-allyl-2-(7-chloro-3-
hydroxymethyl-2-oxo-5-phenyl-2,3-dihydro-benzo[e][1,4]diaze-
pin-1-yl-acetamide (50 mg, 0.12 mmol) in dry CH2Cl2 (5 mL) under
N2 was added 4-Cl-2-CF3C6H3NCO (56 mg, 37 lL, 0.25 mmol) and
the resulting mixture was stirred for 12 h at room temperature.
After this time, the mixture was washed with water (2 ꢁ 10 mL).
The residue was purified by flash chromatography, eluting with
petroleum ether/EtOAc 6:4, to afford compound 31 (55 mg, 71%).
Rf = 0.40 (petroleum ether/EtOAc 4:6). 1H NMR (300 MHz, CDCl3):
3.84 (t, 2H, J = 5.7 Hz, NHCH2), 4.04 (t, 1H, J = 6.5 Hz, CHCH2), 4.24
(d, 1H, J = 15.1 Hz, NCHA), 4.61 (d, 1H, J = 15.1 Hz, NCHB), 4.89–
5.13 (m, 4H, CHCH2 and CH@CH2), 5.72 (m, 1H, CH@CH2), 6.37
(br t, 1H, NH), 6.96 (s, 1H, NH), 7.26–7.62 (m, 9H, Ar), 7.75 (d,
1H, J = 8.8 Hz,), 8.11 (d, 1H, J = 9.1 Hz). 13C NMR (75 MHz, CDCl3):
41.75 (NHCH2), 54.03 (NCH2), 60.38 (CHCH2), 65.86 (CHCH2),
114.62 (Ar), 116.25 (CH@CH2), 121.75 (Ar), 121.98 (Ar), 123.35
4.1.1.15. (R)-N-Allyl-2-(3-((tert-butyldimethylsilyloxy)methyl)-
5-(4-methoxyphenyl)-2-oxo-2,3-dihydro-1H -benzo[e][1,4]dia-
zepin-1-yl)acetamide (29). Compound 29 was obtained from 23
(200 mg, 0.42 mmol) and amine 25 (48 mg, 0.86 mmol) employing
the procedure described for compound 27 The residue was purified
by flash chromatography, eluting with petroleum ether/EtOAc
(4:6), to afford compound 29 (212 mg, 97.8%). Rf = 0.53 (petroleum
ether/EtOAc 4:6). 1H NMR (300 MHz, CDCl3): 0.01 (s, 3H, Si(CH3)2),
0.03 (s, 3H, Si(CH3)2), 0.79 (s, 9H, C(CH3)3), 3.68–3.72 (m, 3H,
NHCH2 and CHCH2), 3.73 (s, 3H, OCH3), 4.09–4.20 (m, 2H, NCHA

F. Bova et al. / Bioorg. Med. Chem. 18 (2010) 4928–4938
4935
(Ar), 124.58 (CF3), 125.25 (Ar), 127.34 (Ar), 128.97 (Ar), 129.08 (Ar),
129.24 (Ar), 129.46 (Ar), 130.27 (Ar), 131.17 (Ar), 131.32 (Ar),
132.45 (Ar), 134.38 (CH@CH2), 139.04 (Ar), 140.75 (Ar), 153.97
(CO), 168.67 (C@N), 171.12 (CO), 171.24 (CO). Anal. Calcd for
4.1.1.20. (R)-(1-(2-(5-methylhex-1-en-3-ylamino)-2-oxoethyl)-2
-oxo-5-phenyl-2,3-dihydro-1H -benzo[e][1,4]diazepin-3-yl)met-
hyl 4-chloro-2-(trifluoromethyl)phenylcarbamate (34). Com-
pound 34 was obtained from 30 (0.31 g, 0.58 mmol) employing
the procedure described for compound 31. The intermediate 2-
(3-hydroxymethyl-2-oxo-5-phenyl-2,3-dihydro-benzo[e][1,4]dia-
zepin-1-yl)-N-3-methyl-1-vinyl-butyl)-acetamide was obtained in
63% yield (155 mg), after purification by flash chromatography
(petroleum ether/EtOAc 2:8). 1H NMR (300 MHz, CDCl3): 0.74 (d,
3H, J = 6.0 Hz, CH(CH3)2), 0.76 (d, 3H, J = 6.0 Hz, CH(CH3)2), 1.27–
1.52 (m, 3H, CH2CH and CH(CH3)2), 3.94–4.07 (m, 3H, CHAOH,
CHCH2OH and NHCH), 4.30–4.35 (m, 2H, CHBOH and NCHA), 4.57
(d, 1H, J = 15.9 Hz, NCHB), 4.85–5.22 (m, 2H, CH@CH2), 5.51–5.60
(m, 1H, CH@CH2), 6.55 (d, 1H, J = 8.5 Hz, NH), 7.11–7.51 (m, 9H,
Ar). Title compound 34 was purified by flash chromatography,
eluting with petroleum ether/EtOAc (5:5) (150 mg, 63.4%).
Rf = 0.31 (petroleum ether/EtOAc 6:4). 1H NMR (300 MHz, CDCl3):
0.85 (d, 3H, J = 6.3 Hz, CH(CH3)2), 0.87 (d, 3H, J = 6.0 Hz, CH(CH3)2),
1.46–1.62 (m, 3H, CH2CH and CH(CH3)2), 4.07 (dd, 1H, J = 6.6 and
J = 6.0 Hz, CHCH2O), 4.30 (d, 1H, J = 15.1 Hz, NCHA), 4.41–4.58 (m,
1H, NHCH), 4.71 (d, 1H, J = 15.1 Hz, NCHB), 4.85–5.17 (m, 4H,
CHCH2O and CH@CH2), 5.66–5.77 (m, 1H, CH@CH2), 6.36 (d, 1H,
J = 8.5 Hz, NHCH), 7.0 (br s, 1H, NHCO), 7.27–7.64 (m, 11H, Ar),
8.15 (d, 1H, J = 9.3 Hz, Ar). 13C NMR (75 MHz, CDCl3): 22.90
(CH(CH3)2), 23.45 (CH(CH3)2), 25.01 (CH(CH3)2), 43.65 (CHCH2),
51.98 (CHCH2), 53.34 (NCH2), 62.38 (CHCH2O), 65.80 (CHCH2O),
111.13 (Ar), 115.36 (Ar), 116.61 (CH@CH2), 120.77 (Ar), 123.27
(Ar), 125.57 (CF3), 126.70 (Ar), 128.92 (Ar), 130.20 (Ar), 131.28
(Ar), 132.35 (Ar), 132.65 (CH@CH2), 133.29 (Ar), 134.60 (Ar),
138.48 (Ar), 139.0 (Ar), 148.28 (Ar), 152.57 (CO), 161.75 (C@N),
168.10 (CO), 170.15 (CO). Anal. Calcd for C33H32ClF3N4O4: C,
61.83; H, 5.03; N, 8.74. Found: C, 61.63; H, 4.89; N, 9.02.
C29H23Cl2F3N4O4: C, 56.25; H, 3.74; N, 9.04. Found: C, 56.03; H,
4.04; N, 8.71.
4.1.1.18. (R)-(1-(2-Allylamino)-2-oxoethyl)-5-(4-bromophenyl)-
2-oxo-2,3-dihydro-1H -benzo[e][1,4]diazepin-3-yl)methyl 4-
chloro-2-(trifluoromethyl)phenylcarbamate (32). Compound 32
was obtained from 28 (250 mg, 0.45 mmol) employing the proce-
dure described for compound 31. Intermediate N-allyl-2-(5-(4-bro-
mo-phenyl)-3-hydroxymethyl-2-oxo-2,3-dihydro-benzo[e][1,4]
diazepin-1-yl-acetamide was obtained in 90.6% yield (180 mg),
after purification by flash chromatography (petroleum ether/EtOAc
2:8). 1H NMR (300 MHz, CDCl3): 3.72–3.93 (m, 3H, NHCH2 and
CHCH2), 4.18–4.37 (m, 3H, NCHA and CHCH2), 4.57 (d, 1H,
J = 15.6 Hz, NCHB), 5.00–5.09 (m, 2H, CH@CH2), 5.70 (m, 1H,
CH@CH2), 6.45 (br t, 1H, NH), 7.19–7.64 (m, 8H, Ar). Title com-
pound 32 was purified by flash chromatography, eluting with
petroleum ether/EtOAc (4:6) (50 mg, 72.5%). Rf = 0.42 (petroleum
ether/EtOAc 4:6). 1H NMR (300 MHz, CDCl3): 3.70–3.84 (m, 2H,
NHCH2), 4.03 (dd, 1H, J = 6.3 and J = 5.8 Hz, CHCH2), 4.31 (d, 1H,
J = 15.4 Hz, NCHA), 4.64 (d, 1H, J = 15.4 Hz, NCHB), 4.87–5.12 (m,
4H, CHCH2 and CH@CH2), 5.73 (m, 1H, CH@CH2), 6.38 (br t, 1H,
NH), 6.94 (s, 1H, NH), 7.27–7.63 (m, 9H, Ar), 7.74 (d, 1H,
J = 8.0 Hz, Ar), 8.09 (d, 1H, J = 8.8 Hz, Ar). 13C NMR (75 MHz, CDCl3):
41.25 (NHCH2), 54.02 (NCH2), 60.35 (CHCH2), 64.86 (CHCH2),
114.60 (Ar), 116.15 (CH@CH2), 121.62 (Ar), 121.99 (Ar), 123.32
(Ar), 124.59 (CF3), 125.27 (Ar), 127.31 (Ar), 127.93 (Ar), 129.12
(Ar), 129.27 (Ar), 129.49 (Ar), 131.27 (Ar), 131.33 (Ar), 131.35
(Ar), 132.45 (Ar), 134.39 (CH@CH2), 139.10 (Ar), 140.73 (Ar),
154.97 (CO), 168.68 (C@N), 171.21 (CO), 171.24 (CO). Anal. Calcd
for C29H23BrClF3N4O4: C, 52.47; H, 3.49; N, 8.44. Found: C, 52.77;
H, 3.80; N, 8.75.
4.1.1.21. Methyl 4-(2-((R)-3-((4-chloro-2-(trifluoromethyl)phe-
nylcarbamoyloxy)methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e]
[1,4]diazepin-1-yl)acetamido)-6-methylhept-2-enoate (2). To a
solution of 34 (41 mg, 0.063 mmol) in dry CH2Cl2 (5 mL) was added
methyl acrylate (55 mg, 0.64 mmol) followed by Hoveyda–Grubbs
2nd generation catalyst [(1,3-bis-(2,4,6-trimethylphenyl)-2-imi-
dazolidinylidene)-dichloro-(o-isopropoxyphenylmethylene)ruthe-
nium] (4.5 mg, 0.0072 mmol). The resulting mixture was heated
under microwave irradiation at 100 °C for 2 h. The solvent was
then removed under reduced pressure and the residue was purified
by preparative HPLC to give the title compound as a white solid
(34 mg, 76.1%). HPLC: retention time 8.76 min. Rf = 0.24 (petro-
leum ether/EtOAc 6:4). 1H NMR (300 MHz, CDCl3): 0.81 (d, 3H,
J = 6.3 Hz, CH(CH3)2), 0.84 (d, 3H, J = 6.0 Hz, CH(CH3)2), 1.33–1.59
(m, 3H, CH2CH and CH(CH3)2), 3.65 (s, 3H, OCH3), 4.04 (dd, 1H,
J = 6.6 and J = 6.0 Hz, CHCH2O), 4.25 (d, 1H, J = 15.1 Hz, NCHA),
4.58–4.68 (m, 2H, NCHB and NHCH), 4.94 (dd, 1H, J = 11.5 and
J = 6.6 Hz, CHCHAO), 5.08 (dd, 1H, J = 11.5 and J = 6.0 Hz, CHCHBO),
5.88 (d, 1H, J = 15.6 Hz, CH@CHCO), 6.54 (d, 1H, J = 8.2 Hz, NHCH),
6.80 (dd, 1H, J = 15.6 and 5.2 Hz, CH@CHCO), 7.20 (br s, 1H, NHCO),
7.37–7.63 (m, 10H, Ar), 7.77 (d, 1H, J = 8.2 Hz, Ar), 8.03 (d, 1H,
J = 8.2 Hz, Ar). 13C NMR (75 MHz, CDCl3): 22.78 (CH(CH3)2), 23.41
(CH(CH3)2), 24.89 (CH(CH3)2), 43.55 (CHCH2), 48.95 (OCH3), 51.89
(CHCH2), 53.29 (NCH2), 62.26 (CHCH2O), 65.70 (CHCH2O), 120.67
(Ar), 123.07 (Ar), 125.50 (Ar), 126.54 (Ar), 128.66 (Ar), 129.48
(CF3), 129.59 (Ar), 130.10 (Ar), 130.73 (Ar), 131.18 (Ar), 132.59
(Ar), 133.17 (Ar), 134.49 (Ar), 138.36 (Ar), 142.41 (Ar), 148.13
(CH@CHCO), 152.43 (CO), 153.57 (Ar), 161.62 (Ar), 167.95
(CH@CHCO), 166.52 (CO), 167.98 (C@N), 170.18 (CO). 170.76
(CO). Anal. Calcd for C35H34ClF3N4O6: C, 60.13; H, 4.90; N, 8.01.
Found: C, 60.33; H, 4.77; N, 7.90.
4.1.1.19. (R)-(1-(2-Allylamino)-2-oxoethyl)-5-(4-methoxyphen
yl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)methyl 4-
chloro-2-(trifluoromethyl)phenylcarbamate (33). Compound 33
was obtained from 29 (106 mg, 0.21 mmol) employing the proce-
dure described for compound 31. Intermediate N-allyl-2-(3-
hydroxymethyl-5-(4-methoxy-phenyl)-2-oxo-2,3-dihydro-benzo[e]
[1,4]diazepin-1-yl-acetamide was obtained in 61% yield (50 mg),
after purification by flash chromatography (petroleum ether/EtOAc
1:9). 1H NMR (300 MHz, CDCl3): 3.68–3.75 (m, 3H, NHCH2 and
CHCH2), 3.77 (s, 3H, OCH3), 4.12–4.28 (m, 3H, NCHA and CHCH2),
4.53 (d, 1H, J = 15.4 Hz, NCHB), 4.94–5.02 (m, 2H, CH@CH2), 5.63
(m, 1H, CH@CH2), 6.37 (br t, 1H, NH), 6.82 (d, 2H, J = 8.5 Hz, Ar),
7.15–7.59 (m, 6H, Ar). Title compound 33 was purified by flash
chromatography, eluting with petroleum ether/EtOAc (5:5)
(40 mg, 51.2%). Rf = 0.36 (petroleum ether/EtOAc 4:6). 1H NMR
(300 MHz, CDCl3): 3.86–3.92 (m 2H, NHCH2), 3.89 (s, 3H, OCH3),
4.05 (dd, 1H, J = 6.3 and J = 5.8 Hz, CHCH2), 4.38 (d, 1H,
J = 15.4 Hz, NCHA), 4.85 (d, 1H, J = 15.4 Hz, NCHB), 4.90–5.13 (m,
4H, CHCH2 and CH@CH2), 5.73 (m, 1H, CH@CH2), 6.41 (br t, 1H,
NH), 6.93–6.99 (m, 3H, NH and Ar), 7.30–7.66 (m, 6H, Ar), 7.76
(d, 1H, J = 8.2 Hz, Ar), 8.13 (d, 1H, J = 8.6 Hz, Ar). 13C NMR
(75 MHz, CDCl3): 41.28 (NHCH2), 55.12 (NCH2), 55.82 (OCH3),
60.25 (CHCH2), 64.73 (CHCH2), 115.60 (Ar), 116.35 (CH@CH2),
122.62 (Ar), 123.97 (Ar), 124.31 (Ar), 124.69 (CF3), 125.33 (Ar),
126.44 (Ar), 127.88 (Ar), 129.34 (Ar), 129.46 (Ar), 129.51 (Ar),
131.27 (Ar), 131.33 (Ar), 131.38 (Ar), 132.56 (Ar), 134.44
(CH@CH2), 139.17 (Ar), 142.38 (Ar), 155.12 (CO), 168.34 (C@N),
171.22 (CO), 171.25 (CO). Anal. Calcd for C30H26ClF3N4O5: C,
58.59; H, 4.26; N, 9.11. Found: C, 58.77; H, 4.57; N, 8.98.

4936
F. Bova et al. / Bioorg. Med. Chem. 18 (2010) 4928–4938
4.1.1.22. (R)-Methyl 4-(2-(7-chloro-3-((4-chloro-2-(trifluoromethyl)
phenylcarbamoyloxy)methyl-2-oxo-5-phenyl-2,3-dihydro-1H-
benzo[e][1,4]diazepin-1-yl)acetamido)but-2-enoate (3). Com-
pound 31 (30 mg, 0.048 mmol) was reacted with methyl acrylate
(41 mg, 0.48 mmol) according to the procedure described for 2.
The title compound was obtained after purification by preparative
HPLC as a white solid (25 mg, 76.2%). HPLC: retention time
51.35 min. Rf = 0.44 (petroleum ether/EtOAc 3:7). 1H NMR
(300 MHz, CDCl3): 3.62 (s, 3H, OCH3), 3.94–4.06 (m, NHCH2 and
CHCH2), 4.16 (d, 1H, J = 15.1 Hz, NCHA), 4.55 (d, 1H, J = 15.1 Hz,
NCHB), 4.83–4.99 (m, 2H, CHCH2) 5.78 (d, 1H, J = 15.6, CH@CHCO),
6.52 (br t, 1H, NH), 6.77 (dr t, 1H, J = 15.6 and J = 4.7 Hz,
CH@CHCO), 6.97 (s, 1H, NH), 7.19–7.54 (m, 9H, Ar), 7.67 (d, 1H,
J = 8.8 Hz, Ar), 7.99 (d, 1H, J = 8.8 Hz, Ar). 13C NMR (75 MHz, CDCl3):
40.51 (NHCH2), 47.80 (OCH3), 52.89 (NCH2), 62.26 (CHCH2), 65.63
(CHCH2), 121.93 (Ar), 121.99 (Ar), 124.56 (CF3), 124.79
(CH@CHCO), 126.56 (Ar), 128.73 (Ar), 128.89 (Ar), 129.79 (Ar),
130.18 (Ar), 130.83 (Ar), 131.17 (Ar), 131.19 (Ar), 131.48 (Ar),
132.67 (Ar), 133.24 (Ar), 137.69 (Ar), 143.34 (Ar), 143.51
(CH@CHCO), 158.24 (CO), 166.34 (CO), 168.10 (C@N), 172.22
(CO), 173.06 (CO). Anal. Calcd for C31H25Cl2F3N4O6: C, 54.96; H,
3.72; N, 8.27. Found: C, 55.22; H, 3.41; N, 8.48.
147.18 (Ar), 161.79 (CO), 169.01 (C@N), 182.26 (CO), 182.31 (CO),
192.70 (CO). Anal. Calcd for C32H28ClF3N4O7: C, 55.11; H, 4.19; N,
8.32. Found: C, 55.35; H, 4.07; N, 8.40.
4.1.1.25. (R)-Methyl 4-(2-(3-((4-methoxyphenylcarbamoyloxy)
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 N-allyl-2-((3R)-2,
3-dihydro-3-hydroxymethyl-2-oxo-5-phenyl-benzo[e][1,4]diaze-
pin-1-yl-acetamide 356 (100 mg, 0.27 mmol) in dry CH2Cl2 (15 mL)
under N2 was added 4-OCH3C6H4NCO (82 mg, 0.071 mL, 0.54 mmol)
and the resulting mixture was stirred for 12 h at room temperature.
After this time, the mixture was washed with water (2 ꢁ 15 mL).
The residue was purified by flash chromatography, eluting with
petroleum ether/EtOAc 4:6, to afford the intermediate (4-meth-
oxy-phenyl)-carbamic acid 1-allylcarbamoylmethyl-2-oxo-5-phe-
nyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-ylmethyl ester (100 mg,
72.2%). Rf = 0.35 (petroleum ether/EtOAc 4:6). 1H NMR (300 MHz,
CDCl3): 3.75 (s, 3H, OCH3), 3.80–3.90 (m, 2H, NHCH2), 4.01 (m, 1H,
CHCH2), 4.35 (d, 1H, J = 15.6 Hz, NCHA), 4.62 (d, 1H, J = 15.6 Hz,
NCHB), 4.90–5.08 (m, 4H, CH@CH2 and CHCH2), 5.68 (m, 1H,
CH@CH2), 6.36 (br s, 1H, NH), 6.75–6.82 (m, 2H, Ar), 7.24–765 (m,
11H, Ar). 13C NMR (75 MHz, CDCl3): 42.17 (NHCH2), 52.56 (OCH3),
55.76 (NCH2), 62.70 (CHCH2), 64.34 (CHCH2), 116.62 (CH@CH2),
122.84 (Ar), 123.11 (Ar), 125.23 (Ar), 128.20 (Ar), 128.53 (Ar),
129.61 (Ar), 129.98 (Ar), 130.56 (Ar), 130.95 (Ar), 132.35 (Ar),
133.76 (CH@CH2), 138.51 (Ar), 114.53 (Ar), 142.47 (Ar), 144.04
(Ar), 157.25 (CO), 169.17 (C@N), 169.82 (CO), 170.02 (CO). To a solu-
tion of the intermediate (50 mg, 0.097 mmol) methyl acrylate
(84 mg, 0.97 mmol) was added followed by Hoveyda–Grubbs 2nd
generation catalyst (12 mg, 0.0195 mmol). The resulting mixture
was heated under microwave irradiation at 100 °C for 2 h. The sol-
vent was then removed under reduced pressure and the residue
was purified by preparative HPLC as a white solid (40 mg, 81%).
HPLC: retention time 20.66 min. Rf = 0.18 (petroleum ether/EtOAc
4:6). 1H NMR (300 MHz, CDCl3): 3.76 (s, 3H, COOCH3), 3.80 (s, 3H,
ArOCH3), 4.07 (m, 1H, CHCH2), 4.15–4.24 (m, 3H, NHCH2 and NCHA),
4.78 (d, 1H, J = 15.2 Hz, NCHB), 4.98–5.11 (m, 2H, CHCH2), 5.93 (d,
1H, J = 16.2 Hz, CH@CHCO), 6.87–7.04 (m, 5H, 2NH, CH@CHCO and
Ar), 7.31–7.98 (m, 11H, Ar). 13C NMR (75 MHz, CDCl3): 40.40
(NHCH2), 53.37 (OCH3), 53.56 (NCH2), 55.76 (ArOCH3), 62.75
(CHCH2), 64.49 (CHCH2), 114.41 (Ar), 114.53 (Ar) (CH@CHCO),
122.84 (Ar), 123.11 (Ar), 125.23 (Ar), 128.20 (Ar), 128.53 (Ar),
129.61 (Ar), 129.98 (Ar), 130.56 (Ar), 130.95 (Ar), 132.35 (Ar),
138.51 (Ar), 142.53 (Ar), 142.47 (Ar), 144.04 (CH@CHCO), 154.25
(CO), 166.57 (CO), 169.17 (C@N), 169.82 (CO), 170.2 (CO). Anal.
Calcd for C31H30N4O7: C, 65.25; H, 5.30; N, 9.82. Found: C, 65.06;
H, 5.52; N, 9.71.
4.1.1.23. (R)-Methyl 4-(2-(5-(4-bromophenyl)-3-((4-chloro-2-(tri-
fluoromethyl)phenylcarbamoyloxy) methyl-2-oxo-2,3-dihydro-
1H-benzo[e][1,4]diazepin-1-yl)acetamido)but-2-enoate (4).
Compound 32 (25 mg, 0.041 mmol) was reacted with methyl acry-
late (35 mg, 0.41 mmol) according to the procedure described for
2. The title compound was obtained after purification by prepara-
tive HPLC as a white solid (29 mg, 98.2%). HPLC: retention time
40.52 min. Rf = 0.42 (petroleum ether/EtOAc 3:7). 1H NMR
(300 MHz, CDCl3): 3.62 (s, 3H, OCH3), 3.93–3.97 (m, NHCH2 and
CHCH2), 4.27 (d, 1H, J = 15.1 Hz, NCHA), 4.58 (d, 1H, J = 15.1 Hz,
NCHB), 4.87–4.96 (m, 2H, CHCH2) 5.83 (d, 1H, J = 15.2, CH@CHCO),
6.56 (br t, 1H, NH), 6.78 (dt, 1H, J = 15.2 and J = 4.7 Hz, CH@CH2CO),
6.96 (s, 1H, NH), 7.19–7.57 (m, 9H, Ar), 7.66 (d, 1H, J = 8.2 Hz, Ar),
7.97 (d, 1H, J = 9.06 Hz, Ar). 13C NMR (75 MHz, CDCl3): 40.51
(NHCH2), 51.86 (OCH3), 52.88 (NCH2), 62.32 (CHCH2), 65.64
(CHCH2), 121.59 (Ar), 121.89 (Ar), 123.18 (CH@CHCO), 124.84
(Ar), 125.21 (Ar), 125.63 (Ar), 125.93 (CF3), 126.48 (Ar), 129.00
(Ar), 129.51 (Ar), 130.42 (Ar), 131.50 (Ar), 131.87 (Ar), 132.86
(Ar), 133.23 (Ar), 134.39 (Ar), 137.21 (Ar), 142.46 (Ar), 143.70
(CH@CHCO), 153.39 (CO), 166.56 (CO), 168.43 (C@N), 169.30
(CO), 171.19 (CO). Anal. Calcd for C31H25BrClF3N4O6: C, 51.58; H,
3.49; N, 7.76. Found: C, 51.73; H, 3.11; N, 7.60.
4.1.1.24. (R)-Methyl 4-(2-(3-((4-chloro-2-(trifluoromethyl)
phenylcarbamoyloxy)-5-(methoxyphenyl)-2-oxo-2,3-dihydro-
1H-benzo[e][1,4]diazepin-1-yl)acetamido)but-2-enoate (5).
Compound 33 (15 mg, 0.024 mmol) was reacted with methyl acry-
late (20 mg, 0.24 mmol) according to the procedure described for 2.
The title compound was obtained after purification by flash chro-
matography, eluting with petroleum ether/EtOAc 2:8 (16 mg,
97.8%). Rf = 0.35 (petroleum ether/EtOAc 3:7). 1H NMR (300 MHz,
CDCl3): 3.61 (s, 3H, OCH3), 3.78 (s, 3H, ArOCH3), 3.85–3.97 (m,
NHCH2 and CHCH2), 4.26 (d, 1H, J = 15.4 Hz, NCHA), 4.59 (d, 1H,
J = 15.4 Hz, NCHB), 4.81–5.01 (m, 2H, CHCH2) 5.75 (d, 1H, J = 15.6,
CH@CHCO), 6.52 (br t, 1H, NH), 6.74 (dt, 1H, J = 15.6 and 4.7 Hz,
CH=CH2CO), 6.83 (d, 2H, J = 8.8 Hz, Ar), 6.98 (s, 1H, NH), 7.19–
7.56 (m, 7H, Ar), 7.65 (d, 1H, J = 8.5 Hz, Ar), 7.99 (d, 1H,
J = 8.5 Hz, Ar). 13C NMR (75 MHz, CDCl3): 40.53 (NHCH2), 51.30
(OCH3), 52.84 (NCH2), 55.75 (ArOCH3), 62.03 (CHCH2), 65.77
(CHCH2), 114.05 (Ar), 121.75 (Ar), 121.85 (Ar), 122.77 (Ar),
123.02 (CH@CHCO), 124.23 (CF3), 124.84 (Ar), 125.58 (Ar), 126.13
(Ar), 126.53 (Ar), 129.66 (Ar), 130.78 (Ar), 131.39 (Ar), 131.69
(Ar), 132.59 (Ar), 133.22 (Ar), 142.38 (Ar), 143.66 (CH@CHCO),
4.1.1.26. (R)-Methyl 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 (7). Compound 35 (100 mg,
0.27 mmol) was reacted with 1-naphthyl isocyanate (93 mg,
0.072 mL, 0.54 mmol) according to the same procedure described
for compound 6. The intermediate naphthalen-2-yl-carbamic acid
1-allylcarbamoylmethyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e]
[1,4]diazepin-3-yl-methyl ester was obtained in 76.5% yield
(110 mg), after purification by flash chromatography (petroleum
ether/EtOAc 4:6). Rf = 0.38 (petroleum ether/EtOAc 4:6). 1H NMR
(300 MHz, CDCl3): 3.81 (dd, 1H, J = 6.0 and J = 5.5 Hz, CHCH2),
4.06–4.12 (m, 2H, NHCH2), 4.34 (d, 1H, J = 15.3 Hz, NCHA), 4.64
(d, 1H, J = 15.3 Hz, NCHB), 4.95–5.13 (m, 4H, CHCH2 and CH@CH2),
5.74 (m, 1H, CH@CH2), 6.43 (br s, 1H, NH), 7.22–7.87 (m, 16H, Ar).
13C NMR (75 MHz, CDCl3): 45.36 (NHCH2), 53.19 (NCH2), 62.66
(CHCH2), 65.00 (CHCH2), 109.42 (Ar), 119.88 (CH@CH2), 121.19
(Ar), 122.84 (Ar), 123.14 (Ar), 124.70 (Ar), 125.53 (Ar), 126.01
(Ar), 126.31 (Ar), 127.50 (Ar), 128.67 (Ar), 128.89 (Ar), 129.38

F. Bova et al. / Bioorg. Med. Chem. 18 (2010) 4928–4938
4937
(Ar), 130.07 (Ar), 130.70 (Ar), 131.15 (Ar), 132.60 (Ar), 134.30
(CH@CH2), 136.53 (Ar), 142.51 (Ar), 144.01 (Ar), 153.90 (CO),
154.70 (C@N), 170.13 (CO), 171.14 (CO). This intermediate
(50 mg, 0.093 mmol) was reacted with methyl acrylate (80 mg,
0.93 mmol). The title compound was obtained after purification
by preparative HPLC (40 mg, 72.4%). HPLC: retention time
25.62 min. Rf = 0.21 (petroleum ether/EtOAc 4:6). 1H NMR
(300 MHz, CDCl3): 3.64 (s, 3H, OCH3), 4.01 (m, 1H, CHCH2), 4.12–
4.20 (m, 2H, NHCH2), 4.29 (d, 1H, J = 15.1 Hz, NCHA), 4.75 (d, 1H,
J = 15.1 Hz, NCHB), 5.06–5.17 (m, 2H, CHCH2), 5.92 (d, 1H,
J = 15.6 Hz, CH@CHCO), 6.85–6.91 (m, 2H, CH@CHCO and NH),
7.28–7.96 (m, 16H, Ar). 13C NMR (75 MHz, CDCl3): 40.46 (NHCH2),
52.01 (OCH3), 53.19 (NCH2), 62.66 (CHCH2), 65.00 (CHCH2), 109.42
(Ar), 119.88 (Ar), 121.29 (Ar), 121.58 (Ar), 123.14 (Ar), 124.70 (Ar),
125.53 (CH@CHCO), 126.01 (Ar), 126.31 (Ar), 128.67 (Ar), 128.89
(Ar), 129.38 (Ar), 130.07 (Ar), 130.70 (Ar), 131.15 (Ar), 132.60,
132.90 (Ar), 134.34 (Ar), 138.42 (Ar), 142.51 (Ar), 144.01
(CH@CHCO), 153.90 (Ar), 154.70 (CO), 166.53 (C@N), 170.13 (CO),
171.14 (CO), 171.25 (CO). Anal. Calcd for C34H30N4O6: C, 69.14; H,
5.12; N, 9.49. Found: C, 69.20; H, 4.90; N, 9.81.
reacted with cyclohexyl isocyanate (67 mg, 0.068 mL, 0.54 mmol)
according to the procedure described for compound 6. The inter-
mediate cyclohexyl-carbamic acid 1-allylcarbamoylmethyl-2-
oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl-methyl
ester was obtained in 53% yield (70 mg), after purification by flash
chromatography (petroleum ether/EtOAc 4:6). Rf = 0.42 (petro-
leum ether/EtOAc 4:6). 1H NMR (300 MHz, CDCl3): 1.04–1.91
(m, 2H, c-C6H11), 1.25–1.37 (m, 4H, c-C6H11), 1.59–1.68 (m, 2H,
c-C6H11), 1.87–1.92 (m, 2H, c-C6H11), 3.45 (m, 1H, c-C6H11),
3.80–3.95 (m, 2H, NHCH2), 4.01 (dd, 1H, J = 6.3 and J = 5.8 Hz,
CHCH2O), 4.34 (d, 1H, J = 15.3 Hz, NCHA), 4.63 (d, 1H, J = 15.3 Hz,
NCHB), 4.72–5.08 (m, 4H, CHCH2 and CH@CH2), 5.74 (m, 1H,
CH@CH2), 6.48 (br s, 1H, NH), 7.24–7.69 (m, 9H, Ar). 13C NMR
(75 MHz, CDCl3): 24.97 (c-C6H11), 25.73 (c-C6H11), 33.54 (c-
C6H11), 33.63 (NHCH2), 42.17 (c-C6H11), 52.56 (NCH2), 62.70
(CHCH2), 64.34 (CHCH2), 116.61 (CH@CH2), 122.84 (Ar), 125.23
(Ar), 128.53 (Ar), 129.61 (Ar), 129.98 (Ar), 130.56 (Ar), 130.95
(Ar), 132.35 (Ar), 133.76 (CH@CH2), 138.51 (Ar), 142.47 (Ar),
157.25 (CO), 169.17 (C@N), 169.82 (CO), 170.02 (CO). This inter-
mediate (50 mg, 0.102 mmol) was reacted with methyl acrylate
(88 mg, 1.02 mmol). The title compound was obtained after puri-
fication by flash chromatography, eluting with petroleum ether/
EtOAc 2:8, (38 mg, 68%). Rf = 0.28 (petroleum ether/EtOAc 4:6).
1H NMR (300 MHz, CDCl3): 1.05–1.34 (m, 6H, c-C6H11), 1.57–
1.64 (m, 2H, c-C6H11), 1.87–1.90 (m, 2H, c-C6H11), 3.45 (m, 1H,
c-C6H11), 3.72 (s, 3H, OCH3), 3.86–4.12 (m, 3H, NHCH2 and
CHCH2), 4.24 (d, 1H, J = 15.1 Hz, NCHA), 4.70 (d, 1H, J = 15.1 Hz,
NCHB), 4.96–5.05 (m, 2H, CHCH2), 5.83 (d, 1H, J = 15.6 Hz,
CH=CHCO), 6.81–6.87 (m, 2H, CH@CHCO and NH), 7.22–7.61 (m,
8H, Ar), 7.74 (d, 1H, J = 8.3 Hz, Ar). 13C NMR (75 MHz, CDCl3):
24.78 (c-C6H11), 29.91 (c-C6H11), 33.51 (c-C6H11), 40.41 (NHCH2),
50.20 (OCH3), 53.05 (c-C6H11), 55.76 (NCH2), 62.81 (CHCH2),
64.15 (CHCH2), 122.14 (Ar), 122.97 (CH@CHCO), 125.53 (Ar),
128.50 (Ar), 128.64 (Ar), 128.92 (Ar), 130.07 (Ar), 131.11 (Ar),
132.55 (Ar), 138.46 (Ar), 142.62 (CH@CHCO), 143.65 (Ar), 166.50
(CO), 168.60 (C@N), 171.10 (CO), 171.27 (CO). Anal. Calcd for
4.1.1.27. (R)-Methyl 4-(2-(3-((butylcarbamoyloxy)methyl)-2-
oxo-5-phenyl-2,3-dihydro-1H -benzo[e][1,4]diazepin-1-yl)acet-
amido)but-2-enoate (8). Compound 35 (100 mg, 0.27 mmol) was
reacted with butyl isocyanate (54 mg, 0.062 mL, 0.54 mmol)
according to the procedure described for compound 6. The interme-
diate butyl-carbamic acid 1-allylcarbamoylmethyl-2-oxo-5-phe-
nyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl-methyl ester was
obtained in 86.4% yield (108 mg), after purification by flash chro-
matography (petroleum ether/EtOAc 4:6). Rf = 0.36 (petroleum
ether/EtOAc 4:6). 1H NMR (300 MHz, CDCl3): 0.94 (t, 3H,
J = 7.1 Hz, CH3CH2), 1.27–1.36 (m, 2H, CH3CH2), 1.37–1.49 (m, 2H,
CH3CH2CH2), 3.10–3.17 (q, 2H, J = 6.5 Hz, NHCH2CH2), 3.79–3.83
(m, 2H, NHCH2CH@CH2), 3.96 (t, 1H, J = 6.8 Hz, CHCH2), 4.34 (d,
1H, J = 15.6 Hz, NCHA), 4.63 (d, 1H, J = 15.6 Hz, NCHB), 4.78–5.09
(m, 4H, CHCH2 and CH@CH2), 5.70 (m, 1H, CH@CH2), 6.44 (br s,
1H, NH), 7.21–7.69 (m, 9H, Ar). 13C NMR (75 MHz, CDCl3): 14.51
(CH3CH2), 20.23 (CH3CH2), 34.21 (CH3CH2CH2), 42.17 (NHCH2CH2),
42.3 (NHCH2CH@CH2), 52.56 (NCH2), 62.70 (CHCH2), 64.34
(CHCH2), 116.62 (CH@CH2), 122.24 (Ar), 125.23 (Ar), 128.53 (Ar),
129.61 (Ar), 129.98 (Ar), 130.56 (Ar), 130.95 (Ar), 132.35 (Ar),
133.76 (CH@CH2), 138.51 (Ar), 142.47 (Ar), 156.21 (CO), 169.17
(C@N), 169.82 (CO), 170.02 (CO). This intermediate (50 mg,
0.108 mmol) was reacted with methyl acrylate (92 mg, 1.08 mmol).
The title compound was obtained after purification by preparative
HPLC (42 mg, 74.6%). HPLC: retention time 19.11 min. Rf = 0.20
(petroleum ether/EtOAc 4:6). 1H NMR (300 MHz, CDCl3): 1.02 (t,
3H, J = 7.4 Hz, CH2CH3), 1.31–1.55 (m, 4H, CH2CH2CH3), 3.18 (q,
2H, J = 6.3 Hz, NHCH2CH2), 3.77 (s, 3H, OCH3), 3.80–4.00 (m, 3H,
CHCH2O and NHCH2), 4.27 (d, 1H, J = 15.1 Hz, NCHACHBCO), 4.76
(d, 1H, J = 15.1 Hz, NCHACHBCO), 4.86–4.97 (m, 2H, CHCH2O), 5.25
(br t, 1H, NH), 5.90 (d, 1H, J = 15.6 Hz, CH@CHCOOCH3), 6.86–6.92
(m, 2H, CH@CHCOOCH3 and NH), 7.31–7.79 (m, 9H, Ar). 13C NMR
(75 MHz, CDCl3): 13.06 (CH2CH3), 19.87 (CH2CH3), 32.36 (CH2CH2
CH3), 40.41 (NHCH2(CH2)2CH3), 40.46 (NHCH2), 52.01 (OCH3),
53.19 (NCH2CO), 62.66 (CHCH2O), 65.00 (CHCH2O), 124.70 (Ar),
125.53 (CH@CHCOOCH3), 128.67 (Ar), 129.38 (Ar), 130.07 (Ar),
130.70 (Ar), 131.15 (Ar), 132.60 (Ar), 132.90 (Ar), 138.42 (Ar),
142.51 (Ar), 144.01 (CH@CHCOOCH3), 154.70 (CO), 166.53 (C@N),
170.13 (CO), 171.14 (CO), 171.25 (CO). Anal. Calcd for C28H32
N4O6: C, 64.60; H, 6.20; N, 10.76. Found: C, 64.37; H, 6.33; N, 10.98.
C
30H34N4O6: C, 65.92; H, 6.27; N, 10.25. Found: C, 65.60; H,
6.48; N, 10.09.
4.1.1.29. (R)-Methyl 4-(2-(3-((adamantan-1-ylcarbamoyloxy)
methyl)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-
1-yl)acetamido)but-2-enoate (10). To a solution of 35 (100 mg,
0.27 mmol) in dry CH2Cl2 (15 mL) adamantyl isocyanate (96 mg,
0.54 mmol) and Et3N (54.6 mg, 0.075 mL, 0.54 mmol) were added
under N2. The resulting mixture was stirred for 48 h at room tem-
perature. After this time, the mixture was washed with citric acid
solution and water (2 ꢁ 15 mL). The residue was purified by flash
chromatography, eluting with petroleum ether/EtOAc 5:5, to
afford the intermediate adamantan-1-yl-carbamic acid 1-ally-
lcarbamoylmethyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]
diazepin-3-yl-methyl ester (60 mg, 41.2%). Rf = 0.58 (petroleum
ether/EtOAc 4:6). 1H NMR (300 MHz, CDCl3): 1.14–1.18 (m, 4H,
Ad), 1.40–1.56 (m, 9H, Ad), 1.70–1.73 (m, 2H, Ad), 3.78–3.82 (m,
2H, NHCH2), 3.94 (m, 1H, CHCH2), 4.35 (d, 1H, J = 15.3 Hz, NCHA),
4.64 (d, 1H, J = 15.3 Hz, NCHB), 4.75–4.91 (m, 2H, CHCH2), 4.98–
5.08 (m, 2H, CH@CH2), 5.67 (m, 1H, CH@CH2), 6.38 (br s, 1H,
NH), 7.22–7.70 (m, 9H, Ar). 13C NMR (75 MHz, CDCl3): 29.68
(Ad), 36.54 (Ad), 42.07 (Ad), 42.85 (NHCH2), 46.83 (Ad), 52.65
(NCH2), 62.77 (CHCH2), 63.30 (CHCH2), 116.68 (CH@CH2), 122.84
(Ar), 125.30 (Ar), 128.58 (Ar), 129.60 (Ar), 130.01 (Ar), 130.65
(Ar), 131.00 (Ar), 132.40 (CH@CH2), 138.49 (Ar), 142.31 (Ar),
142.42 (Ar), 155.36 (CO), 168.62 (C@N), 178.20 (CO), 180.20
(CO). This intermediate (50 mg, 0.092 mmol) was reacted with
methyl acrylate (71 mg, 0.92 mmol). The title compound was
obtained after purification by preparative HPLC (34 mg, 61.7%).
4.1.1.28. (R)-Methyl 4-(2-(3-((cyclohexylcarbamoyloxy)methyl)-
2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)acet
amido)but-2-enoate (9). Compound 35 (100 mg, 0.27 mmol) was

4938
F. Bova et al. / Bioorg. Med. Chem. 18 (2010) 4928–4938
HPLC: retention time 68.87 min. Rf = 0.38 (petroleum ether/EtOAc
4:6). 1H NMR (300 MHz, CDCl3): 1.22–1.40 (m, 4H, Ad), 1.58–1.79
(m, 9H, Ad), 1.87–2.00 (m, 2H, Ad), 3.75 (s, 3H, OCH3), 3.97–4.15
(m, 3H, NHCH2 and CHCH2), 4.30 (d, 1H, J = 15.0 Hz, NCHA), 4.71
(d, 1H, J = 15.3 Hz, NCHB), 4.85–4.91 (m, 2H, CHCH2), 5.70 (d,
1H, J = 15.6 Hz, CH@CHCO), 6.84–6.93 (m, 2H, CH@CHCO and
NH), 7.25–7.50 (m, 7H, Ar) 7.64 (d, 1H, J = 7.1 Hz, Ar), 7.75 (d,
1H, J = 7.9 Hz, Ar). 13C NMR (75 MHz, CDCl3): 29.69 (Ad), 36.74
(Ad), 40.47 (Ad), 42.85 (NHCH2), 50.10 (Ad), 52.89 (OCH3), 56.20
(NCH2), 62.86 (CHCH2), 63.84 (CHCH2), 121.76 (Ar), 122.92
(CH@CHCO), 125.44 (Ar), 126.90 (Ar), 128.61 (Ar), 129.98 (Ar),
131.04 (Ar), 132.48 (Ar), 138.46 (Ar), 138.93 (Ar), 142.41 (Ar),
143.72 (CH@CHCO), 155.81 (CO), 166.52 (C@N), 169.87 (CO),
178.54 (CO), 179.20 (CO). Anal. Calcd for C34H38N4O4: C, 68.21;
H, 6.40; N, 9.36. Found: C, 68.51; H, 6.77; N, 9.12.
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
100–0.0488 lM. P. falciparum (strain FCBR) was maintained in a
continuous culture basically according to Trager and Jensen.24 Par-
asites were cultured in human red blood cells (RBC) (blood group
ARh+) in RPMI 1640 medium supplemented with 25 mM HEPES
(Molecular Probes, Invitrogen), 20 mM sodium bicarbonate, and
0.5% AlbuMAX I (Molecular Probes, Invitrogen) instead of human
serum at 2.5% (v/v) hematocrit. Cultures were maintained at
37 °C with a gaseous phase of 94% N2, 1% O2, and 5% CO2. Early
staged parasites of strain FCBR were plated in 96-well plates at a
parasitemia 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 etha-
nol alone was used as a negative control. As a positive control, the
parasites were incubated in presence of 2 lM chloroquine (0% via-
4.2. Pharmacology
bility). The viability of the parasites was assayed according to Evers
et al. (2008).19
4.2.1. Enzyme assays
The preliminary screening was performed with 100
lM inhibi-
Acknowledgments
tor concentrations using an equivalent amount of DMSO as
negative control. Product release from substrate hydrolysis (Cbz-
Phe-Arg-AMC, 40 lM for FP-2 and 10 lM for rhodesain) was deter-
mined continuously over a period of 10 min. Compounds showing
at least 50% inhibition were subjected to detailed assays. These
were performed in a 100 mM sodium acetate buffer, pH 5.5 con-
We would like to thank the DAAD Vigoni project (2009/10) for
partial support of this work. T.S. thanks the German Research Soci-
ety (DFG, SFB630) for financial support.
References and notes
taining 10 mM DTT with Cbz-Phe-Arg-AMC (40 or 10
strate.15 The Km values used to correct Kiapp values was
determined to 21.5 M (FP-2) and 0.9
M (rhodesain).21 Inhibitor
solutions were prepared from stocks in DMSO. Each assay was per-
formed twice in 96-well plates in a total volume of 300 L. A Var-
lM) as sub-
1. World Malaria Report 2008 http://malaria.who.int/wmr2008.
2. Sahu, N. K.; Sahu, S.; Veer Kohli, D. Chem. Biol. Drug Des. 2008, 71, 287.
3. Shenai, B. R.; Sijwali, P. S.; Singh, A.; Rosenthal, P. J. J. Biol. Chem. 2000, 275,
29000.
4. Dua, M.; Raphael, P.; Sijwali, P. S.; Rosenthal, P. J.; Hanspal, M. Mol. Biochem.
Parasitol. 2001, 116, 95.
l
l
l
ian Cary Eclipse spectrofluorometer Varian, Darmstadt, Germany
with a microplate reader (excitation 365 nm, emission 460 nm)
was used. Standard units of FP-2 were added to the reaction mix-
ture containing the fluorogenic substrate and increasing from 0 to
5. Ettari, R.; Bova, F.; Grasso, S.; Zappalà, M.; Micale, N. Med. Res. Rev. 2010, 1, 136.
6. Ettari, R.; Nizi, E.; Di Francesco, M. E.; Dude, M. A.; Pradel, G.; Vicik, R.;
Schirmeister, T.; Micale, N.; Grasso, S.; Zappalà, M. J. Med. Chem. 2008, 51, 988.
7. Ettari, R.; Nizi, E.; Di Francesco, M. E.; Micale, N.; Grasso, S.; Zappalà, M.; Vicik,
R.; Schirmeister, T. ChemMedChem 2008, 3, 1030.
8. Ettari, R.; Micale, N.; Schirmeister, T.; Gelhaus, C.; Leippe, M.; Nizi, E.; Di
Francesco, M. E.; Grasso, S.; Zappalà, M. J. Med. Chem. 2009, 52, 2157.
9. Micale, N.; Kozikowski, A. P.; Ettari, R.; Grasso, S.; Zappalà, M.; Jeong, J.-J.;
Kumar, A.; Hanspal, M.; Chishti, A. H. J. Med. Chem. 2006, 49, 3064.
10. Albeck, A.; Persky, R. J. Org. Chem. 1994, 59, 653.
11. (a) Kingsbury, J. S.; Harrity, J. P. A.; Bonitatebus, P. J.; Hoveyda, A. H. J. Am. Chem.
Soc. 1999, 121, 791; (b) Garber, S. B.; Kingsbury, J. S.; Gray, B. L.; Hoveyda, A. H.
J. Am. Chem. Soc. 2000, 122, 8168; (c) Hoveyda, A. H.; Gillingham, D. G.; Van
Veldhuizen, J. J.; Kataoka, O.; Garber, S. B.; Kingsbury, J. S.; Harrity, J. P. A. Org.
Biomol. Chem. 2004, 2, 1; (d) Michrowska, A.; Bujok, R.; Harutyunyan, S.;
Sashuk, V.; Dolgonos, G.; Grela, K. J. Am. Chem. Soc. 2004, 126, 9318.
12. Bargiggia, F. C.; Murray, W. V. J. Org. Chem. 2005, 70, 9636.
13. Shenai, B. R.; Lee, B. J.; Alvarez-Hernandez, A.; Chong, P. Y.; Emal, C. D.; Neitz, R.
J.; Roush, W. R.; Rosenthal, P. J. Antimicrob. Agents Chemother. 2003, 47, 154.
14. Sijwali, P. S.; Brinen, L. S.; Rosenthal, P. J. Protein Expr. Purif. 2001, 22, 128.
15. Pandey, K. C.; Wang, S. X.; Sijwali, P. S.; Lau, A. L.; McKerrow, J. H.; Rosenthal, P.
J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9138.
25 or 50 lM concentrations of compounds. To determine first-or-
der inactivation rate constants (kobs) for the time dependent inhi-
bition, progress curves (fluorescence (F) versus time) were
analyzed by non-linear regression analysis using the equation
F = A(1 ꢀ exp(ꢀkobs t)) + B.16 Product formation was monitored
continuously for 15 min at room temperature. Fitting of the kobs
values against the inhibitor concentrations to the hyperbolic equa-
tion kobs = Kinac [I]/(Kiapp + [I]) gave the individual values of Kiapp
16
and kinac
.
The Kiapp values were corrected to zero substrate con-
centration by the term (1 + [S]/Km) in equation Ki = Kiapp/(1 + [S]/
Km). The second-order rate constants k2nd = kinac/Ki were directly
calculated from the individual constants. Ki and kinac values were
calculated by non-linear regression analyses using the program
GraFit.22 In cases where the kobs versus [I] plots were restricted
to the linear range k2nd was calculated by using the equation
k2nd ꢂ kobs [I]ꢀ1 (1 + [S] Kmꢀ1). In case of time independent inhibi-
tion Ki was obtained by a Dixon plot17 using equation [E]0/
[E]a = 1 + [I]/Kiapp and correction to zero substrate concentration
from Ki = Kiapp/(1 + [S] Kmꢀ1) with [E]0 as enzyme activity in the ab-
sence, and [E]a as residual enzyme activities in the presence of the
inhibitor. Assays with cathepsins B and L were performed as de-
scribed previously.23 Cbz-Phe-Arg-AMC was used as substrate
16. Tian, W. X.; Tsou, C. L. Biochemistry 1982, 21, 1028.
17. Henderson, P. J. F. Biochem. J. 1972, 127, 321.
18. Otto, H. H.; Schirmeister, T. Chem. Rev. 1997, 97, 133.
19. Evers, A.; Heppner, S.; Leippe, M.; Gelhaus, C. Biol. Chem. 2008, 389, 1523.
20. Frye, S. V.; Jhonson, M. C.; Valvano, N. L. J. Org. Chem. 1991, 56, 3750.
21. (a) Gelhaus, C.; Vicik, R.; Hilgenfeld, R.; Schmidt, C. L.; Leippe, M.; Schirmeister,
T. Biol. Chem. 2004, 385, 435; (b) Vicik, R.; Hoerr, V.; Glaser, M.; Schultheis, M.;
Hansell, E.; McKerrow, J. H.; Holzgrabe, U.; Caffrey, C. R.; Ponte-Sucre, A.; Moll,
H.; Stich, A.; Schirmeister, T. Bioorg. Med. Chem. Lett. 2006, 16, 2753.
22. GraFit, Version 3.0, Erithacus Software Ltd, London, 1992.
23. Vicik, R.; Busemann, M.; Gelhaus, C.; Stiefl, N.; Scheiber, J.; Schmitz, W.; Schulz,
F.; Mladenovic, M.; Engels, B.; Leippe, M.; Baumann, K.; Schirmeister, T.
ChemMedChem 2006, 1, 1126.
(80 lM for cathepsin B, 5 lM for cathepsin L). The Km values used
to correct Kiapp values were 150 lM (cathepsin B) and 6.5 lM
24. Trager, W.; Jensen, J. B. Science 1976, 193, 673.
(cathepsin L).