Synthesis and modifications of a small library of 1,4-benzodiazepin-3-ones toward potential inhibitors of the collagen-von Willebrand Factor interaction

Article abstract of DOI:10.1016/j.tet.2009.03.095

A library of 1,4-benzodiazepin-3-one-based turn mimetics were synthesized and tested for their antithrombotic abilities. These mimetics are designed to incorporate amino acid side chains of the previously reported paratope of the inhibitory anti-von Wille

Full text of DOI:10.1016/j.tet.2009.03.095

Tetrahedron 65 (2009) 4521–4529
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Synthesis and modifications of a small library of 1,4-benzodiazepin-3-ones
toward potential inhibitors of the collagendvon Willebrand Factor interaction
Tiny Deschrijver a, Peter Verwilst a, Katleen Broos b, Hans Deckmyn b, Wim Dehaen a,
,
Wim M. De Borggraeve a
*
a Molecular Design and Synthesis, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, bus 2404, 3001 Heverlee, Belgium
b Laboratory for Thrombosis Research, Katholieke Universiteit Leuven Campus Kortrijk, E. Sabbelaan 53, 8500 Kortrijk, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A library of 1,4-benzodiazepin-3-one-based turn mimetics were synthesized and tested for their an-
tithrombotic abilities. These mimetics are designed to incorporate amino acid side chains of the pre-
viously reported paratope of the inhibitory anti-von Willebrand Factor antibody 82D6A3. Modifications
were performed on the scaffolds by alkylation of the N1-position and acylation of the reduced nitro
group. Hereby the number of functional groups incorporated on the core was expanded.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 6 February 2009
Received in revised form 22 March 2009
Accepted 30 March 2009
Available online 5 April 2009
1. Introduction
with shear-independent platelet aggregation causing increased
bleeding risk and resulting in the need for close patient moni-
The process of platelet adhesion, activation and aggregation
(primary hemostasis), necessary for the cessation of bleeding at
sites of vascular injury, is also responsible for pathological
thrombus formation, particularly at sites of high shear. This un-
controlled formation of such aggregates, can lead to vascular oc-
clusion resulting in serious ischemic disorders such as acute
myocardial infarction (AMI), unstable angina, or thrombotic
stroke. Upon injury subendothelial compounds such as collagen
become exposed on cell walls of arteries. The blood plasma pro-
tein von Willebrand Factor (VWF) is immobilized by binding to
the collagen with its A3-domain. Then blood platelets interact
reversibly with the immobilized VWF, which causes them to slow
down.1–5 This allows them to bind irreversibly with collagen.
After this adhesion other platelets are activated and aggregate at
the place of injury, forming the fatal thrombus. Without VWF the
platelets are unable to bind directly to the collagen because of the
high blood shear in the arteries. This VWF–collagen interaction,
crucial in thrombus formation, is an interesting therapeutical
target. Recently, a monoclonal antibody binding to the A3-domain
of human VWF called 82D6A3, which blocks the VWF–collagen
interaction has been characterized.6–8 Its antithrombotic effect
has already been proven in vivo in a baboon arterial thrombosis
model in which no bleeding side effects were detected even at
high doses.9,10 Therefore, the antibody is superior to known
antithrombotics such as Aspirin and Reopro, which both interfere
toring.11–13 An orally available drug, with the same antithrombotic
characteristics and advantages as the 82D6A3 antibody would be
a considerable advancement for cardiovascular treatments.
To this end, only the minimum active sequence and (stereo)
structural requirements of the antibody are required. Staelens et al.
characterized the paratope of the antibody by X-ray crystallography
and mutagenesis studies.14 The paratope comprises two short se-
quences of the heavy chain, CDR 1 (Asn 31 and Tyr 32) and CDR3
(Asp 99, Pro 101, Tyr 102, and Tyr 103), which are discontinuous in
primary structure, but form one continuous surface patch. Two
turns of type I and type IV were detected in the paratope confor-
mation.15–18
-Turns are one of the secondary elements of peptides,
b-
b
which play next to a structural role, also a functional role as mo-
lecular recognition sites in many biological processes. Previously
the binding site for collagen within VWF was located on the A3-
domain of VWF.19,20 By comparing binding studies of VWF mutants
to collagen, with the Ab 82D6A3-VWF crystal structure, a sub-
stantial overlap of the binding site for collagen and the binding site
for the Ab on the A3-domain was confirmed. Hereby it was con-
cluded that direct competition for the same interaction site ex-
plains the potent action of the Ab. Creating peptidomimetics
mimicking the charge distribution and structure of this paratope is
the next goal in the development of this new class of potential
antithrombotics.
The basic molecular scaffold discussed in this article is the 1,4-
benzodiazepin-3-one (Fig. 1). 1,4-Benzodiazepines are privileged
scaffolds mimicking peptide secondary structures such as
-turns making them interesting candidates to mimick the struc-
tural features of the paratope.21–27 Numerous synthetic approaches
g-and
b
* Corresponding author. Tel.: þ32 16 327693; fax: þ32 16 327990.
E-mail address: wim.deborggraeve@chem.kuleuven.be (W.M. De Borggraeve).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.03.095

4522
T. Deschrijver et al. / Tetrahedron 65 (2009) 4521–4529
R1
N
The first modification conducted was the deprotection of the
10
7
R2
groups R2 and R4. Because the arylmethylether requires the
harshest deprotection conditions, the methyl group was first re-
moved to form the phenol moiety using BBr3 (Scheme 2). Yields
obtained were moderate and unidentified side products were
formed. Fortunately extra deprotection steps were avoided since
the other two protecting groups, tBu and Trt, also were removed in
the same step (Table 2).
9
O
N
O2N
5
R4
Figure 1. Structure of 1,4-benzodiazepin-3-ones.
have been developed for the preparation of 1,4-benzodiazepin-3-
ones.28–36 Hence these compounds seemed to be an interesting
synthetic starting point for this work for which at present no small
molecule lead compounds are available.
H
N
H
N
R2
R2'
O
We applied the strategy of the group of Prof. A. Hallberg because
it allows extensive modification of the scaffold.25,37 The scaffold
incorporates the side chains on position R2 and R4. With further
modifications, the number of side chains can be expanded by
R1-alkylation on nitrogen and by acylation of the latent amine
group obtained after reduction of the nitro function (Fig. 1). The
basic targets are mimics of the Tyr–Tyr and Asn–Tyr patterns
present in a beta turn conformation in the paratope. We also de-
scribe a third derivative, which is a homologue of the Tyr–Tyr
pattern by incorporating 2-(4-methoxyphenyl)ethylamine instead
of 4-methoxybenzylamine as an R4-group (Fig. 1).
BBr3, CH2Cl2
O
N
N
O2N
O2N
R4
R4'
7-9
4-6
Scheme 2. Deprotection of the R2 and R4 groups.
Table 2
Yields of the deprotection of the R2 and R4 groups
0
0
Compound
R2
R4
%
7
8
9
CH2(C6H4)OH
CH2(C6H4)OH
CH2CONH2
CH2(C6H4)OH
(CH2)2(C6H4)OH
CH2(C6H4)OH
33
45
52
2. Results and discussion
A second modification is the alkylation of the N1-position in
which the number of functional groups on the scaffold is expanded.
Compound 4 was alkylated with four different halides using so-
dium hydride (NaH) as a base in dry DMF (Scheme 3 and Table 3).
Formation of compound 13 was difficult to monitor. After purifi-
cation, starting material 4 was still recovered.
The synthetic pathway toward these bicyclic core structures is
outlined in Scheme 1. Reductive amination of 2-fluoro-5-nitro-
benzaldehyde with 4-methoxybenzylamine or 2-(4-methoxy-
fphenyl)ethylamine, was followed by coupling the secondary amine
with the fluorenylmethyloxycarbonyl (Fmoc) protected amino
acids Fmoc-L L-Asn(NHTrt)-OH. To ensure
-Tyr(OtBu)-OH or Fmoc-
a complete reaction, 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetra-
methyl uronium hexafluorophosphate (HATU) was used as cou-
pling reagent. To prevent nucleophilic aromatic substitution on the
activated benzene, the following Fmoc-deprotection was con-
ducted with 8-diazabicyclo[5.4.0]undec-7-ene (DBU). The final key
cyclization step, a nucleophilic aromatic substitution of the free
amine, proceeded smoothly with triethylamine in DMSO.
OtBu
OtBu
OMe
R1
N
H
N
NaH, R1-X
DMFdry
O
O
N
O2N
N
10-13
O2N
OMe
4
Scheme 3. N1-Alkylation.
4-methoxybenzylamine or
F
Table 3
Yields of the alkylation of the N1-position
2-(4-methoxyphenyl)ethylamine
HOAc, NaCNBH3, MeOH
F
O2N
CHO
O2N
NH
R4
Compound
R1-X
%
1a-b
10
11
12
13
CH3I
81
54
59
32
Fmoc-L-Tyr(OtBu)-OH or
Fmoc-L-Asn(NHTrt)-OH
HATU, DIEA, CH2Cl2
F
BrCH2(C6H5)
BrCH2CHCH2
BrCH2CO2Me
O
NHFmoc
R2
O2N
N
R4
Et3N, DMSO
2a-c
H
N
R2
F
O
NH2
R2
DBU, THF
O
N
O2N
O2N
N
Different attempts for the alkylation of derivative 6 under pre-
viously mentioned conditions produced in each case product 14
(Scheme 4). Only when the solvent was degassed, methylated
product 15 was obtained in good yield. We believe that compound
14 is formed because of the acidity of the protons next to the amide.
R4
R4
3a-c
4,5,6
Scheme 1. Synthesis of the basic scaffolds 4–6.
In Table 1 the three different benzodiazepines and their overall
yields are given. No significant problems were encountered during
their synthesis. The NMR spectra of compounds 2a–2c and 3a–3c
showed two rotamers, the signals of which coalesced at elevated
temperature.37
H
N
CONHTrt
NaH, MeI
DMFdry
O
H
N
CONHTrt
N
O2N
O2N
OMe
Table 1
14 (99%)
O
Overall yields of the synthesis of the basic scaffolds
N
O2N
CONHTrt
OMe
6
NaH, MeI,
DMFdry + degas
N
Compound
R2
R4
%
O
N
4
5
6
CH2(C6H4)OtBu
CH2(C6H4)OtBu
CH2CONHTrt
CH2(C6H4)OMe
(CH2)2(C6H4)OMe
CH2(C6H4)OMe
27
37
15
OMe
15 (60%)
Scheme 4. N1-Alkylation of compound 6.

T. Deschrijver et al. / Tetrahedron 65 (2009) 4521–4529
4523
Instead of deprotonation of N1, one of these protons is deprotonated
to form an anion, which reacts with oxygen.
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Another way to produce more diversity on the scaffold is to
reduce the nitro group and further functionalize the resulting
amine. Reduction was effected in a Parr apparatus with palla-
dium/carbon under hydrogen in a quantitative manner. To in-
crease the stability of this electron rich aniline derivate, the
amine was immediately capped with acetic anhydride (16–18)
(Scheme 5).
H
N
H
N
R2
O
R2
O
1) Pd/C, H2, MeOH
2) Ac2O, CH2Cl2
N
N
O2N
AcHN
R4
R4
16 (72%)
17 (79%)
18 (79%)
4-6
Figure 2. Effect of 82D6A3 and the benzodiazepines on VWF–collagen binding as de-
termined by ELISA. NHP, 82D6A3, 7–16, and 18 are representing the VWF control, the
inhibitory antibody and the tested benzodiazepines, respectively. Absorbance (490 nm)
is shown in function of the highest concentration tested (0.100 mM). All data point the
mean and standard error of single measurements in two independent experiments.
Scheme 5. Reduction of the nitro-group followed by acylation.
Compound 5 was coupled with another Fmoc-protected amino
acids of the antibody paratope sequence (19–21) after reduction of
the nitro group 22 (Scheme 6). Because of the low reactivity of the
aromatic amine, benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (Pybop) was used as the coupling reagent.
After coupling overnight the products were obtained in moderate
yields. Side products could not be identified or purified. The Fmoc-
group was then removed with piperidine in DMF to yield the
compounds 23–25, which were difficult to purify (Table 4).
absorbance was determined at 490 nm. After each incubation step,
plates were washed with phosphate buffer solution (PBS), 0.1%
Tween-20, 3 times after coating and blocking steps and 12 times
elsewhere. In Figure 2 the effect of 82D6A3 and some of benzodi-
azepines on binding of plasma VWF to collagen is shown as de-
termined by ELISA. Absorbance is plotted against the highest
concentration compound tested (0.100 mM). As can be derived
from the figure 4 mg/ml of the 82D6A3 antibody completely blocks
the VWF–collagen interaction. No inhibitory effect on the collagen–
VWF binding could be detected after pre-incubation of NHP with
the compounds.
OtBu
OMe
OtBu
H
N
H
N
Pd/C, H2, MeOH
H2N
O
O
N
N
O2N
OMe
OtBu
2
2
3. Conclusions
5
22
H
In this article we have discussed the synthesis of a small library
of 1,4-benzodiazepin-3-one turn mimics based on the synthesis of
the group of Prof. A. Hallberg. By this we try to mimic the confor-
mation and the charge distribution of the paratope of the mono-
clonal antibody 82D6A3 against the A3-domain of VWF. This
plasma protein plays a crucial role in the formation of fatal
thrombosis in the arteries. Each synthesized molecule was tested
for inhibition of the collagen–VWF interaction with Enzyme-Linked
Immuno Sorbent Assay (ELISA) in cooperation with the KULAK
research group of the Lab of Thrombosis Research. None of these
compounds showed the ability to block the interaction.
N
O
1) Fmoc-L-AA-OH, PyBop, DIEA, DMF
O
H2N
N
N
H
2) Piperidine, DMF
OMe
R
2
23-25
Scheme 6. Coupling of the reduced nitro-group with AA’s.
Table 4
Yields of the coupling and the Fmoc-deprotection
Compound
Fmoc-
L-AA-OH
%
19
20
21
23
24
25
Fmoc-Asn(NHTrt)-OH
Fmoc-Asp(OtBu)-OH
Fmoc-Tyr(OtBu)-OH
Fmoc-Asn(NHTrt)-OH
Fmoc-Asp(OtBu)-OH
Fmoc-Tyr(OtBu)-OH
35
68
57
45
21
42
4. Experimental section
4.1. General methods
All chemical reagents were used without further purification.
Solvents for column chromatography and TLC were laboratory
grade and distilled before use. TLC was performed on Fluka Chemica
Kiezelgel glass plates, column chromatography with silica gel SI 60
(230–400 mesh) and HPLC was performed on an Altech column
2.1. Determination of inhibition of the VWF–collagen binding
The enzyme-linked immunosorbent assay (ELISA) was per-
formed essentially as described.38 Briefly, microtiter plates were
coated with human collagen type III (Sigma, St. Louis, MO) and
blocked with 3% milk powder solution. A 1:80 dilution of human
normal plasma (NHP) (prewarmed for 5 min at 37 ꢀC) was pre-in-
cubated (30 min at 37 ꢀC) in presence of 1% DMSO with a serial
twofold dilution of either the antibody 82D6A3 or the synthesized
(25ꢁ2.5 cm, 5
mm). NMR spectra were recorded on a Bruker Avance
300 (300 MHz) or a Bruker Avance 400 (400 MHz) spectrometer.
NMR samples were run in the indicated solvents and were refer-
enced internally. Chemical shifts (d) are reported relative to TMS
and coupling constants (J) in hertz (Hz). Low-resolution mass
spectra were recorded on a HEWLETT–PACKARD instrument (CI-EI)
and a Thermo Finnigan LCQ Advantage (ESI). High-resolution mass
spectra were recorded on a KRATOS MS50TC instrument. IR spectra
were obtained using KBr pellets or NaCl plates. Melting points were
determined using a Reichert-Jung Thermovar apparatus and were
uncorrected.
compounds, starting at a concentration of 4 mg/ml and 0.100 mM,
respectively. Plasma VWF was then allowed to bind collagen for
1.5 h at 37 ꢀC. Rabbit antihuman VWF labeled with horseradish
peroxidase was added for 1 h at room temperature. Visualization
was obtained with H2O2 and ortho-phenylenediamine (OPD, Sigma)
and the coloring reaction was stopped with 4 M H2SO4. The

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T. Deschrijver et al. / Tetrahedron 65 (2009) 4521–4529
4.2. Synthesis of the basic scaffolds
chromatography (1% MeOH in DCM). MS (ESI, MeOH): 746.7 [MHþ],
768.8 [MNaþ], 1508.5 [2MHþ], 1513.5 [2MNaþ].
4.2.1. (2-Fluoro-5-nitrophenyl)-N-(4-methoxybenzyl)-
methanamine (1a)
4.2.5. 9H-Fluoren-9-ylmethyl-2-[(2-fluoro-5-nitrobenzyl)(4-
methoxybenzyl)amino]-2-oxo-1-[(tritylamino)carbonyl]-
ethylcarbamate (2c)
To a solution of 2-fluoro-5-nitrobenzaldehyde (2 g, 11.83 mmol)
in MeOH (20 ml), acetic acid (1.02 ml, 17.74 mmol), and 4-methoxy-
benzylamine (3.08 ml, 23.66 mmol) was added. After stirring the
mixture for 8 h under Ar at room temperature, NaCNBH3 (1.49 g,
23.66 mmol) was added. The solution was stirred another 8 h under
the same conditions and afterward quenched by H2O (100 ml).
MeOH was evaporated and the aqueous layer was extracted with
EtOAc (3ꢁ20 ml). The organic layers were collected, dried (MgSO4),
and evaporated. The crude product was purified by column chro-
matography (1% MeOH in DCM) to afford product 1a as a yellow oil
Amine 1a (2.27 g, 7.82 mmol) was coupled with Fmoc-L-
Asn(NHTrt)-OH by a procedure similar to that used for compound
2a. Product 2c was obtained as a yellow foam (3.01 g, 43%) after
column chromatography (0.5% MeOH in DCM). MS (ESI, MeOH):
869.5 [MHþ], 891.4 [MNaþ], 1738.2 [2MHþ], 1759.3 [2MNaþ].
4.2.6. 2-Amino-3-(4-tert-butoxyphenyl)-N-(2-fluoro-5-
nitrobenzyl)-N-(4-methoxybenzyl)propanamide (3a)
(2.06 g, 60%). 1H NMR (300 MHz, CDCl3):
8.7 Hz, 1H, 6-H), 8.03 (dd, J¼2.8, 6.2 Hz, 1H, 4-H), 7.21 (d, J¼8.3 Hz,
2H), 7.08 (t, J¼8.9 Hz, 1H, 3-H), 6.80 (d, J¼8.3 Hz, 2H), 3.82 (s, 2H,
C1CH2), 3.70 (s, 5H, OCH3, NHCH2),1.30 (s,1H, NHCH2) ppm.13C NMR
d
8.31 (ddd, J¼2.8, 4.2,
Fmoc-protected compound 2a (3.59 g, 4.76 mmol) was dis-
solved in dry THF (30 ml) and DBU (0.47 ml, 4.76 mmol) was added.
After 2 h stirring at room temperature under Ar, THF was evapo-
rated. The crude residue was purified with column chromatography
(gradient 0.5–2% MeOH in DCM) and product 3a was obtained as
yellow oil (1.87 g, 74%). Two rotamers were detected. 1H NMR
(75 MHz, CDCl3):
d
45.78 (d, JC–F¼2.3 Hz, C1CH2), 53.00 (NHCH2),
55.47 (OCH3), 114.18 (2CH), 116.45 (d, JC–F¼24.7 Hz, CH3), 124.73 (d,
JC–F¼10.2 Hz, 4-CH), 126.17 (d, JC–F¼6.6 Hz, 6-CH), 129.70 (2CH),
129.96 (d, JC–F¼16.2 Hz, 1-C), 132.20 (CCH2), 144.64 (5-C), 159.16
(COCH3), 164.77 (d, JC–F¼257.2 Hz, 2-C) ppm. MS (EI): m/z (%)¼289
(300 MHz, DMSO): d 8.18–8.16 (m, 2H, 6-H, 4-H) (major), 7.97–8.03
(m, 2H, 6-H, 4-H) (minor), 7.46–7.37 (m,1H, 3-H), 7.10–6.92 (m, 4H),
6.81 (d, J¼7.7 Hz, 2H), 6.76 (d, J¼7.7 Hz, 2H), 4.75 (d, J¼14.5 Hz, 1H,
C1CH2) (minor), 4.61 (d, J¼15.3 Hz, 1H, C1CH2) (major), 4.40–4.34
(m, 3H, NCH2, C1CH2) (major), 4.15 (d, J¼14.5 Hz, 1H, C1CH2)
(95) [MꢂH]þ, 259 (95) [MꢂOCH3]þ,169 (25) [-CH2-
f
-OMe]þ. HRMS
(EI) calculated for C15H15O3N2F: 290.1066; found: 290.1062.
(minor), 3.97–3.12 (m, 1H,
a
-H) (major), 3.84–3.80 (m, 1H,
a-H)
4.2.2. N-(2-Fluoro-5-nitrobenzyl)-2-(4-methoxyphenyl)-
ethanamine (1b)
(minor), 3.71 (s, 3H, OCH3), 2.95–2.84 (m, 1H, b-H), 2.77–2.68 (m,
t
1H,
(75 MHz, DMSO):
(major), 43.89 (CH2NH) (minor), 48.46 (NHCH2) (minor), 50.62
(NHCH2) (major), 53.68 ( -CH) (minor), 53.76 ( -CH) (major), 55.67
b
-H), 2.51–1.9 (br s, 2H, NH2), 1.24 (s, 9H, Bu) ppm. 13C NMR
Compound 1b was synthesized by the procedure described for
d
29.30 (C(CH3)3), 42.55 ( -CH2), 42.82 (CH2NH)
b
compound
1a.
2-(4-Methoxyphenyl)ethylamine
(3.47 ml,
23.66 mmol) was used in the reaction. The crude product was pu-
rified by column chromatography (1% MeOH in DCM) to afford
product 1b as a yellow oil (2.27 g, 64%). 1H NMR (300 MHz, CDCl3):
a
a
(OCH3) (major), 55.77 (OCH3) (minor), 78.34 (C(CH3)3), 114.50
(2CH) (minor), 114.77 (2CH) (major), 117.45 (d, JC–F¼25.2 Hz, 3-CH)
(major), 117.65 (d, JC–F¼24.5 Hz, 3-CH) (minor), 124.28 (2CH) (ma-
jor), 124.38 (2CH) (minor), 125.15 (d, JC–F¼6.6 Hz, 6-CH) (minor),
125.83 (d, JC–F¼10.7 Hz, 4-CH) (major), 126.06 (d, JC–F¼10.7 Hz, 4-CH)
(minor), 126.85 (d, JC–F¼6.3 Hz, 6-CH) (major), 127.30 (d, JC–F¼17.5 Hz,
1-C) (minor), 127.44 (d, JC–F¼17.0 Hz, 1-C) (major), 128.89 (2CH) (ma-
jor), 129.25 (CCH2) (major), 129.79 (CCH2) (minor), 130.25 (2CH) (mi-
nor), 130.56 (2CH) (minor), 130.62 (2CH) (major), 133.66 (CCH2)
(minor), 133.79 (CCH2) (major), 144.72 (5-C) (major), 145.00 (5-C)
(minor), 154.25 (COtBu), 159.34 (COMe), 159.64 (COMe) (major), 164.30
(d, JC–F¼255.3 Hz, 2-C) (minor), 164.57 (d, JC–F¼255.7 Hz, 2-C) (major),
176.06 (CO) (minor), 176.25 (CO) (major) ppm. MS (EI): m/z (%)¼436
d
8.26–8.24 (ddd, J¼2.8, 4.5, 8.3 Hz, 1H, 6-H), 8.03–8.00 (dd, J¼2.8,
6.3 Hz, 1H, 4-H), 7.09–7.03 (m, 3H, 3-H, 2H), 6.77 (d, J¼8.1 Hz, 2H),
3.84 (s, 2H, CH2NH), 3.68 (s, 3H, OCH3), 2.86–2.82 (m, 2H,
NHCH2CH2), 2.75–2.73 (m, 2H, NHCH2CH2), 1.46 (s, 1H,
NHCH2CH2) ppm. 13C NMR (75 MHz, CDCl3):
d 35.73 (NHCH2CH2),
46.40 (d, JC–F¼2.5 Hz, C1CH2), 51.14 (NHCH2CH2), 55.34 (OCH3),
114.23 (2CH), 116.38 (d, JC–F¼24.8 Hz, 3-CH), 124.64 (d, JC–F¼11.1 Hz,
4-CH),126.4 (d, JC–F¼7.4 Hz, 6-CH),129.95 (2CH),130.19 (1-C),132.11
(CCH2), 144.68 (5-C), 158.46 (COCH3), 164.68 (d, JC–F¼257.6 Hz, 2-
C) ppm. MS (EI): m/z (%)¼183 (100) [MꢂCH2-
f
-OMe]þ. HRMS (EI)
calculated for C16H17O3N2F: 304.1223; found: 304.1215.
(22) [MꢂOtBu]þ, 346 (15) [MꢂCH2-
f
-OtBu]þ. MS (ESI, MeOH): 510.5
4.2.3. 9H-Fluoren-9-ylmethyl-1-(4-tert-butoxybenzyl)-2-[(2-
fluoro-5-nitro-benzyl)(4-methoxybenzyl)amino]-2-
oxoethylcarbamate (2a)
[MHþ], 732.1 [MNaþ], 1019.6 [2MHþ], 1041.3 [2MNaþ].
4.2.7. 2-Amino-3-(4-tert-butoxyphenyl)-N-(2-fluoro-5-
To a solution of amine 1a (2.06 g, 7.10 mmol) in DCM (35 ml),
nitrobenzyl)-N-[2-(4-methoxyphenyl)ethyl]propanamide (3b)
Fmoc-protected compound 2b (4.69 g, 6.29 mmol) was depro-
tected by a procedure similar to that used for compound 3a. The
crude residue was purified with column chromatography (gradient
0.5–2% MeOH in DCM) and product 3a was obtained as yellow oil
(2.86 g, 87%). Two conformers were detected. 1H NMR (400 MHz,
1 equiv Fmoc-L
-Tyr(OtBu)-OH (3.26 g, 7.10 mmol), 1 equiv HATU
(2.69 g, 7.10 mmol), and 2.7 equiv DIPEA (1.95 ml, 21.30 mmol) was
added. After stirring the reaction mixture overnight under N2 at
room temperature, H2O (50 ml) and EtOAc (50 ml) were added. The
layers were separated and the aqueous layer was further extracted
with EtOAc (3ꢁ10 ml). The organic layers were collected, washed
with 1 M KHSO4, H2O, saturated NaHCO3, brine, dried (MgSO4), and
evaporated. Purification with column chromatography (1% MeOH
in DCM) afforded product 2a as yellow foam (3.59 g, 67%). MS (ESI,
MeOH): 754.5 [MNaþ], 1485.1 [2MNaþ].
CDCl3):
d 8.09–8.06 (m, 1H, 6-H), 7.91–7.87 (m, 1H, 4-H) (major),
7.82–7.80 (m, 1H, 6-H) (minor), 7.00–6.86 (m, 5H), 6.86–6.75 (m,
4H), 4.53 (d, J¼15.3 Hz, 1H, C1CH2) (major), 4.21 (d, J¼15.3 Hz, 1H,
C1CH2) (major), 4.15 (d, J¼17.2 Hz, 1H, C1CH2) (minor), 3.97 (d,
J¼17.2 Hz, 1H, C1CH2) (minor), 3.55–3.47 (br s, 4H, OCH3,
a-CH),
3.13–3.02 (m, 2H, NHCH2CH2), 2.81–2.32 (m, 4H, b-H, NHCH2CH2),
4.2.4. 9H-Fluoren-9-ylmethyl-1-(4-tert-butoxybenzyl)-2-{(2-
fluoro-5-nitro-benzyl)[2-(4-methoxyphenyl)ethyl]amino}-2-
oxoethylcarbamate (2b)
1.12 (s, 2H, NH2), 1.12 (s, 9H, tBu) (minor), 1.04 (s, 9H, tBu)
(major) ppm. 13C NMR (100 MHz, CDCl3):
28.59 (C(CH3)3) (major),
28.66 (C(CH3)3) (minor), 32.55 (NCH2CH2) (minor), 34.14
(NCH2CH2) (major), 42.05 ( -CH2) (major), 42.35 ( -CH2) (minor),
42.53 (NCH2CH2) (major), 44.60 (NCH2CH2) (minor), 48.63 (C1CH2)
(minor), 49.06 (C1CH2) (major), 53.24 (
-CH) (major), 53.46 ( -CH)
d
Amine 1b (2.27 g, 7.47 mmol) was coupled with Fmoc-
L-
b
b
Tyr(OtBu)-OH by a procedure similar to that used for compound 2a.
Product 2b was obtained as a yellow foam (4.69 g, 84%) after column
a
a

T. Deschrijver et al. / Tetrahedron 65 (2009) 4521–4529
4525
(minor), 54.95 (OCH3) (major), 53.61 (OCH3) (minor), 77.89
(C(CH3)3), 113.87 (2CH) (minor), 114.09 (2CH) (major), 116.13 (d,
JC–F¼15.1 Hz, 3-CH) (major), 116.54 (d, JC–F¼15.1 Hz, 3-CH) (minor),
123.90 (2CH) (major), 124.02 (2CH) (minor), 124.29 (d, JC–F¼5.8 Hz,
6-CH) (minor), 124.71 (d, JC–F¼10.8 Hz, 4-CH) (major), 125.17 (d,
JC–F¼10.8 Hz, 4-CH) (minor), 126.21 (d, JC–F¼16.2, 1-C) (minor),
126.44 (d, JC–F¼9.2 Hz, 4-CH) (major), 126.49 (d, JC–F¼16.2 Hz, 1-C)
(major), 129.52 (2CH), 129.59 (2CH) (major), 130.50 (CCH2), 132.42
(CCH2) (minor), 132.50 (CCH2) (major), 144.20 (5-C) (major), 144.45
(5-C) (minor), 154.02 (COtBu) (major), 154.21 (COtBu) (minor),
158.15 (COMe) (minor), 158.41 (COMe) (major), 163.88
(JC–F¼256.6 Hz, 2-C), 164.04 (d, JC–F¼256.6 Hz, 2-C), 175.11 (CO)
(minor), 175.77 (CO) (major) ppm. MS (EI): m/z (%)¼508 (14)
[MꢂCH3]þ, 450 (30) [MꢂOtBu]þ. HRMS (EI) calculated for
C29H34O5N3F: 523.2482; found: 523.2469.
(C(CH3)3), 35.07 (
b
-CH2), 48.71 (NCH2), 49.40 (5-CH2), 54.53 (2-CH),
55.02 (OCH3), 77.64 (C(CH3)3), 113.57 (2CH), 115.05 (10-CH), 118.78
(6-C), 123.27 (2CH), 124.66 (9-CH), 126.26 (7-CH), 129.25 (2CH),
129.82 (CCH2), 129.84 (2CH), 132.61 (CCH2), 135.57 (8-C), 152.71
(11-C), 153.47 (COtBu), 158.47 (COMe), 168.98 (3-C) ppm. MS (EI):
m/z (%)¼326 (64) [MꢂCH2-
f
-OtBu]þ. HRMS (EI) calculated for
C28H31O3N5: 489.2263; found: 489.2261. Melting point: 166 ꢀC.
4.2.10. 2-(4-tert-Butoxybenzyl)-4-[2-(4-methoxyphenyl)ethyl]-7-
nitro-1,2,4,5-tetrahydro-3H-1,4-benzodiazepin-3-one (5)
Amine 3b (2.86 g, 5.46 mmol) was cyclized by a procedure
similar as described for compound 4. After column chromatography
(1% MeOH in DCM), product 5 was obtained as a yellow solid
(2.19 g, 80%). 1H NMR (400 MHz, CDCl3):
d
7.82 (dd, J¼2.5, 9.0 Hz,
1H, 9-H), 7.53 (d, J¼2.5 Hz, 1H, 7-H), 7.18 (d, J¼8.4 Hz, 2H), 6.95 (d,
J¼8.4 Hz, 4H), 6.66 (d, J¼8.4 Hz, 2H), 6.31 (d, J¼9.0 Hz, 1H, 10-H),
5.21 (d, J¼16.9 Hz, 1H, 5-H), 4.90–4.85 (m, 1H, 2-H), 4.51 (d,
J¼2.7 Hz, 1H, 1-NH), 3.90–3.83 (m, 1H, NHCH2CH2), 3.70 (s, 3H,
OCH3), 3.65–3.50 (m, 2H, 5-HþNHCH2CH2), 3.36 (dd, J¼5.5, 14.5 Hz,
4.2.8. 2-Amino-N1-(2-fluoro-5-nitrobenzyl)-N1-(4-methoxybenzyl)-
N4-trityl-succinamide (3c)
Fmoc-protected compound 2c (3.01 g, 3.38 mmol) was depro-
tected by a procedure similar to that used for compound 3a. The
crude residue was purified with column chromatography (gradient
1–10% MeOH in DCM) and product 3c was obtained as yellow oil
(1.31 g, 60%). Two conformers were detected. 1H NMR (400 MHz,
1H,
b
-H), 2.87 (dd, J¼8.7, 14.5 Hz, 1H,
b
-H), 2.82–2.72 (m, 2H,
28.87
-CH2), 51.14 (NHCH2CH2), 52.56
NCH2CH2), 1.33 (s, 9H, tBu) ppm. 13C NMR (100 MHz, CDCl3):
(C(CH3)3), 33.64 (NCH2CH2), 63.18 (
d
b
(5-CH2), 55.06 (OCH3), 55.26 (2-CH), 78.48 (C(CH3)3), 113.81 (2CH),
115.28 (10-CH),118.31 (6-C),124.44 (2CH),125.20 (9-CH),125.86 (7-
CH), 129.60 (2CH), 129.65 (2CH), 130.40 (CCH2), 131.14 (CCH2),
137.85 (8-C), 151.16 (11-C), 154.64 (COtBu), 158.27 (COMe), 168.81
CDCl3):
d 8.29 (s, 1H, NHTrt) (major), 8.26 (s, 1H, NHTrt) (minor),
8.11–8.03 (m, 2H, 4-H, 6-H) (major), 8.00–7.97 (m, 1H) (minor),
7.22–7.19 (m, 16H, Trt, 3-H), 7.11–6.98 (m, 2H), 6.80 (d, J¼8.1 Hz, 2H)
(major), 6.75 (d, J¼8.1 Hz, 2H) (minor), 4.69 (d, J¼14.7 Hz, 1H,
C1CH2) (minor), 4.63 (d, J¼15.6 Hz, 1H, NCH2) (major), 4.57 (d,
J¼16.4 Hz,1H, C1CH2) (major), 4.50 (d, J¼15.6 Hz,1H, NCH2) (major),
4.45 (d, J¼16.8 Hz,1H, C1CH2) (major), 4.23 (d, J¼14.8 Hz,1H, C1CH2)
(3-C) ppm. MS (EI): m/z (%)¼447 (120) [Mꢂ BuþH]þ, 369 (19)
t
[MꢂCH2CH2-
f
-OMeþH]þ, 340 (85) [CH2-
f
-OtBu]. HRMS (EI) cal-
culated for C29H33O5N3: 503.2420; found: 503.2425. Melting point:
147 ꢀC.
(minor), 4.17 (t, J¼6.4 Hz, 1H,
(s, 3H, OCH3) (minor), 2.56 (d, J¼6.4 Hz, 2H,
NH2) ppm. 13C NMR (100 MHz, CDCl3):
42.81 (
42.99 (NHCH2) (major), 43.09 ( -CH2) (minor), 43.96 (NHCH2)
(minor), 48.39 (C1CH2) (minor), 49.27 (C1CH2) (major), 50.82 (
-CH)
(major), 53.61 ( -CH) (minor), 55.35 (OCH3), 70.56 (C( )3), 114.31
(2CH) (minor), 114.56 (2CH) (major), 116.42 (d, JC–F¼24.5 Hz, 3-CH)
(major), 116.81 (d, JC–F¼25.7 Hz, 3-CH) (minor), 124.81 (d, JC–F
a
-H), 3.73 (s, 3H, OCH3) (major), 3.71
-H), 1.77 (s, 2H,
-CH2) (major),
b
4.2.11. 2-[4-(4-Methoxybenzyl)-7-nitro-3-oxo-2,3,4,5-tetrahydro-
1H-1,4-benzo-diazepin-2-yl]-N-tritylacetamide (6)
d
b
b
Amine 3c (1.31 g, 2.03 mmol) was cyclized by a procedure
similar as described for compound 4. After column chromatography
(1% MeOH in DCM), product 6 was obtained as a yellow solid (1.16 g,
a
a
f
92%). 1H NMR (400 MHz, CDCl3):
d 7.92 (s, 1H, NHTrt), 7.74 (dd,
¼
J¼2.3, 9.1 Hz,1H, 9-H), 7.49 (d, J¼2.3 Hz,1H, 7-H), 7.23–7.16 (m,15H,
Trt), 7.04 (d, J¼8.6 Hz, 2H), 6.70 (d, J¼8.6 Hz, 2H), 6.22 (d, J¼9.1 Hz,
1H, 10-H), 5.61 (d, J¼2.8 Hz, 1H, 1-NH), 5.10 (d, J¼16.4 Hz, 1H, 5-H),
5.12–5.05 (m, 1H, 2-H), 4.57 (dd, J¼14.8 Hz, 2H, NCH2), 3.73–3.70
6.5 Hz, 6-CH) (minor), 125.00 (d, JC–F¼10.2 Hz, 4-CH) (major),
125.48 (d, JC–F¼10.0 Hz, 4-CH) (minor), 125.79 (d, JC–F¼15.2 Hz, 1-C)
(minor), 126.00 (d, JC–F¼6.2 Hz, 6-CH) (major), 126.24 (JC–F¼17.2 Hz,
1-C) (major), 126.91 (3CH, Trt), 127.56 (CCH2), 127.86 (6CH, Trt),
128.28 (2CH) (major), 128.94 (6CH, Trt), 129.47 (2CH) (minor),
144.72 (5-C), 144.89 (3C, Trt), 159.27 (COMe) (minor), 159.47
(COMe) (major), 164.07 (d, JC–F¼258.5 Hz, 2-C) (minor), 164.17 (d,
JC–F¼258.3 Hz, 2-C) (major),169.51 (CO),174.92 (CO) (minor),175.21
(CO) (major) ppm. MS (EI): m/z (%): 403 (6) [MꢂTrt]þ. HRMS (EI)
calculated for C38H35O5N4F: 646.2591; found: 646.2573.
(m, 4H, OCH3, 5-H), 2.83–2.73 (m, 2H,
(100 MHz, CDCl3): 38.00 (
52.17 (2-CH), 55.29 (OCH3), 70.69 (C(
b
-H) ppm. 13C NMR
d
b
-CH2), 49.8 (NCH2), 49.91 (5-CH2),
)3), 114.15 (2CH), 115.65 (10-
f
CH), 117.82 (6-C), 125.21 (7-CH), 126.00 (9-CH), 127.01 (3CH, Trt),
127.92 (6CH, Trt), 128.44 (CCH2), 128.72 (6CH, Trt), 129.54 (2CH),
137.47 (8-C), 144.48 (3C, Trt), 151.31 (11-C), 159.35 (COMe), 169.36
(CO), 169.39 (CO) ppm. HRMS (EI) calculated for C38H34O5N4:
626.2529; found: 626.2516. MS (EI): m/z (%)¼383 (37) [MꢂTrt]þ.
Melting point: 117 ꢀC.
4.2.9. 2-(4-tert-Butoxybenzyl)-4-(4-methoxybenzyl)-7-nitro-
1,2,4,5-tetrahydro-3H-1,4-benzodiazepin-3-one (4)
Amine 3a (1.87 g, 3.67 mmol) was dissolved in DMSO (10 ml),
triethylamine (0.19 ml, 11.02 mmol), and H2O (0.72 ml,
40.41 mmol). After stirring the mixture overnight at room tem-
perature under Ar, EtOAc (50 ml) and saturated NaHCO3 (50 ml)
were added. The layers were separated and the organic layer was
washed with brine, dried (MgSO4), and evaporated. After column
chromatography (1% MeOH in DCM), product 4 was obtained as
4.3. Deprotection of the side chain groups R2 and R4
4.3.1. 2-[4-(4-Hydroxybenzyl)-7-nitro-3-oxo-2,3,4,5-tetrahydro-
1H-1,4-benzo-diazepin-2-yl]acetamide (7)
To a cooled solution of benzodiazepine 4 (50 mg, 0.1 mmol) in
dry DCM (2 ml), BBr3 (0.51 ml of 1 M solution in DCM, 0.51 mmol)
was added. After stirring 1 h at 0 ꢀC under Ar, MeOH (5 ml) and H2O
(5 ml) were added. The organic solvents were evaporated and the
aqueous layer was extracted with EtOAc (3ꢁ2 ml). The organic layer
was dried (MgSO4) and evaporated. Product 7 was obtained after
purification with column chromatography (EtOAc/heptane 90:10)
a yellow solid (1.59 g, 89%). 1H NMR (400 MHz, DMSO):
d 7.78 (dd,
J¼2.6, 9.1 Hz, 1H, 9-H), 7.71 (d, J¼2.6 Hz, 1H, 7-H), 7.30 (d, J¼8.3 Hz,
2H), 7.18 (d, J¼4.1 Hz, 1H, 1-NH), 7.13 (d, J¼8.6 Hz, 2H), 6.89 (d,
J¼8.3 Hz, 2H), 6.74 (d, J¼8.6 Hz, 2H), 6.63 (d, J¼9.1 Hz, 1H, 10-H),
5.45 (d, J¼17.1 Hz, 1H, 5-H), 5.24–5.19 (m, 1H, 2-H), 4.52 (dd,
J¼15.1 Hz, 2H, NCH2), 4.03 (d, J¼17.1 Hz, 1H, 5-H), 3.66 (s, 3H,
as a yellow solid (13.83 mg, 33%). 1H NMR (400 MHz, DMSO):
d 9.22
(s, 1H, OH), 9.17 (s, 1H, OH), 7.80 (dd, J¼2.6, 9.0 Hz, 1H, 9-H), 7.75 (d,
J¼2.6 Hz, 1H, 7-H), 7.19 (d, J¼8.5 Hz, 2H), 7.13 (d, J¼4.2 Hz, 1H, 1-
NH), 7.03 (d, J¼8.5 Hz, 2H), 6.68 (d, J¼8.5 Hz, 2H), 6.63 (d, J¼9.0 Hz,
OCH3), 3.20 (dd, J¼6.6, 14.3 Hz, 1H,
b
-H), 2.84 (dd, J¼7.1, 14.3 Hz, 1H,
t
b
-H), 1.27 (s, 9H, Bu) ppm. 13C NMR (100 MHz, DMSO):
d 28.60

4526
T. Deschrijver et al. / Tetrahedron 65 (2009) 4521–4529
1H, 10-H), 6.59 (d, J¼8.5 Hz, 2H), 5.41 (d, J¼16.6 Hz, 1H, 5-H), 5.17–
5.10 (m, 1H, 2-H), 4.47 (d, J¼14.6 Hz, 2H, NCH2), 4.04 (d, J¼16.6 Hz,
afford product 10 as a orange solid (42.82 mg, 81%). 1H NMR
(300 MHz, CDCl3):
d
7.93 (dd, J¼2.5, 9.3 Hz, 1H, 9-H), 7.62 (d,
1H, 5-H), 3.15 (dd, J¼6.3, 14.3 Hz, 1H,
b-H), 2.76 (dd, J¼7.3, 14.3 Hz,
J¼2.5 Hz, 1H, 7-H), 7.06 (d, J¼8.2 Hz, 2H), 6.95 (d, J¼8.5 Hz, 2H),
6.84 (d, J¼8.2 Hz, 2H), 6.73 (d, J¼9.3 Hz, 1H, 10-H), 6.69 (d,
J¼8.5 Hz, 2H), 4.67–4.43 (m, 4H, 2-H, N4CH2, 5-H), 4.16 (d,
1H, b d 35.42 (b-CH2), 49.20
-H) ppm. 13C NMR (100 MHz, DMSO):
(NCH2), 49.81 (5-CH2), 55.26 (2-CH), 115.32 (2CH), 115.37 (2CH),
115.46 (10-CH), 119.19 (6-C), 125.13 (9-CH), 126.74 (7-CH), 128.41
(CCH2), 128.53 (CCH2), 129.70 (2CH), 130.72 (2CH), 135.91 (8-C),
153.20 (11-C), 156.24 (COH), 156.95 (COH), 169.47 (3-C) ppm. MS
J¼16.8 Hz, 1H, 5-H), 3.68 (s, 3H, OCH3), 3.50–3.26 (m, 3H,
b
-H,
t
CH2CH3), 3.03 (dd, J¼6.43, 13.6 Hz, 1H,
b-H), 1.26 (s, 9H, Bu), 1.07
(t, J¼7.1 Hz, 3H, CH2CH3) ppm. 13C NMR (75 MHz, CDCl3):
d 14.29
(EI): m/z (%): 312 (66) [MꢂCH2-
f
-OH]þ, 313 (71) [MꢂCH2-
f
-
(CH2CH3), 29.49 (C(CH3)3), 36.27 (b-CH2), 46.07 (CH2CH3), 50.27
OHþH], 206 (71) [Mꢂ2(CH2-
f
-OH)þH]þ. HRMS (EI) calculated for
(N4CH2), 51.67 (5-CH2), 55.67 (2-CH), 64.89 (OCH3), 78.84
(C(CH3)3), 114.45 (2CH), 117.04 (10-C), 122.49 (6-C), 124.73 (2CH),
125.16 (9-CH), 126.52 (7-CH), 128.95 (CCH2), 129.81 (2CH), 130.13
(2CH), 132.27 (CCH2), 138.48 (8-C), 153.90 (11-C), 154.69 (COtBu),
159.60 (COMe), 169.82 (CO) ppm. MS (EI): m/z (%)¼354 (76)
C23H21O5N3: 419.1481; found: 419.1461. Melting point: 115 ꢀC.
4.3.2. 2-(4-Hydroxybenzyl)-4-[2-(4-hydroxyphenyl)ethyl]-7-nitro-
1,2,4,5-tetra-hydro-3H-1,4-benzodiazepin-3-one (8)
Benzodiazepine 5 (50 mg, 0.1 mmol) was deprotected by a pro-
cedure similar to that used for product 7. The crude residue was
purified by column chromatography (EtOAc/heptane 80:20) to af-
ford product 8 as a yellow solid (20 mg, 45%). 1H NMR (400 MHz,
[MꢂCH2-
f
-OtBu]þ. HRMS (EI) calculated for C30H35O5N3:
517.2576; found: 517.2570. Melting point: 65 ꢀC.
4.4.2. 1-Benzyl-2-(4-tert-butoxybenzyl)-4-(4-methoxybenzyl)-7-
nitro-1,2,4,5-tetrahydro-3H-1,4-benzodiazepin-3-one (11)
Benzodiazepine 4 (50 mg, 0.1 mmol) was alkylated with benzyl
bromide (0.1 mmol, 0.011 ml) by a procedure similar to that used
for product 10. After purification by column chromatography
(EtOAc/heptane 30:70), product 11 was obtained as a brown solid
DMSO):
d
9.17 (s, 1H, OH), 9.11 (s, 1H, OH), 7.96 (d, J¼1.8 Hz, 1H, 7-
H), 7.84 (dd, J¼1.8, 8.9 Hz, 1H, 9-H), 7.20–7.09 (m, 3H, 2H, 1-NH),
6.90 (d, J¼8.1 Hz, 2H), 6.72 (d, J¼8.5 Hz, 2H), 6.71–5.66 (m, 3H, 2H,
10-H), 5.37 (d, J¼16.6 Hz, 1H, 5-H), 5.09–5.00 (m, 1H, 2-H), 4.14 (d,
J¼16.6 Hz, 1H, 5-H), 3.64–3.43 (m, 2H, NCH2CH2), 3.09 (dd, J¼6.2,
13.9 Hz, 1H,
b
-H), 2.71 (dd, J¼6.4, 13.9 Hz, 1H,
b
-H), 2.56 (t, J¼7.2 Hz,
34.04 (NCH2CH2),
-CH2), 49.47 (NCH2CH2), 50.70 (5-CH2), 55.65 (2-CH), 115.76
(34.93 mg, 59%). 1H NMR (400 MHz, CDCl3):
d
7.89 (dd, J¼2.6,
9.1 Hz, 1H, 9-H), 7.69 (d, J¼2.6 Hz, 1H, 7-H), 7.37–7.32 (m, 2H, ),
7.28–7.23 (m, 3H,
2H, NCH2CH2) ppm. 13C NMR (100 MHz, DMSO):
35.73 (
d
f
b
f
), 7.08 (d, J¼8.4 Hz, 2H), 7.01 (d, J¼8.5 Hz, 2H),
(2CH), 115.84 (2CH), 116.91 (10-CH), 119.86 (6-C), 125.69 (9-CH),
126.97 (CCH2), 127.02 (7-CH), 128.94 (CCH2), 129.59 (2CH), 130.52
(2CH), 136.42 (8-C), 153.13 (11-C), 156.45 (COH), 156.60 (COH),
169.43 (3-C) ppm. MS (EI): m/z (%)¼383 (37) [MꢂTrt]þ. HRMS (EI)
calculated for C24H23O5N3: 447.1794; found: 447.1796. Melting
point: 105 ꢀC.
6.90 (d, J¼8.4 Hz, 2H), 6.75 (d, J¼8.5 Hz, 2H), 6.69 (d, J¼9.1 Hz, 1H,
10-H), 4.94 (dd, J¼5.2, 8.9 Hz, 1H, 2-H), 4.85 (d, J¼17.2 Hz, 1H, 5-H),
4.71 (d, J¼17.5 Hz,1H, CH2f), 4.66 (d, J¼14.5 Hz,1H, N4CH2), 4.57 (d,
J¼14.5 Hz, 1H, N4CH2), 4.43 (d, J¼17.5 Hz, 1H, CH2f), 4.15 (d,
J¼17.2 Hz, 1H, 5-H), 3.78 (s, 3H, OCH3), 3.42 (dd, J¼8.9, 13.7 Hz, 1H,
t
b
-H), 2.95 (dd, J¼5.2, 13.7 Hz, 1H,
b
-H), 1.33 (s, 9H, Bu) ppm. 13C
NMR (100 MHz, CDCl3):
d 28.87 (C(CH3)3), 35.37 (b-CH2), 49.87
4.3.3. 2-[4-(4-Hydroxybenzyl)-7-nitro-3-oxo-2,3,4,5-tetrahydro-
1H-1,4-benzo-diazepin-2-yl]acetamide (9)
(N4CH2), 51.45 (5-CH2), 55.24 (CH2f), 55.26 (OCH3), 63.72 (2-CH),
78.37 (C(CH3)3), 114.11 (2CH), 117.38 (10-C), 122.39 (6-C), 124.21
Benzodiazepine 6 (50 mg, 0.08 mmol) was deprotected by
a procedure similar to that used for product 7. The crude residue
was purified by column chromatography (8% MeOH in DCM) to
afford product 9 as a yellow solid (15.37 mg, 52%). 1H NMR
(2CH), 124.74 (CH,
128.62 (CCH2), 128.91 (CH,
f), 125.83 (9-C), 126.30 (CH, f), 127.43 (7-C),
f
), 129.38 (2CH), 129.79 (2CH), 131.89
(CCH2), 138.03 (CCH2f), 138.86 (8-C), 154.33 (11-C), 154.44 (COtBu),
159.28 (COMe), 168.74 (3-C) ppm. MS (EI): m/z (%)¼416 (100)
(400 MHz, DMSO):
d
9.22 (s, 1H, OH), 7.81 (dd, J¼9.0, 2.5 Hz, 1H, 9-
[MꢂCH2-
f
-OtBu]þ. HRMS (EI) calculated for C35H37O5N3: 579.2733;
H), 7.78 (d, J¼2.5 Hz, 1H, 7-H), 7.44 (s, 1H, NH2), 7.25 (d, J¼3.2 Hz,
1H, 1-NH), 7.04 (d, J¼8.3 Hz, 2H), 6.87 (s, 1H, NH2), 6.64 (d, J¼9.1 Hz,
1H, 10-H), 6.60 (d, J¼8.3 Hz, 2H), 5.36 (d, J¼16.6 Hz, 1H, 5-H), 5.26
(m, 1H, 2-H), 4.47 (dd, J¼14.5 Hz, 2H, NCH2), 4.02 (d, J¼16.6 Hz, 1H,
found: 579.27460. Melting point: 57 ꢀC.
4.4.3. 1-Allyl-2-(4-tert-butoxybenzyl)-4-(4-methoxybenzyl)-7-
nitro-1,2,4,5-tetrahydro-3H-1,4-benzodiazepin-3-one (12)
5-H), 2.80 (dd, J¼15.8, 8.2 Hz, 1H,
b
-H), 2.43 (dd, J¼15.8, 2.5 Hz, 1H,
36.29 ( -CH2), 49.10
Benzodiazepine 4 (50 mg, 0.1 mmol) was alkylated with allyl
bromide (0.1 mmol, 0.008 ml) by a procedure similar to that used for
product 10. After purification by column chromatography (EtOAc/
heptane 30:70), product 12 was obtained as a brown oil (28.21 mg,
b
-H) ppm. 13C NMR (100 MHz, DMSO):
d
b
(NCH2), 49.77 (5-CH2), 50.96 (2-CH), 115.34 (10-CH), 115.38 (2CH),
119.05 (6-C), 125.23 (9-CH), 126.76 (7-CH),128.28 (CCH2), 129.60
(2CH), 135.95 (8-C), 153.33 (11-C), 156.96 (C-OH), 169.33 (CONH2),
170.71 (3-C) ppm. MS (EI): m/z (%): 353 (46) [MꢂOH]þ, 263 (78)
52%). 1H NMR (400 MHz, CDCl3):
d
7.88 (dd, J¼2.7, 9.2 Hz, 1H, 9-H),
7.59 (d, J¼2.7 Hz, 1H, 7-H), 7.06 (d, J¼8.4 Hz, 2H), 6.93 (d, J¼8.6 Hz,
2H), 6.85 (d, J¼8.4 Hz, 2H), 6.71 (d, J¼9.2 Hz, 1H, 10-H), 6.67 (d, J¼8.6
Hz, 2H), 5.82–5.71 (m, 1H, N1CH2CHCH2), 5.19–5.12 (m, 2H,
N1CH2CHCH2), 4.74 (dd, J¼5.3, 8.7 Hz,1H, 2-H), 4.68 (d, J¼16.8 Hz, 1H,
5-H), 4.50 (dd, J¼14.6 Hz, 2H, N4CH2), 4.07 (d, J¼16.8 Hz, 1H, 5-H),
4.05–3.99 (m, 1H, N1CH2CHCH2), 3.78–3.72 (m, 1H, N1CH2CHCH2),
[MꢂCH2-
f
-OH]þ, 247 (69) [MꢂNH2, ꢂCH2-
f
-OH]þ. HRMS (EI)
calculated for C18H18O5N4: 370.1277; found: 370.1261. Melting
point: 142 ꢀC.
4.4. Alkylation of the N1-position
3.69 (s, 3H, OCH3), 3.37 (dd, J¼13.6, 8.7 Hz, 1H,
b
-H), 3.03 (dd, J¼13.6,
-H), 1.26 (s, 9H, Bu) ppm. 13C NMR (100 MHz, CDCl3):
28.87 (C(CH3)3), 35.32 (
-CH2), 49.81 (N4CH2), 51.29 (5-CH2), 53.70
t
4.4.1. 2-(4-tert-Butoxybenzyl)-1-ethyl-4-(4-methoxybenzyl)-7-
nitro-1,2,4,5-tetrahydro-3H-1,4-benzodiazepin-3-one (10)
5.3 Hz, 1H, b
d
b
Benzodiazepine 4 (50 mg, 0.1 mmol) was dissolved in dry DMF
(5 ml) at 0 ꢀC. NaH (60%) (4 mg, 0.1 mmol) was added and the
reaction was stirred for 10 min at 0 ꢀC under Ar. After adding
iodoethane (0.01 ml, 0.1 mmol), the solution was further stirred
for 4 h at 50 ꢀC. Brine (20 ml) was added and the aqueous layer
was extracted with EtOAc (3ꢁ5 ml). The organic layers were col-
lected, dried (MgSO4), and evaporated. The crude residue was
purified by column chromatography (EtOAc/heptane 50:50) to
(N1CH2CHCH2), 55.25 (OCH3), 63.50 (2-CH), 78.38 (C(CH3)3), 114.08
(2CH), 117.30 (10-CH), 117.39 (N1CH2CHCH2), 122.18 (6-C), 124.22
(2CH), 124.64 (9-CH), 125.76 (7-CH), 128.61 (N4CH2), 129.39 (2CH),
129.77 (2CH), 131.91 (CCH2), 134.20 (N1CH2CHCH2), 138.61 (8-C),
154.25 (11-C), 154.35 (COtBu), 159.27 (COMe), 168.87 (3-C) ppm. MS
(EI): m/z (%)¼514 (12) [MꢂCH3]þ, 487 (10) [MꢂCH2CHCH2-H]þ, 367
(100) [MꢂCH2CHCH2, ꢂCH2-
C31H35O5N3: 529.2576; found: 529.2577.
f
-OMe]þ. HRMS (EI) calculated for

T. Deschrijver et al. / Tetrahedron 65 (2009) 4521–4529
4527
4.4.4. 1-Phenyl-2-(4-tert-butoxybenzyl)-4-(4-methoxybenzyl)-7-
nitro-1,2,4,5-tetrahydro-3H-1,4-benzodiazepin-3-one (13)
Benzodiazepine 4 (50 mg, 0.1 mmol) was alkylated with meth-
ylbromoacetate (0.1 mmol, 0.010 ml) by a procedure similar to that
used for product 10. After purification by HPLC (1% MeOH in DCM),
product 13 was obtained as a yellow-brown oil (17.95 mg, 32%). 1H
155.70 (11-C),159.20 (COMe),168.29 (CO),169.65 (CO) ppm. MS (EI):
m/z (%)¼397 (33) [MꢂTrt]þ, 354 (17) [MꢂCONHTrt]þ, 243 (100)
[Trt]þ. HRMS (EI) calculated for C39H36O5N4: 640.2685; found:
640.2656. Melting point: 93 ꢀC.
4.5. Acylation of the reduced nitro group
NMR (400 MHz, CDCl3):
d
7.99 (dd, J¼2.3, 9.1 Hz, 1H, 9-H), 7.70 (d,
J¼2.3 Hz, 1H, 7-H), 7.19 (d, J¼8.3 Hz, 2H), 7.00 (d, J¼8.4 Hz, 2H), 6.92
(d, J¼8.3 Hz, 2H), 6.73 (d, J¼8.4 Hz, 2H), 6.69 (d, J¼9.1 Hz, 1H, 10-H),
4.94 (d, J¼17.2 Hz,1H, 5-H), 4.88 (dd, J¼4.4, 9.4 Hz,1H, 2-H), 4.73 (d,
J¼14.5 Hz, 1H, N4CH2), 4.43 (d, J¼14.5 Hz, 2H, N4CH2), 4.33 (d,
J¼18.6 Hz, 1H, N1CH2), 4.00 (d, J¼17.2 Hz, 1H, 5-H), 3.90 (d,
J¼18.6 Hz, 1H, N1CH2), 3.78 (s, 3H, OCH3), 3.73 (3H, OCH3), 3.44 (dd,
4.5.1. N-[2-(4-tert-Butoxybenzyl)-4-(4-methoxybenzyl)-3-oxo-
2,3,4,5-tetra-hydro-1H-1,4-benzodiazepin-7-yl]acetamide (16)
To a solution of benzodiazepinone 4 (30 mg, 0.06 mmol) in
MeOH (3 ml), 3 mg Pd (10% on carbon) catalyst was added. After 3 h
shaking under hydrogen (2.7 atm) in the Parr apparatus, the catalyst
was filtrated off and washed. MeOH was evaporated and a white oil
was obtained. The aniline derivate was dissolved without further
purification in DCM (3 ml) and acetic anhydride (0.05 ml,
0.06 mmol) was added. After stirring the reaction mixture for 2 h,
DCM was evaporated and the residue was redissolved in EtOAc. The
solution was washed with saturated NaHCO3, brine, dried (MgSO4),
and evaporated. Purification with column chromatography (EtOAc/
heptane 95:5) afforded 16 as a light yellow solid (22.14 mg, 72%). 1H
J¼9.4, 12.9 Hz, 1H,
b
-H), 3.02 (dd, J¼4.4, 13.2 Hz, 1H,
9H, Bu) ppm. 13C NMR (100 MHz, CDCl3):
28.86 (C(CH3)3), 34.14
-CH2), 49.61 (N4CH2), 51.46 (5-CH2), 52.65 (N1CH2), 52.60 (OCH3),
b-H), 1.31 (s,
t
d
(b
55.25 (OCH3), 61.64 (2-CH), 78.42 (C(CH3)3), 114.06 (2CH), 117.32
(10-CH), 122.61 (6-C), 124.28 (2CH), 124.74 (9-CH), 125.90 (7-CH),
128.55 (CCH2N), 129.34 (2CH), 129.91 (2CH), 131.42 (CCH2), 139.3
(8-C), 154.16 (11-C), 154.34 (COtBu), 159.22 (COMe), 168.54 (3-C),
170.86 (CO2CH3) ppm. MS (EI): m/z (%)¼398 (94) [MꢂCH2-
f
-
NMR (400 MHz, CDCl3):
d
7.20 (d, J¼8.3 Hz, 2H), 7.19–7.17 (m, 1H, 7-
OtBu]þ. HRMS (EI) calculated for C31H35O7N3: 561.2475; found:
561.2491.
H), 7.13 (d, J¼8.3 Hz, 2H), 7.01 (d, J¼0.8 Hz, 1H, NH), 6.94 (d,
J¼8.3 Hz, 2H), 6.95–6.93 (m,1H, 9-H), 6.79 (d, J¼8.3 Hz, 2H), 6.33 (d,
J¼8.5 Hz, 1H, 10-H), 5.07 (d, J¼16.4 Hz, 1H, 5-H), 4.90 (d, J¼14.6 Hz,
1H, NCH2), 4.79–4.76 (m, 1H, 2-H), 4.30 (d, J¼14.6 Hz, 1H, NCH2),
3.77 (s, 3H, OCH3), 3.74 (d, J¼16.4 Hz, 1H, 5-H), 3.64 (s, 1H, NH), 3.36
4.4.5. 2-[4-(4-Methoxybenzyl)-7-nitro-3-oxo-1,3,4,5-tetrahydro-
2H-1,4-benzodiazepin-2-ylidene]-N-tritylethanamide (14)
Product 14 was obtained by attempts of alkylation of benzodi-
(dd, J¼14.4, 4.9 Hz, 1H,
b
-H), 2.89 (dd, J¼14.4, 8.6 Hz, 1H,
b-H), 2.08
t
azepine
6
(50 mg, 0.08 mmol) with iodomethane (0.015 ml,
(s, 3H, CH3CO), 1.34 (s, 9H, Bu) ppm. 13C NMR (100 MHz, CDCl3):
0.08 mmol) by a procedure similar to that used for product 10. After
purification by column chromatography (0.5% MeOH in DCM),
product 14 was obtained as a yellow solid (32.40 mg, 65%). 1H NMR
d
24.18 (CH3CO), 28.88 (C(CH3)3), 36.92 ( -H), 49.73 (NCH2), 50.34
b
(5-CH2), 55.27 (OCH3), 56.30 (2-CH), 78.41 (C(CH3)3), 114.03 (2CH),
117.19 (10-CH), 120.40 (6-C), 121.43 (9-CH), 122.00 (7-CH), 124.32
(2CH), 128.80 (CCH2), 129.24 (CCH2), 129.50 (2CH), 129.69 (2CH),
132.21 (8-C), 142.58 (11-C), 154.30 (COtBu), 159.07 (COMe), 168.07
(400 MHz, CDCl3):
d
11.54 (s, 1H, NHTrt), 7.99 (dd, J¼2.3, 8.7 Hz, 1H,
9-H), 7.52 (d, J¼2.3 Hz, 1H, 7-H), 7.35–7.10 (m, 15H, Trt), 7.15 (d,
J¼8.5 Hz, 2H), 6.90 (s, 1H, NH), 6.85 (d, J¼8.7 Hz, 1H, 10-H), 6.83 (d,
J¼8.5 Hz, 2H), 5.85 (s, 1H, C2CH), 4.59 (s, 2H, 5-H), 4.19 (s, 2H,
(CO), 170.56 (CO) ppm. MS (EI): m/z (%)¼338 (68) [MꢂCH2-
f-
OtBu]þ. HRMS (EI) calculated for C30H35O4N3: 501.2627; found:
501.2634. Melting point: 183 ꢀC.
N4CH2), 3.79 (s, 3H, OCH3) ppm. 13C NMR (100 MHz, CDCl3):
d
47.22
(N4CH2), 50.05 (5-CH2), 55.33 (OCH3), 70.78 (C(
f
)3), 98.06 (C2CH),
114.32 (2CH), 120.02 (10-CH), 123.39 (7-CH), 123.74 (6-C), 125.01
(9-CH), 127.11 (3CH, Trt), 127.47 (CCH2), 128.04 (6CH, Trt), 128.25
(6CH, Trt), 129.97 (2CH), 141.76 (2-C), 144.48 (8-C), 145.80 (3C, Trt),
149.11 (11-C), 159.77 (COMe), 162.93 (CONHTrt), 168.52 (3-C) ppm.
MS (EI): m/z (%)¼381 (15) [MꢂTrt]þ. HRMS (EI) calculated for
C38H32O5N4: 624.2372; found: 624.2362. Melting point: 133 ꢀC.
4.5.2. N-{2-(4-tert-Butoxybenzyl)-4-[2-(4-methoxyphenyl)-
ethyl]-3-oxo-2,3,4,5-tetrahydro-1H-1,4-benzodiazepin-7-yl}-
acetamide (17)
Benzodiazepinone 5 (30 mg, 0.06 mmol) was reduced and ac-
ylated by the same procedure as described for product 16. After
column chromatography (EtOAc/heptane 95:5), product 17 was
obtained as a light yellow solid (24.27 mg, 79%). 1H NMR (400 MHz,
4.4.6. 2-[4-(4-Methoxybenzyl)-1-methyl-7-nitro-3-oxo-
CDCl3):
d
7.24–7.23 (m, 1H, 7-H), 7.17 (d, J¼8.4 Hz, 2H), 7.03 (d,
tetrahydro-1H-1,4-benzodiazepin-2-yl]-N-tritylacetamide (15)
Benzodiazepine 6 (50 mg, 0.08 mmol) was dissolved in degassed
and dry DMF (5 ml) at 0 ꢀC. NaH (60%) (8 mg, 0.16 mmol) was added
and the reaction was stirred for 10 min at 0 ꢀC under Ar. After adding
iodomethane (0.015 ml, 0.08 mmol), the solutionwas further stirred
for 4 h at 50 ꢀC. Brine (20 ml) was added and the aqueous layer was
extracted with EtOAc (3ꢁ5 ml). The organic layers were collected,
dried (MgSO4), and evaporated. The crude residue was purified by
column chromatography (0.5% MeOH in DCM) to afford product 15
J¼1.5 Hz, 1H, NH), 7.00 (d, J¼8.4 Hz, 2H), 6.93–6.90 (m, 1H, 9-H),
6.94 (d, J¼8.4 Hz, 2H), 6.73 (d, J¼8.4 Hz, 2H), 6.32 (d, J¼8.5 Hz, 1H,
10-H), 5.14 (d, J¼16.4 Hz, 1H, 5-H), 4.72–4.66 (m, 1H, 2-H), 3.78–
3.70 (m, 1H, NCH2CH2), 3.76 (s, 3H, OCH3), 3.66–3.59 (m, 3H, NH,
NCH2CH2, 5-H), 3.30 (dd, J¼5.1, 14.3 Hz, 1H,
14.3 Hz, 1H,
-H), 2.74 (t, J¼6.9 Hz, 2H, NCH2CH2), 2.09 (s, 3H,
CH3CO), 1.33 (s, 9H, Bu) ppm. 13C NMR (100 MHz, CDCl3):
(CH3CO), 28.87 (C(CH3)3), 33.78 (NCH2CH2), 36.78 ( -CH2), 50.78 (5-
CH2), 52.73 (NCH2CH2), 55.23 (OCH3), 56.15 (2-CH), 78.39 (C(CH3)3),
113.92 (2CH), 117.16 (10-CH), 120.70 (6-C), 121.40 (9-CH), 121.90 (7-
CH), 124.32 (2CH), 129.68 (2CH), 129.74 (2CH), 130.99 (CCH2),
132.24 (8-C), 142.50 (11-C), 150.73 (CH2C), 154.27 (COtBu), 158.12
(COMe), 168.13 (3-C), 170.23 (CH3CO) ppm. MS (EI); m/z (%): 352
b
-H), 2.82 (dd, J¼8.4,
b
t
d
24.18
b
as a yellow solid (30.67 mg, 60%). 1H NMR (400 MHz, CDCl3):
d 7.98
(dd, J¼2.7, 9.2 Hz, 1H, 9-H), 7.70 (d, J¼2.7 Hz, 1H, 7-H), 7.37–7.18 (m,
15H, Trt), 7.10 (s,1H, NH), 7.02 (d, J¼8.5 Hz, 2H), 6.79 (d, J¼9.2 Hz,1H,
10-H), 6.73 (d, J¼8.6 Hz, 2H), 5.10 (d, J¼17.0 Hz, 1H, 5-H), 5.11–5.06
(m, 1H, 2-H), 4.86 (d, J¼14.6 Hz, 1H, N4CH2), 4.37 (d, J¼14.7 Hz, 1H,
N4CH2), 3.89 (d, J¼17.0 Hz, 1H, 5-H), 3.78 (s, 3H, OCH3), 3.31 (dd,
(100) [MꢂCH2-
f
-OtBu]þ. HRMS (EI) calculated for C31H37O4N3:
515.2784; found: 515.2788. Melting point: 174 ꢀC.
J¼13.8,10.0 Hz,1H,
1H,
b
-H), 2.99 (s, 3H, N1CH3), 2.64 (dd, J¼13.8, 3.5 Hz,
b
-H) ppm. 13C NMR (100 MHz, CDCl3):
d
36.90 (
b
-CH2), 37.21
4.5.3. 2-[7-(Acetylamino)-4-(4-methoxybenzyl)-3-oxo-2,3,4,5-
tetrahydro-1H-1,4-benzodiazepin-2-yl]-N-tritylacetamide (18)
Benzodiazepinone 6 (30 mg, 0.05 mmol) was reduced and acy-
lated by the same procedure as described for product 16. After
column chromatography (EtOAc/heptane 95:5), product 18 was
(N1CH3), 49.58 (NCH2), 51.68 (5-CH2), 55.29 (2-CH), 55.74 (OCH3),
70.68 (C( )3), 114.21 (2 CH), 118.98 (10-C), 122.77 (6-C), 124.42 (7-
f
CH), 125.79 (9-CH), 127.09 (3CH, Trt), 128.02 (6CH, Trt), 128.35
(CCH2),128.62 (6CH, Trt),129.32 (2CH),139.07 (8-C),144.44 (3C, Trt),

4528
T. Deschrijver et al. / Tetrahedron 65 (2009) 4521–4529
obtained as a light yellow solid (24.15 mg, 79%). 1H NMR (400 MHz,
CDCl3): 8.10 (s,1H, NHTrt), 7.58 (s,1H, 7-H), 7.24–7.18 (m,15H, Trt),
(COtBu), 158.15 (COMe), 168.49 (CO), 170.17 (CO) ppm. MS (ESI,
d
MeOH): 1074.9 [MNaþ]. Melting point: 131 ꢀC.
7.02 (d, J¼8.1 Hz, 2H), 6.99 (s, 1H, NH), 6.73 (d, J¼8.1 Hz, 2H), 6.67
(d, J¼8.1 Hz, 1H, 9-H), 6.07 (d, J¼8.2 Hz, 1H, 10-H), 4.96 (d,
J¼16.6 Hz, 1H, 5-H), 4.89–4.86 (m, 1H, 2-H), 4.83 (d, J¼14.6 Hz, 1H,
NCH2), 4.14 (d, J¼14.6 Hz, 1H, NCH2), 3.6 (s, 1H, NH), 3.74 (s, 3H,
4.5.6. Compound (20)
Aniline derivate 22 (88.97 mg, 0.19 mmol) was coupled to Fmoc-
L
-Asp(OtBu)-OH (0.078 mg, 0.19 mmol) by a procedure similar to
OCH3), 3.59 (d, J¼16.6 Hz, 1H, 5-H), 2.86–2.74 (m, 2H,
b
-H), 1.93 (s,
24.01 (CH3CO), 39.24
-CH2), 49.74 (NCH2), 50.62 (5-CH2), 52.64 (OCH3), 55.27 (2-CH),
70.63 (C( )3), 114.10 (2CH), 117.34 (10-CH), 119.42 (6-C), 121.75
that used for product 23. After purification by column chroma-
tography (EtOAc/DCM 10:90), product 23 was obtained as a pale
3H, CH3CO) ppm. 13C NMR (100 MHz, CDCl3)
d
(b
white solid (111 mg, 68%). 1H NMR (400 MHz, CDCl3):
d 8.24 (br s,
f
1H), 7.75 (d, J¼7.4 Hz, 2H), 7.57 (d, J¼7.4 Hz, 2H), 7.37 (t, J¼7.4 Hz,
2H), 7.32–7.24 (m, 3H), 7.18 (d, J¼8.5 Hz, 2H), 7.02–6.92 (m, 6H),
6.73 (d, J¼8.5 Hz, 2H), 6.33 (d, J¼8.8 Hz, 1H), 6.14–6.03 (br s, 1H),
(9-CH), 122.11 (7-CH), 126.90 (3CH, Trt), 127.85 (6CH, Trt), 128.73
(6CH, Trt), 128.88 (CCH2), 129.34 (2CH), 134.82 (8-C), 141.18 (11-C),
144.59 (3C, Trt), 159.07 (COMe), 168.43 (CO), 169.74 (CO),
170.71 (CO) ppm. MS (EI): m/z (%): 395 (100) [MꢂTrt]þ. HRMS (EI)
calculated for C40H38O4N4: 638.2893; found: 638.2905. Melting
point: 132 ꢀC.
5.13 (d, J¼16.6 Hz, 1H, 5-H), 4.72–4.66 (m, 1H,
a-H), 4.66–4.57 (m,
1H,
3.74 (s, 3H, OCH3), 3.68 (dd, J¼7.1, 14.5 Hz, 2H, NCH2CH2), 3.60 (d,
J¼16.6 Hz, 1H, 5-H), 3.31 (dd, J¼5.4, 14.2 Hz, 1H, -H), 2.92 (dd,
J¼3.3, 16.9 Hz, 1H, -H), 2.83 (dd, J¼8.3, 14.2 Hz, 1H, -H), 2.75 (t,
J¼7.1 Hz, 2H, NCH2CH2), 2.67 (dd, J¼7.1,16.9 Hz,1H, -H),1.44 (s, 9H,
tBu), 1.33 (s, 9H, tBu) ppm. 13C NMR (100 MHz, CDCl3):
28.05
(C(CH3)3), 28.87 (C(CH3)3), 33.75 (NCH2CH2), 36.71 ( -CH2), 37.53
-CH2), 47.15 (CH, Fmoc), 50.74 (NCH2CH2), 51.75 ( -CH), 52.68 (5-
CH2), 55.22 (OCH3), 56.01 -CH), 67.35 (CH2, Fmoc), 78.41
a
-H), 4.45 (d, J¼6.8 Hz, 2H, Fmoc), 4.21 (t, J¼6.8 Hz, 1H, Fmoc),
b
b
b
4.5.4. 7-Amino-2-(4-tert-butoxybenzyl)-4-[2-(4-methoxyphenyl)-
ethyl]-1,2,4,5-tetrahydro-3H-1,4-benzodiazepin-3-one (22)
b
d
Benzodiazepinone 5 (100 mg, 0.19 mmol) was dissolved in
MeOH (5 ml) and 5 mg Pd (10% on carbon) was added. After 3 h
shaken under hydrogen (40 atm) in the Parr apparatus, the cat-
alyst was filtrated off and washed. MeOH was evaporated and
product 22 was obtained as a white oil (88.97 mg, 99%). 1H NMR
b
(b
a
(
a
(C(CH3)3), 82.11 (C(CH3)3), 113.89 (2CH), 117.16 (10CH), 120.06 (2CH,
Fmoc), 120.52 (6-C), 121.23 (9-CH), 121.50 (7-CH), 124.34 (2CH),
124.97 (2CH, Fmoc), 127.11 (2CH, Fmoc), 127.81 (2CH, Fmoc), 128.30
(CCH2), 129.71 (2CH), 129.75 (2CH), 130.09 (CCH2), 132.27 (8-C),
141.34 (2C, Fmoc), 142.65 (11-C), 143.63 (2C, Fmoc), 154.23 (COMe),
158.12 (COtBu), 163.38 (CO), 171.3 (CO), 170.18 (CO), 168.19
(CO) ppm. MS (ESI, MeOH): 867.7 [MHþ], 889.7 [MNaþ], 1733.5
[2MHþ]. Melting point: 83 ꢀC.
(400 MHz, CDCl3):
d
7.18 (d, J¼8.3 Hz, 2H), 7.03 (d, J¼8.4 Hz, 2H),
6.93 (d, J¼8.4 Hz, 2H), 6.77 (d, J¼8.4 Hz, 2H), 6.42 (d, J¼8.1 Hz, 1H,
7-H), 6.31 (d, J¼8.1 Hz, 1H, 9-H), 6.17 (s, 1H, 10-H), 4.93 (d,
J¼16.3 Hz, 1H, 5-H), 4.57–4.46 (m, 1H, 2-H), 3.75 (s, 3H, OCH3),
3.74–3.54 (m, 3H, 5-H, NCH2CH2), 3.30 (dd, J¼5.0, 14.2 Hz, 1H,
CH2), 3.16–2.76 (br s, 2H, NH2), 2.82 (dd, J¼8.4, 14.2 Hz, 1H, CHb2),
2.75 (t, J¼6.8 Hz, 2H, NCH2CH2), 1.28 (s, 9H, Bu) ppm. 13C NMR
t
(100 MHz, CDCl3):
d
28.87 (C(CH3)3), 33.75 (NCH2CH2), 37.29 (
b
-
4.5.7. Compound (21)
CH2), 50.91 (5-CH2), 52.56 (NCH2CH2), 55.22 (OCH3), 57.66 (2-
CH), 78.38 (C(CH3)3), 113.87 (2CH), 116.14 (9-CH), 116.41 (10-CH),
119.20 (7-CH), 123.34 (6-C), 124.26 (2CH), 129.79 (4CH), 131.07
(CCH2),132.79 (CCH2),138.12 (8-C),138.71 (11-C),154.07 (COtBu),
158.11 (COMe), 170.81 (CO) ppm. HRMS (EI) calculated for
C29H35O3N3: 473.2678; found: 473.2672.
Aniline derivate 22 (87.32 mg, 0.19 mmol) was coupled to Fmoc-
L
-Tyr(OtBu)-OH (0.078 mg, 0.19 mmol) bya procedure similar to that
used for product 23. After purification by column chromatography
(EtOAc/heptane 50:50), product 23 was obtained as a pale white
solid (79.88 mg, 46%). 1H NMR (400 MHz, CDCl3):
d
7.73 (d, J¼7.3 Hz,
2H), 7.52 (d, J¼7.3 Hz, 2H), 7.47–7.42 (br s,1H), 7.36 (t, J¼7.4 Hz, 2H),
7.29–7.25 (m, 4H), 7.17 (d, J¼8.4 Hz, 2H), 7.12–7.04 (m, 2H), 7.01 (d,
J¼8.2 Hz, 2H), 6.97 (d, J¼8.4 Hz, 2H), 6.94 (d, J¼8.4 Hz, 2H), 6.80 (s,
2H), 6.70 (d, J¼8.4 Hz, 2H), 6.24 (d, J¼8.8 Hz, 1H), 5.17 (d, J¼16.5 Hz,
4.5.5. Compound (19)
Aniline derivate 22 (88.97 mg, 0.19 mmol) was dissolved with-
out further purification in dry DMF (10 ml). Pybop (97.87 mg,
1H, 5-H), 4.70–4.62 (m, 1H, a-H), 4.51–4.26 (m, 3H, a-H, Fmoc), 4.16
0.19 mmol), Fmoc-
L
-Asn(NHTrt)-OH (0.113 mg, 0.19 mmol), and
(t, J¼6.7 Hz, 1H, Fmoc), 3.71 (s, 3H, OCH3), 3.69–3.58 (m, 2H,
DIPEA (0.017 ml, 0.19 mmol) were added. After stirring the solution
overnight under Ar at room temperature, DMF was evaporated and
the crude mixture was purified by column chromatography (EtOAc/
heptane 50:50). Product 23 was obtained as a pale white solid
NCH2CH2), 3.52 (d, J¼16.5 Hz, 1H, 5-H), 3.30 (dd, J¼5.3, 14.4 Hz, 1H,
b
-H), 3.16–2.96 (m, 2H,
b
-H), 2.81(dd, J¼8.4,14.4 Hz,1H,
b-H), 2.72 (t,
t
t
J¼7.2 Hz, 2H, NCH2CH2), 1.33 (s, 9H, Bu), 1.28 (s, 9H, Bu) ppm. 13
C
NMR (100 MHz, CDCl3):
d 28.83 (C(CH3)3), 28.87 (C(CH3)3), 33.74
(69.89 mg, 35%). 1H NMR (400 MHz, CDCl3):
d
8.57 (s, 1H), 7.71 (d,
(NCH2CH2), 36.70 (
(NCH2CH2), 52.60 (5-CH2), 55.24 (OCH3), 55.96 (
b
-CH2), 38.18 (
b
-CH2), 47.12 (CH, Fmoc), 50.72
-CH), 57.03 ( -CH),
J¼7.2 Hz, 2H), 7.53 (d, J¼7.2 Hz, 2H), 7.35 (t, J¼7.2 Hz, 2H), 7.28–7.04
(m, 21H), 6.98 (d, J¼8.1 Hz, 2H), 6.97–6.91 (m, 4H), 6.72 (d,
J¼8.4 Hz, 2H), 6.48 (d, J¼5.3 Hz, 1H), 6.31 (d, J¼9.1 Hz, 1H), 5.12 (d,
a
a
67.19 (CH2, Fmoc), 78.41 (C(CH3)3), 78.47 (C(CH3)3), 113.91 (2CH),
117.05 (10-CH), 120.02 (2CH, Fmoc), 120.40 (9-CH), 121.56 (7-CH),
121.81 (6-C), 124.34 (2CH), 124.37 (2CH), 124.98 (2CH), 127.12 (2CH,
Fmoc), 127.79 (2CH, Fmoc), 128.23 (CCH2), 128.45 (CCH2), 129.70
(2CH), 129.75 (2CH), 129.84 (2CH), 130.91 (CCH2), 132.25 (8-C),
141.31 (2C, Fmoc), 142.77 (11-C), 143.66 (2C, Fmoc), 154.25 (COtBu),
154.57 (COtBu), 158.07 (COMe), 168.89 (CO), 170.13 (CO) ppm. MS
(ESI, MeOH): 937.9 [MNaþ], 1851.4 [2MNaþ]. Melting point: 110 ꢀC.
J¼16.2 Hz, 1H, 5-H), 4.74–4.66 (m, 1H,
a-H), 4.66–4.56 (m, 1H, a-H),
4.38 (d, J¼6.8 Hz, 2H, Fmoc), 4.15 (t, J¼6.8 Hz, 1H, Fmoc), 3.70 (s, 3H,
OCH3), 3.67–3.61 (m, 2H, NCH2CH2), 3.56 (d, J¼16.2 Hz, 1H, 5-H),
3.32 (dd, J¼5.3, 14.4 Hz, 1H,
b-H), 3.14–3.04 (m, 1H, b-H), 2.82 (dd,
J¼8.4, 14.4 Hz, 1H,
J¼6.6, 15.5 Hz, 1H,
b-H), 2.78–2.68 (m, 2H, NCH2CH2), 2.63 (dd,
t
b
-H), 2.33 (s, 9H, Bu) ppm. 13C NMR (100 MHz,
CDCl3):
d
28.92 (C(CH3)3), 33.78 (NCH2CH2), 36.76 (
b
a
-CH2), 38.97 (
-CH), 52.62 (5-
)3),
b-
CH2), 47.15 (CH-Fmoc), 50.62 (NCH2CH2), 51.93 (
CH2), 55.23 (
4.5.8. N1-{2-(4-tert-Butoxybenzyl)-4-[2-(4-methoxyphenyl)ethyl]-
3-oxo-2,3,4,5-tetrahydro-1H-1,4-benzodiazepin-7-yl}-N4-
tritylaspartamide (23)
Fmoc-protected compound 19 (69.89 mg, 0.06 mmol) was dis-
solved in a DMF/piperidine (4:1) mixture and stirred for 1 h at room
temperature. After evaporation of the solvent, purification by col-
umn chromatography (2% MeOH in DCM) and preparative TLC (1%
MeOH in DCM), compound 23 was obtained as an oil (24.80 mg,
a
-CH), 56.06 (OCH3), 67.25 (CH2-Fmoc), 71.00 (C(
f
78.43 (C(CH3)3), 113.92 (2CH), 117.14 (7-CH), 120.08 (2CH, Fmoc),
120.36 (6-C), 121.46 (2CH, 9-CH, 7-CH), 124.37 (2CH, Fmoc), 125.09
(2CH), 127.13 (2CH, Fmoc), 127.19 (2CH, Fmoc), 127.80 (3CH, Trt),
128.06 (3CH, Trt), 128.37 (CCH), 128.64 (6CH, Trt), 129.79 (4CH),
130.86 (CCH2), 132.36 (8-C), 141.34 (2C, Fmoc), 142.58 (11C), 143.71
(2C, Fmoc), 144.16 (CO), 145.54 (3C, Trt), 154.26 (CO), 156.30

T. Deschrijver et al. / Tetrahedron 65 (2009) 4521–4529
4529
45%). 1H NMR (400 MHz, CDCl3):
7.04–6.92 (m, 5H), 6.74 (d, J¼8.6 Hz, 2H), 6.31 (d, J¼8.6 Hz, 1H), 5.13
(d, J¼16.1 Hz, 1H, 5-H), 4.74–4.65 (m, 1H, -H), 3.74 (s, 3H, OCH3),
3.67 (t, J¼7.2 Hz, 2H, NCH2CH2), 3.70–3.63 (m, 1H, -H), 3.60 (d,
J¼16.1 Hz, 1H, 5-H), 3.32 (dd, J¼5.1, 14.4 Hz, 1H, -H), 2.88–2.63 (m,
5H, -H, -H, NCH2CH2), 2.32–2.07 (br s, 2H, NH2), 1.34 (s, 9H,
tBu) ppm. 13C NMR (100 MHz, CDCl3):
29.00 (C(CH3)3), 33.73
(NCH2CH2), 36.78 ( -CH2), 41.05 ( -CH2), 50.10 (NCH2CH2), 52.62
(5-CH2), 52.73 ( -CH), 55.22 (OCH3), 56.14 ( -CH), 70.62 (C( )3),
d
9.32 (s, 1H), 7.43–7.08 (m, 18H),
MS (EI); m/z (%): 529 (100) [MꢂCH2-
f
-OtBu]þ. HRMS (EI) calculated
for C42H52O5N3: 693.4016; found: 693.3996.
a
Acknowledgements
a
b
This research was funded by a Ph.D. grant of the institute for the
Promotion of Innovation through Science and Technology in Flan-
ders (IWT-Vlaanderen) and by IOF-KP 06/001 from the KULeuven.
W.M.D.B. thanks the F.W.O.-Vlaanderenfora ‘Krediet aan Navorsers’.
b
b
d
b
b
a
a
f
78.38 (C(CH3)3), 113.87 (2CH), 117.26 (10CH), 120.59 (6-C), 120.68
(10-CH),120.73 (9-CH), 124.31 (2CH), 127.01 (3CH, Trt), 127.94 (6CH,
Trt), 128.54 (6CH, Trt), 128.76 (CCH2), 129.81 (2CH), 129.73 (2CH),
130.90 (CCH2), 132.34 (8-C), 142.27 (11-C), 144.41 (NHCO), 144.45
(3C, Trt), 154.23 (COtBu), 158.11 (COMe), 170.16 (CO), 170.24
References and notes
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(CO) ppm. MS (EI); m/z (%): 529 (100) [MꢂCH2-
f
-OtBu]þ. MS (ESI,
3. Savage, B.; Saldivar, E.; Ruggeri, Z. M. Cell 1996, 84, 289–297.
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MeOH): 852.6 [MNaþ], 1762.3 [2MNaþ].
4.5.9. N1-{2-(4-tert-Butoxybenzyl)-4-[2-(4-methoxyphenyl)ethyl]-
3-oxo-2,3,4,5-tetrahydro-1H-1,4-benzodiazepin-7-yl}-O-(tert-
butyl)asparaginate (24)
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in a DMF/piperidine (4:1) mixture and stirred for 1 hour at room
temperature. After evaporation of the solvent, purification by column
chromatography (10% EtOAc in DCM) and preparative TLC (1% MeOH
in DCM), compound 24 was obtained as an oil (17.33 mg, 21%). 1H
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NMR (400 MHz, CDCl3):
d
9.24 (s, 1H), 7.22 (d, J¼1.7 Hz, 1H), 7.19 (d,
J¼8.2 Hz, 2H), 7.06–7.00 (m, 3H), 6.94 (d, J¼8.3 Hz, 2H), 6.75 (d,
J¼8.3 Hz, 2H), 6.35 (d, J¼8.5 Hz, 1H), 5.16 (d, J¼16.5 Hz, 1H, 5-H), 4.74–
4.65 (m, 1H, a-H), 3.76 (s, 3H, OCH3), 3.76–3.70 (m, 2H, NCH2CH2),
3.66 (d, J¼16.5 Hz, 1H, 5H), 3.57–3.53 (m, 1H,
a
b
-H), 3.32 (dd, J¼5.1,
-H), 2.83 (dd, J¼8.7,
-H), 2.76 (t, J¼7.0 Hz, 2H, NCH2CH2), 2.60 (dd, J¼8.1,
-H), 1.93–1.70 (br s, 2H, NH2), 1.45 (s, 9H, tBu), 1.34 (s, 9H,
28.11 (C(CH3)3), 28.86
-CH2), 40.36 ( -CH2), 50.71
-CH), 52.70 (5-CH2), 55.22 (OCH3), 56.12 ( -CH),
14.3 Hz, 1H,
14.3 Hz, 1H,
b
b
-H), 2.89 (dd, J¼3.6, 16.5 Hz, 1H,
16.6 Hz, 1H,
tBu) ppm. 13C NMR (100 MHz, CDCl3):
(C(CH3)3), 33.76 (NCH2CH2), 36.77 (
(NCH2CH2), 52.40 (
b
d
b
b
a
a
78.38 (C(CH3)3), 81.33 (C(CH3)3), 113.85 (2CH), 117.25 (10-CH), 120.42
(9-CH), 120.67 (7-CH), 120.71 (6-C),124.31 (2CH), 129.71 (2CH),129.75
(2CH), 128.89 (CCH2), 130.92 (CCH2), 132.33 (8-C), 142.20 (11-C),
154.21 (COtBu), 158.10 (COMe), 170.02 (CO), 171.04 (CO), 171.16
23. Ripka, W. C.; Delucca, G. V.; Bach, A. C.; Pottorf, R. S.; Blaney, J. M. Tetrahedron
1993, 49, 3593–3608.
(CO) ppm. MS (EI); m/z (%): 481 (26) [MꢂCH2-
f
-OtBu]þ. HRMS (EI)
24. Ro¨mer, D.; Bu¨scher, H. H.; Hill, R. C.; Maurer, R.; Petcher, T. J.; Zeugner, H.;
Benson, W.; Finner, E.; Milkowski, W.; Thies, P. W. Nature 1982, 298, 759–760.
25. Rosenstro¨m, U.; Sko¨ld, C.; Plouffe, B.; Beaudry, H.; Lindeberg, G.; Botros, M.;
Nyberg, F.; Wolf, G.; Karlen, A.; Gallo-Payet, N.; Hallberg, A. J. Med. Chem. 2005,
48, 4009–4024.
calculated for C37H48O6N4: 644.3574; found: 644.3583.
4.5.10. N-{2-(4-tert-butoxybenzyl)-4-[2-(4-methoxyphenyl)ethyl]-
3-oxo-2,3,4,5-tetrahydro-1H-1,4-benzodiazepin-7-yl}-O-(tert-
butyl)tyrosinamide (25)
Fmoc-protected compound 21 (79.88 mg, 0.08 mmol) was dis-
solved in a DMF/piperidine (4:1) mixture and stirred for 1 h at room
temperature. After evaporation of the solvent, purification by column
chromatography (EtOAc/heptane 80:20) and HPLC (100% EtOAC),
compound 23 was obtained as an oil (25.40 mg, 42%). 1H NMR
26. Rosenstro¨m, U.; Sko¨ld, C.; Lindeberg, G.; Botros, M.; Nyberg, F.; Karlen, A.;
Hallberg, A. J. Med. Chem. 2006, 49, 6133–6137.
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Hwang, S. M.; Jakas, D. R.; Keenan, R. M.; Ku, T. W.; Kwon, C.; Lee, C. P.; Miller,
W. H.; Newlander, K. A.; Nichols, A.; Parker, M.; Peishoff, C. E.; Rhodes, G.; Ross,
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L. C.; DeBrosse, C.; Eggleston, D. S.; Haltiwanger, R. C.; Hwang, S. M.; Jakas, D. R.;
Ku, T. W.; Miller, W. H.; Newlander, K. A.; Nichols, A.; Parker, M. F.; Southhall, L.
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(400 MHz, CDCl3):
d
9.06 (s, 1H), 7.23 (d, J¼1.0 Hz, 1H), 7.18 (d,
J¼8.4 Hz, 2H), 7.11 (d, J¼8.4 Hz, 2H), 7.05–6.98 (m, 3H), 6.97–6.91 (m,
4H), 6.75 (d, J¼8.4 Hz, 2H), 6.35 (d, J¼8.5 Hz, 1H), 5.15 (d, J¼16.4 Hz,
1H, 5-H), 4.75–4.65 (m,1H, a
ꢂH), 3.75 (s, 3H, OCH3), 3.74–3.64 (m, 4H,
5-H,
14.1 Hz, 1H,
2H, NCH2CH2), 2.73 (dd, J¼9.3, 14.1 Hz, 1H,
a
ꢂH, NCH2CH2), 3.32 (dd, J¼5.3,14.4 Hz,1H,
b
-H), 3.27 (dd, J¼3.8,
-H), 2.76 (t, J¼7.0 Hz,
-H), 1.98–1.81 (br s, 2H,
NH2), 1.45 (s, 9H, Bu), 1.34 (s, 9H, Bu) ppm. 13C NMR (100 MHz,
CDCl3): 28.11 (C(CH3)3), 28.86 (C(CH3)3), 33.78 (NCH2CH2), 36.80 (
CH2), 40.06 ( -CH2), 50.71 (NCH2CH2), 52.71 (5-CH2), 55.20 (OCH3),
56.21 ( -CH), 56.72 ( -CH), 78.35 (C(CH3)3), 78.39 (C(CH3)3), 113.89
b
-H), 2.83 (dd, J¼8.3, 14.4 Hz, 1H,
b
b
t
t
d
b-
b
a
a
(2CH), 117.27 (10-CH), 120.53 (6-C), 120.79 (7-CH), 120.81 (9-CH),
124.26 (2CH),124.33 (2CH),129.67 (2CH),129.70 (2CH),129.75 (2CH),
128.89 (CCH2), 130.95 (CCH2), 132.31 (CCH2),132.33 (8-C), 142.26 (11-
C), 154.27 (COMe), 154.44 (COtBu), 158.14 (COMe), 170.22 (2CO) ppm.
38. Van Weemen, B. K.; Schuurs, A. H. W. M. FEBS Lett. 1971, 15, 232–236.