Influence of ring substitution on the conformation and β-turn mimicry of 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one peptide mimetics

Article abstract of DOI:10.1016/j.tet.2009.01.057

Analogs of 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-ones, containing a methyl substituent at the 4- or 5-position, or a phenyl substituent at C-1, were prepared. Conformational analysis of tetrapeptide models containing these analogs indicated different

Full text of DOI:10.1016/j.tet.2009.01.057

Tetrahedron 65 (2009) 2266–2278
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Influence of ring substitution on the conformation and b-turn mimicry of 4-
amino-1,2,4,5-tetrahydro-2-benzazepin-3-one peptide mimetics
Rien De Wachter a, Luc Brans a, Steven Ballet a, Isabelle Van den Eynde a, Debby Feytens a,
b
b
c
a,
*
´
Attila Keresztes , Geza Toth , Zofia Urbanczyk-Lipkowska , Dirk Tourwe
a Organic Chemistry Department, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
b Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary
c Institute of Organic Chemistry, Polish Academy of Sciences, Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Analogs of 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-ones, containing a methyl substituent at the 4- or
5-position, or a phenyl substituent at C-1, were prepared. Conformational analysis of tetrapeptide models
containing these analogs indicated different conformations of the benzazepinone ring, and extended
backbone conformations, except for the 4-methyl-substituted analog. The latter was shown to have
a strong preference for a turn conformation. Incorporation into the N-terminal tetrapeptide sequence of
dermorphin resulted in potent opioid analogs and an indication that the receptor-bound conformation
might not adopt a turn structure.
Received 3 December 2008
Received in revised form 8 January 2009
Accepted 12 January 2009
Available online 20 January 2009
Keywords:
Benzazepinones
Dermorphin
Ó 2009 Elsevier Ltd. All rights reserved.
b-Turn
Peptidomimetics
Conformational analysis
Molecular modeling
to provide potent therapeutic agents, much effort has been devoted
to the design and synthesis of small mimetics of turn structures.3–7
Different types of tight turns have been defined, depending on
1. Introduction
It is estimated that almost half of the current drugs targets G-
protein coupled receptors (GPCRs). Designing ligands for these
receptors therefore constitutes a major method toward new phar-
maceuticals. Many of their natural ligands are peptides. Tyndall et al.
demonstrated that most peptide-activated GPCRs bind to their li-
gands through a turn structure.1 Also, turns usually occur on the
exposed surface of proteins and are likely to be involved in many of
the molecular recognition events, such as interactions between
antibodies and antigens, and regulatory enzymes and their corre-
sponding substrates.2 Therefore, to better understand the molecular
mechanism of peptide–protein or protein–protein interactions, and
the number of amino acid residues in the turn:
d-, g-, b-, a-, and p-
turns, involving 2–6 residues, respectively.2 A general definition of
a b-turn stated that any tetrapeptide chain in which the distance
between the Ca(i) and the Ca(iþ3) was <7 Å and which occurs in
a non-helical region constitutes a
b
-turn.8,9 The presence of an
intramolecular H-bond between the CO of the first residue (i) and
the amide NH of the fourth residue (iþ3) to form a pseudo 10-
membered ring is often, though not always, observed.10
One of the first examples of
b-turn mimicry was reported by
Freidinger who introduced a -lactam 1 into the luteinizing hor-
g
mone-releasing hormone sequence (Fig. 1). The modified peptide
turned out to be more potent than the parent hormone.11 Frei-
dinger also developed six- and seven-membered
and 3 as molecular scaffolds,12 which were proposed to stabilize
turns of type II.13 Dehydro-Freidinger lactams 4, synthesized by
ring-closing metathesis reactions, were also claimed to be -turn
d- and 3-lactam 2
Abbreviations: Aba, 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one; Ac, acetyl;
b
-
Boc, tert-butyloxycarbonyl; CCK, cholecystokinin; DAMGO, [
ol]enkephalin; DIPEA, diisopropylethylamine; Dmt, 20,60-dimethyl-
D
-Ala2,NMePhe4,Gly5-
L-tyrosine; DCC,
N,N0-dicyclohexylcarbodiimide; EDC, 1-ethyl-3-(30-dimethylaminopropyl)carbodi
imide; GPCR, G-protein coupled receptor; Hba, 4-amino-1,2,4,5-tetrahydro-8-hy-
droxy-3-oxo-2H-2-benzazepine; Moc, methyloxycarbonyl; MSB, methyl-2-((succi-
nimidooxy)carbonyl)benzoate; NOE, Nuclear Overhauser Effect; SUMM, Systematic
Unbounded Multiple Minimum search; TBTU, O-benzotriazol-1-yl-N,N,N0,N0-tetra-
methyluronium tetrafluoroborate; TFMSA, trifluoromethanesulfonic acid.
* Corresponding author. Tel.: þ32 2 629 3295; fax: þ32 2 629 3304.
b
mimetics.14,15 The application of these lactams and of their hetero-
or benzo-fused analogs has been very successful for the design of
inhibitors of angiotensin converting enzyme, neutral endopepti-
dase, renin, thrombin, and others, as well as for analogs of the
dopamine modulator PLG, CCK and gastrin, bradykinin, neurokinin
A, and growth hormone-releasing hormone peptides.16
´
E-mail address: datourwe@vub.ac.be (D. Tourwe).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.01.057

R. De Wachter et al. / Tetrahedron 65 (2009) 2266–2278
2267
2. Results and discussion
2.1. Synthesis of the tetrapeptide mimetics
The synthesis of the racemic erythro- and threo-5-Me-Aba-Gly
building blocks for derivatives 12 and 13 was performed using an
acyliminium ion cyclization pathway as was previously published
for the unsubstituted analog 6 (Scheme 1).17 The required (ꢁ)
erythro-b-Me-Phe-OH 16 was obtained by crystallization from the
erythro/threo mixture according to the procedure reported by
Kataoka.26 Catalytic hydrogenation of Z-2-benzamido-3-phenyl-2-
butenoic acid methyl ester, which was obtained by Erlenmeyer
condensation between acetophenone and hippuric acid, followed
by isomerization of the E-isomer in pyridine, provided the pre-
cursor of (ꢁ) threo-
b
-Me-Phe-OH 17.27,28
erythro- -Me-Phe-OH 16 and threo-b-Me-Phe-OH 17 were
Figure 1. Examples of Freidinger lactams proposed as b-turn mimics.
b
phthaloyl protected (Scheme 1) by means of methyl-2-((succini-
midooxy)carbonyl)benzoate (MSB) to afford 19 and 20, re-
spectively.29 threo-isomer 20 was purified by flash column
chromatography to remove the open derivative 18. This side
product was not observed during phthaloyl protection of the
erythro derivative.
The structural resemblance of the dehydro-Freidinger lactam 4
with the 4-amino-1,2,3,4-tetrahydro-2-benzazepin-3-one (Aba,
Hba) dipeptidic motif 5 has stimulated us to investigate its potential
to induce a b-turn conformation. The Aba and Hba structures have
been applied successfully as conformational constrained Phe and
Tyr analogs, respectively. The Ca–Cb dihedral angle (c1) is fixed to
þ60ꢀ or 180ꢀ, which corresponds to a gauche(þ) and a trans con-
formation, respectively.17 It has proven to influence receptor se-
lectivity in opioid peptides,17–19 and to provide selective enzyme
inhibitors20–23 and antigenic peptides.24
Coupling of Phth-erythro-b-Me-Phe-OH 19 and Phth-threo-b-
Me-Phe-OH 20 with methyl or ethyl glycinate, respectively, using
TBTU as a coupling reagent yielded 21 and 22, which were sub-
sequently hydrolyzed to 23 and 24, respectively. Following forma-
tion of oxazolidinone 25 and 26 by reaction with formaldehyde
using an azeotropic distillation, ring closure was effected by for-
mation of an acyliminium ion intermediate using tri-
fluoromethanesulfonic acid (TFMSA) to provide compounds 27 and
28.30,31 Phthaloyl deprotection by hydrazinolysis17 led to erythro-
and threo-5-Me-Aba-Gly-OH, which were acetylated with acetic
anhydride in water,32 and further transformed into methyl amides
12 and 13 using N,N-dicyclohexylamine (DCC) as a coupling re-
agent. The compounds were purified by preparative HPLC. Any
epimerization at C-4 during these reaction steps would result in the
formation of the threo-isomer from the erythro-isomer and vice
versa. Since these stereoisomers were well separated in the HPLC
chromatogram (see Experimental section), epimerization could be
excluded.
In an earlier report, the synthesis and evaluation of the b-turn
properties of the tetrapeptide models Ac-Aba-Gly/Ala-NHMe 6–8,
Ac-MeAba-Gly-NHMe 9, and of their spirocyclic derivative 10
(Fig. 2) were investigated by NMR spectroscopy and molecular
modeling. Interestingly, all lactams 6–9 adopt extended confor-
mations, only the spiro-benzazepinone-containing peptide analog
10 showed a strong preference for the formation of a type II0
b-
turn.25 It was noticed that the azepinone ring conformation was
different in lactams 6–9 where it adopts a chair-like conformation,
from that in spiro-analog 10 where it adopts a boat-like confor-
mation. This suggested that the propensity of these 2-benzazepi-
nones for adopting a turn conformation might depend on the ring
conformation. The ring conformation, on the other hand, might be
influenced by the introduction of substituents at position 1, 4 or 5.
In this paper, we report on the synthesis and conformational
analysis by NMR spectroscopy and molecular modeling for the
The starting material for Ac-4-Me-Aba-Gly-NHMe 11, (R,S)-2-
amino-3-(2-cyanophenyl)-2-methylpropanoic acid hydrochloride
33 (Scheme 2), was prepared via a phase transfer catalyzed alkyl-
ation of N-benzylidene alanine ethyl ester 2933,34 with o-cyano-
benzyl bromide, benzyltriethylammonium chloride as a catalyst and
KOH/K2CO3 as a base, resulting in compound 30.35,36 Hydrolysis of
evaluation of the
b-turn mimicry of the ring substituted N-acetyl
dipeptide N0-methyl amides 11–15 tetrapeptide models.
7
8
6
9
5a
9a
5
4
1
N
2
CH3
O
N
3
CH3
O
N
N
H3C
H3C
N
HN
HN
H3C
HN
N
N
O
O
O
O
O
HN
CH3
HN
CH3
O
O
O
H3C
H3C
HN
CH3
O
HN
CH3
O
H3C
O
HN
CH3
O
O
6
10
8
9
7
7
8
6
9
9a
1
5a
5
4
H3C
HN
H3C
H3C
HN
2
N
N
N
N
N
3
HN
HN
HN
H3C
O
O
O
O
O
H3C
H3C
HN
CH3
O
HN
CH3
15 (1S,4S)
O
O
H3C
O
HN
CH3
12 erythro (4S*,5S*)
HN
CH3
13 threo (4S*,5R*)
O
O
O
O
O
HN
CH3
H3C
O
14 (1R,4S)
11
Figure 2. Tetrahydro-2-benzazepinones as potential
b
-turn mimetics.

2268
R. De Wachter et al. / Tetrahedron 65 (2009) 2266–2278
H3C
H2N
H3C
H3C
H3C
c
a
b
H
N
H
N
H
COOH
COOR
Phth=N
COOH
Phth=N
COOH
Phth=N
O
O
(+) erythro-23
(+) threo-24
(+) erythro-21 (R=Me)
(+) threo-22 (R=Et)
(+) erythro-19
(+) threo-20
(+) erythro-16
(+) threo-17
d
H3C
O
COOH
N
H
H3C
Phth=N
O
H3C
HN
H3C
O
e
COOCH3
f,g,h
O
N
N
N
OH
Phth=N
(+) threo-18
O
O
O
OHN
O
(+) erythro-27
(+) threo-28
(+) erythro-25
(+) threo-26
(+) erythro-12
(+) threo-13
Scheme 1. Synthesis of Ac-erythro- and threo-5-Me-Aba-Gly-NHMe 12 and 13. (a) MSB, H2O, CH3CN, overnight, rt, 19: 65%, 20: 72%; (b) HCl$Gly-OMe/OEt, TBTU, Et3N, CH2Cl2, 1 h, rt,
21: 64%, 22: 30%; (c) 1 N HCl, acetone, 19.5 h, , 23: 100%, 24: 100%; (d) benzene, 5 h reflux, (CH2O)n, p-Tos-OH, 25: 89%, 26: 89%; (e) CF3SO3H, CH2Cl2, overnight, rt, 27: 100%, 28:
100%; (f) NH2-NH2$H2O, EtOH; 1,5 h reflux; (g) H2O, Et3N, Ac2O; (h) CH3CN/H2O (3:1), pyridine, DCC, CH3-NH2$HCl.
D
the imine protecting group using a 2 N HCl solution provided the
aminoester, which was hydrolyzed with a 6 N HCl solution at 65 ꢀC
to obtain (R,S)-2-amino-3-(2-cyanophenyl)-2-methylpropanoic
acid hydrochloride 33. Other acidolytic conditions (6 N HCl/acetone,
reflux) resulted in the formation of the side product 1-imino-3-
methyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid 31.
Compound33was methyloxycarbonyl (Moc) protectedbymeans
of MocCl. The use of 4.0 equivof MocCl and a reaction temperature of
0 ꢀC were necessary to avoid the formation of the side product 31.
Reduction of the nitrile in 34 with H2/10% Pd on C provided the
primary amine. Intramolecular cyclization was performed using 1-
ethyl-3-(30-dimethylaminopropyl)carbodiimide (EDC) as a coupling
reagent. Alkylation of (R,S)-Moc-4-Me-Aba 36 with tBu-bromoace-
tate and NaH as a base provided ester 37. Moc deprotection and ester
hydrolysis were performed using a 33% solution of HBr in AcOH to
provide compound 38, which was further transformed into N-acetyl
methyl amide 11, as described above for 12 and 13.
The 1-phenyl-Aba-Gly isomers for the tetrapeptide models 14
and 15 were synthesized by reaction of N-phthaloyl-phenylalanine
chloride with N-benzylideneglycinate ethyl ester, as reported ear-
lier.37 The resulting trans and cis isomers 41 and 40 were separated
using silica gel chromatography (Scheme 3). Further trans-
formation of the N-phthaloyl-protected ethyl esters into pseudo-
tetrapeptides 14 and 15 was performed as described above.
2.2. Molecular modeling results
The conformational preferences for the different tetrapeptide
models 11–15 were investigated using an identical procedure as the
one described for the non-ring substituted analogs 6–10.25 In order
to find the most relevant minimum-energy conformations of the
ring structure in the five substituted 4-amino-2-benzazepin-3-ones
11–15 (Fig. 2), a random pure low mode search38 and energy
minimization in vacuo with the Macromodel 5.039 MM3*40 force
field were carried out on the different 4-formylamino-2-N-Me-2-
benzazepinones (4-Me-, erythro/threo-5-Me-, 1-phenyl-Aba). Sec-
ondly, the exocylic functions were added in each of the low energy
ring conformations obtained to yield the pseudotetrapeptide
structures Ac-4-Me-Aba-Gly-NHMe 11, Ac-erythro-5-Me-Aba-Gly-
NHMe 12 and threo-5-Me-Aba-Gly-NHMe 13, (1R,4S)-4-acetamido-
1-phenyl-Aba-Gly-NHMe 14, and (1S,4S)-4-acetamido-1-phenyl-
Aba-Gly-NHMe 15. Subsequently, the peptide backbone was sub-
jected to a systematic unbounded multiple minimum (SUMM)
search41 and energy minimization in combination with the GB/SA
solvatation model (water).42 All conformations within 50 kJ/mol of
the global minimum were clustered into families based on geo-
metrical similarity.
The
b-turn mimicry of the different tetrapeptide mimetics was
evaluated using various criteria (Fig. 3): (1) the presence of
d
a
b,c
O
N
N
N
N
O
O
O
H
O
H
Et
N
MocHN
HCl.H2N
O
O
O
Et
NH
NH
34
29
30
e,f
33
31
COOH
g
h
i,j
NH
N
N
N
O
COOH
COOtBu
HBr.H2N
HN
MocHN
MocHN
O
O
O
O
HN
O
38
36
37
11
Scheme 2. Synthesis of Ac-4-(R,S)-Me-Aba-Gly-NHMe 11. (a) KOH, K2CO3, (Et)3BnNCl o-CN-BnBr; (b) 2 N HCl, 63% (four steps); (c) 6 N HCl, 65 ꢀC, 81%; (d) 1 N NaOH, MocCl, 69%; (e)
Pd/C, H2, 74%; (f) Py, EDC, 97%; (g) NaH, BrCH2COOtBu, 79%; (h) 33% HBr/AcOH; (i) Et3N, Ac2O; (j) Py, DCC, CH3-NH2$HCl, 60% (three steps).

R. De Wachter et al. / Tetrahedron 65 (2009) 2266–2278
2269
N
COOEt
N
COOEt
N
HN
Phth=N
Phth=N
Phth=N
O
O
O
O
HN
CH3
O
39
40
H3C
14
N
Cl
COOEt
N
COOEt
N
HN
Phth=N
O
O
O
O
HN
CH3
O
H3C
41
15
Scheme 3. Synthesis of tetrapeptide models 14 and 15.37
a hydrogen bond in type I and II
b-turns is indicated by a distance
2.3. NMR analysis
between the N-acetyl carbonyl oxygen atom and the methyl car-
boxamide proton smaller than 2.5 Å and an NH–O angle greater
than 120ꢀ,43 (2) an interatomic distance between Ca1 and Ca4 less
The structural confirmation and the conformational analysis
of the tetrapeptide models were accomplished by 1H NMR and
13C NMR. The chemical shift assignments were supported by
heteronuclear 1H–13C HMQC45 and HMBC46 spectra. The confor-
mation of the benzazepinone ring was examined by 2D 1H NOESY
NMR.47
than 7 Å,8,9 (3) the virtual torsion angle
b
, defined by C1,Ca2,Ca3,
-turn class (Table
j-angle of the central residues,
and N4 of the tetrapeptide model defines the
1),44 (4) the characteristic
- and
which define the turn type.2
b
4
In Table 2 and Figure 4 the lowest-energy conformers together
with the numerical values for the various criteria are given.
The lowest-energy conformer 11a of Ac-4-Me-Aba-Gly-NHMe
As shown in Table 3, the conformation of the amino-2-benza-
zepinone ring in 11–13 was clearly identified by a set of NOE data.
The conformations are characterized by the value of c1 angle of the
Aba amino acid structure. All compounds were shown to adopt
a trans conformation. For the 4-methyl-substituted isomer 11, ev-
11 structure corresponds to a
b
-turn of type I, with a gauche(þ) ring
conformation. The second lower energy conformer 11b is only
2.34 kJ/mol (0.56 kcal/mol) above the absolute minimum and
idence (Table 3) is given by the NOE cross-peaks between the
atom and the methyl hydrogens, and the strong NOE between H
and the aromatic H6. In 11, a strong NOE between Hb0 and H30 and
between H and the aromatic H9 indicates a preferred boat-like
b-H
b
adopts a trans1 ring conformation. A
For both Ac-erythro-5-Me-Aba-Gly-NHMe 12 and threo-5-Me-Aba-
Gly-NHMe 13 none of the lowest-energy conformers show a -turn
b
-turn of type II0 is induced.
b
3
structure. In both cases the ring adopts the same trans2 confor-
mation, irrespective of the orientation of the 5-methyl substituent.
The first conformation of Ac-erythro-5-Me-Aba-Gly-NHMe 12,
which adopts a turn (type I) is 17.87 kJ/mol above the minimum and
adopts a gauche(þ) ring conformation. For threo-isomer 13, the first
turn conformation is found to be 10.54 kJ/mol above the global
minimum.
trans1 conformation. This ring conformation is not the lowest-en-
ergy conformer that was found by the molecular modeling (Table
2), but corresponds to that found as the second lowest one 11b
(2.34 kJ/mol above minimum). The 5-methyl-substituted isomers
12 and 13 both show a different ring conformation compared to
11b. Indeed, in 12 a strong NOE is observed between H
a and H3, and
between H
3
0 and H9, which corresponds to the calculated chair-like
The lowest-energy conformer of (1R,4S)-4-acetamido-1-phenyl-
trans2 conformation with a pseudo-axial methyl group. A similar
trans2 conformation was found for threo-isomer 13, which also
corresponds to the calculated preferred structure.
Evidence for the presence of an intramolecular hydrogen bond
between the carbonyl oxygen atom of the first residue and the
amide NH proton of the fourth residue, which is present in most
Aba-Gly-NHMe 14 adopts
a trans1 ring conformation, with
a pseudo-axial phenyl orientation. No turn formation is observed
for the low energy conformers. The conformer of 14, which adopts
a turn (type II0) is 29.44 kJ/mol above the minimum and adopts also
a trans1 ring conformation. The lowest-energy conformation of
1(S)-isomer 15 adopts a trans2 conformation, without turn prop-
erties. A turn (type II0) conformation is found to be 25.38 kJ/mol
of the
b-turns, was evaluated in scaffolds 11–15. Intramolecular
hydrogen bonds are very little influenced by external factors such
as temperature and solvent in comparison with intermolecular
hydrogen bonds. Therefore, to evaluate the effect of switching
from a non-hydrogen bond forming solvent to a strong hydrogen
bond forming solvent on the chemical shift of the NH-Me and
Ac-NH protons, the 1H NMR spectra of the benzazepinone
compounds were measured in CDCl3 and DMSO-d6. In a second
above the minimum in
a
gauche(þ) ring conformation. In-
terestingly, the preferred ring conformations calculated for 14 and
15 correspond to those found in the crystal structure of the
phthaloyl-protected precursors.37
Table 1
Virtual torsion angle
b
defining
b
-turn classes44
Virtual torsion angle
10< <80
Type of
b-turn
b
I
I0
II
II0
b
ꢂ79<
ꢂ69<
ꢂ59<
b
b
b
<ꢂ10, 20<
b
<40
b
<ꢂ60, ꢂ49<
<ꢂ40, ꢂ29<
b<40
<ꢂ50, ꢂ29<
b
<30
Figure 3. Criteria used to evaluate b-turn mimicry.

2270
R. De Wachter et al. / Tetrahedron 65 (2009) 2266–2278
Table 2
The lowest-energy conformers and values for the various criteria
Structure
Ac-4-Me-
Aba-Gly-
NHMe 11a
Ac-4-Me-
Aba-Gly-
NHMe 11b
Ac-erythro-
(4S ,5S )-5-
Me-Aba-Gly-
Ac-threo-
(4S ,5R )-
5-Me-Aba-
(1S,4S)-Ac-
phenyl-Aba-
Gly-NHMe 15
(1R,4S)-Ac-
phenyl-Aba-
Gly-NHMe 14
*
*
*
*
NH-Me 12
Gly-NH-Me 13
Ring conformationa
Turn
d(NH-CO) (Å)
NHO angle (ꢀ)
gauche (þ)
trans1
trans2
d
trans2
d
trans2
d
trans1
d
I
II0
2.1
163
5.1
17
2.1 [2.08(4)]b
163 [161(3)]b
5.4 [5.505(4)]b
ꢂ10 [ꢂ17.4(2)]b
60 [50.7(4)]b
ꢂ121 [-131.5(3)]b
ꢂ98 [ꢂ93.6(3)]b
17 [14.3(4)]b
6.6
147
9.6
ꢂ150
ꢂ163
161
6.6
140
9.3
ꢂ147
ꢂ131
159
6.5
149
9.3
ꢂ137
ꢂ163
164
6.3
157
9.6
146
ꢂ153
ꢂ120
102
d(C
a
1-C
a
4) (Å)
b
(ꢀ)
2 (ꢀ)
2 (ꢀ)
3 (ꢀ)
3 (ꢀ)
E of first
turn (kJ/mol)
F
ꢂ52
J
ꢂ51
F
ꢂ100
ꢂ114
ꢂ100
ꢂ107
J
21
24
26
27
ꢂ22
2.34, t1 (¼11b),
17.87, g(þ),
type I
10.54, g(þ),
type I
25.38, g(þ),
type II0
29.44, t1,
type II0
type II0
a
The ring conformations are classified by the c1 torsion angle of the Aba amino acid, which can be either gauche(þ) or trans. For each of these conformations, two ori-
entations of C are possible. In the trans1 conformation, the C –H is in close proximity of C –H, whereas in the trans2 conformation the C –H is in close proximity of C ’-H.
Experimental values found in the x-ray structure (Fig. 5).
3
b
3
a
3
b
experiment, samples in DMSO-d6 were heated to examine the
effect of the temperature increase on the chemical shift of amide
protons.
exocyclic part of the molecule, which supports the presence
a pseudo 10-membered ring. The NMR data are therefore in com-
plete accordance with the second lowest-energy conformer of the
Ac-4-Me-Aba-Gly-NHMe tetrapeptide 11b, which was obtained
through molecular modeling.
For the 5-methyl-substituted isomers 12 and 13, the NOE data in
Table 3 indicate the presence of a chair-like trans2 conformation of
the 5-methyl-Aba ring, in agreement with the molecular modeling
results. The large shifts of the NH signal found in the solvent study
and in the temperature study for compounds 12–15 (Table 4) do not
support the presence of an intramolecular hydrogen bond. These
data indicate that, in agreement with the theoretical prediction,
For the 4-Me-Aba derivative 11, the NH-Me amide resonance is
only very little influenced by switching from CDCl3 to DMSO
(ꢂ0.08 ppm), in contrast to that of the NH-Ac (ꢂ2.15 ppm). More-
over, its temperature coefficient is ꢂ3.7 ppb/K, indicating the
presence of an intramolecular hydrogen bond for this compound.
Further evidence of the intramolecular hydrogen bond was given
by the presence of NOE correlations between the Aba ring protons
and those of the glycine Ca. The observation of a strong NOE be-
tween one of the glycine Ca hydrogens and C–H
the other pair, indicates a limited rotational freedom in the
3, and not between
compounds 12–15 do not adopt a b-turn conformation.
Figure 4. 3D representation of the lowest-energy conformers.

R. De Wachter et al. / Tetrahedron 65 (2009) 2266–2278
2271
Table 3
NMR data for determination of the conformation of the Aba ring
Hβ'
O
β'CH3
Hβ'
Hα
Hε'
O
N
O
H
N
H
H
Hε'
H9
N
N
Hε'
N
N
Ac
Ac
Hβ
H6
Ac
Hε
H9
Hβ
H3C
H6
H9
Hε
Hα
12
H3αC
H6
Hε
13
11b
Ac-erythro-(4S*,5S*)-5-
Me-Aba-Gly-NH-Me
Ac-4-Me-Aba-Gly
-NHMe
Ac-threo-(4S*,5R*)-5-
Me-Aba-Gly-NH-Me
3J (H
3J (H
NOE
a
a
,H
b
)
)
d
d
2.6 Hz
d
d
,Hb0
11.0 Hz
Ca(CH3)–Cb(H)
Strong
d
d
d
Ca(H)–Cb(H/CH3)
Strong
Strong
NOE
0
Ca(CH3)–Cb (H)
0
Absent
d
d
d
Ca(H)–Cb (H/CH3)
Absent
Absent
NOE
NHAc/Cb(H/CH3)
Strong
Weak
Weak
Weak
NOE
0
NHAc/Cb (H/CH3)
Strong
Strong
NOE
H
H
b
b
/H6
0/H6
Strong
Absent
Strong
d
d
Strong
NOE
Ca(H/CH3)/H
NOE
3
Absent
Absent
Strong
Strong
Absent
Absent
Strong
Absent
Weak
Ca(H/CH3)/H30
NOE
0
Cb (H/CH3)/H30
NOE
H
H
3
3
/H9
0/H9
Strong
Absent
Absent
Strong
Weak
Strong
2.4. X-ray diffraction
m
-opioid receptor agonists among the naturally occurring opioids.49
The minimal sequence required for opiate-like activity in vivo has
been shown to be the N-terminal tetrapeptide.50,51 Moreover, re-
placement of Tyr1 in opioid peptides with the unnatural amino acid
Crystals suitable for X-ray diffraction were obtained for Ac-4-
Me-Aba-Gly-NHMe 11. Experimental values of the torsion angles
and intramolecular H-bond geometry shown in Table 2 confirm
that the crystal structure (Fig. 5) is completely identical with that of
conformation 11b, which was observed in solution, and which
corresponds to the calculated second lowest-energy one. The
seven-membered ring conformation is an ideal boat.
C8
C7
C9
C6
2.5. Biological application
The heptapeptide dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-
C10
C11
C5
Ser-NH2) was isolated from the skin of the South American frog
Phyllomedusa sauvagei.48 It is one of the most potent and selective
C4
Table 4
C16
C12
Solvent and temperature effect on amide protons in compounds 11–15
Dd
/
D
T (ppb/K)
D
CDCl3 (ppm)
d
DMSO (ppm)
Dd
N2
C13
N3
(R,S)-4-Me-Aba-Gly 11
Ac-NH
NH-Me
ꢂ7.0
ꢂ3.7
6.62
7.60
8.77
7.68
ꢂ2.15
ꢂ0.08
C3
O2
O3
Ac-erythro-(4S*,5S*)-5-Me-Aba-Gly-NH-Me12
C14
H14
C2
Ac-NH
NH-Me
ꢂ7.0
6.94
6.08
7.87
7.71
ꢂ0.93
ꢂ1.63
N1
ꢂ6.5
Ac-threo-(4R*,5S*)-5-Me-Aba-Gly-NH-Me 13
2.08(4) Å
O1
Ac-NH
NH-Me
ꢂ5.4
6.40
5.95
8.18
7.75
ꢂ1.78
ꢂ1.80
ꢂ6.1
C15
(1S,4S)-Ac-1-phenyl-Aba-Gly-NHMe 15
Ac-NH
NH-Me
ꢂ7.0
6.86
6.38
7.84
7.83
ꢂ0.98
ꢂ1.45
C1
ꢂ7.0
(1R,4S)-Ac-1-phenyl-Aba-Gly-NHMe] 14
Ac-NH
NH-Me
ꢂ7.2
6.60
6.28
8.45
7.75
ꢂ1.85
ꢂ1.47
Figure 5. Crystal structure of 11 showing thermal ellipsoids at 30% probability level
and intramolecular H-bond: O3/N1; O3/H14; angle 161; d(C1/C15) 5.505(5) Å.
ꢂ6.1

2272
R. De Wachter et al. / Tetrahedron 65 (2009) 2266–2278
Table 5
CH3CN, t¼20 min, 97% CH3CN; flow rate: 1.0 mL/min, ¼215 nm.
l
Afinities for the m- and d
-opioid receptors65
Preparative HPLC was performed using an RP C-18 column (Supelco
Discovery ÒBIO Wide Pore C18, l¼25 cm, d¼2.12 cm, PS¼10
mm)
Peptide sequence
Kia (nM)
Kib (nM)
with the above mentioned gradient at a flow rate of 20.0 mL/min.
TLC analysis was performed on plastic sheets precoated with silica
gel 60F254 (Merck). Silica gel 60 (0.040–0.063 mm) from Merck was
used for flash column chromatography (w/w, 60:1). Melting points
were measured with a Bu¨chi B 540 melting point apparatus, with
a temperature increment of 1 ꢀC/min. 1H and 13C NMR spectra were
recorded at 250.13 and 62.90 MHz, respectively, with a Bruker
Avance DRX250 spectrometer, using TMS or the residual solvent
signal as internal reference. For some advanced NMR analyses
samples were measured with the Bruker AMX500 spectrometer,
using pulse sequences of the Bruker program library. The temper-
ature study was performed in DMSO-d6, with a temperature in-
crement of 5 K between 298 K and 343 K. Mass spectra were
recorded on a VG Quattro II spectrometer using electrospray ioni-
zation (positive ion mode).
H-Dmt-D-Ala-(S)-Aba-Gly-NH2 42
H-Dmt-D-Ala-(4S)-Me-Aba-Gly-NH2 43
H-Dmt-D-Ala-(S)-spiroAba-Gly-NH2 44
0.047ꢁ0.0012
4.6ꢁ1.7
2.4ꢁ0.6
859ꢁ107
308ꢁ21
3.2ꢁ0.4
a
Displacement of [3H]DAMGO in rat brain homogenate.
b
Displacement of [3H] [Ile5,6]deltorphin 2 in rat brain homogenate.
20,60-dimethyltyrosine (Dmt) resulted in compounds with in-
creased agonist potency.52–54 We therefore prepared analogs of H-
Dmt-D-Ala-Phe-Gly-NH2 in which we have substituted the Phe-Gly
dipeptide by the constrained Aba-Gly (as in 6), 4-Me-Aba-Gly (as in
11) and by the previously reported spiro-Aba-Gly25,55 (as in 10). The
peptides were prepared by standard solid phase peptide synthesis
as described before55 and their affinities for the
m- and d-opioid
receptors were determined. As shown in Table 5, analog 42 con-
taining the Aba-Gly scaffold displayed the highest affinity for both
receptors. Whereas analog 43 containing 4-Me-Aba-Gly showed
a much reduced affinity compared to 42, it still has low nanomolar
affinity for the
spiro-Aba-Gly containing analog 44. The latter scaffold was shown
to induce a
-turn in the opioid peptide endomorphin-2.55 This is
4.2. Molecular modeling
m-opioid receptor, which is comparable to that of the
The calculations were carried out with Macromodel 5.039 with
Maestro 8.0 as graphic interface. The MM3
force field40 was used
*
b
consistent with the fact that the (4S)-Me-Aba-Gly scaffold induces
a similar turn conformation in tetrapeptide 43. The lower affinities
of the two analogs having a turn conformation compared to that of
the analog having the Aba-Gly scaffold, which adopts an extended
conformation, might point out that the latter is more able to adopt
the optimal binding conformation. This also suggests that the turn
conformation induced by the (4S)-Me-Aba-Gly and spiro-Aba-Gly
scaffolds in these tetrapeptides would not be the bioactive one for
in vacuo for the energy minimizations on the formyl-Xxx-NMe
rings (Xxx¼4-Me-, erythro/threo-5-Me-, 1-Ph-Aba). The confor-
mational analyses of the ring structures were carried out with the
Pure Low Mode search;38 1000 structures were generated and
`
minimized by means of the Polak–Ribiere conjugate gradient
method as implemented in Macromodel, using a gradient conver-
gence criterion of 0.02 kJ/mol Å. For the tetrapeptide mimetics, the
MM3 force field was used as well, but this time in combination
*
with the GB/SA solvation model of Still et al.,42 using Macromodel’s
default parameters for an aqueous medium. The conformational
space was sampled by use of a systematic unbounded multiple
binding to the m-opioid receptor. Further application of all stereo-
isomers of the scaffolds reported here in opioid peptides is cur-
rently ongoing and will be reported separately.
mimimum search41 in which three internal coordinates (
j
4
2,
3) were varied. Here the generation of 2000 structures was con-
ducted and the conformers were minimized to an energy conver-
43, and
3. Conclusions
The attempts to influence the ring conformation of the 1,2,4,5-
tetrahydro-4-amino-2-benzazepin-3-one scaffold by the in-
troduction of ring substituents were only successful for the 4-methyl
analog 11. In this case the preferred chair-like trans2 conformation
found for the unsubstituted Aba-Gly in 6 shifted to a boat-like trans1
conformation, as was observed for spiro-analog 10.25 At the same
`
gence of 0.1 kJ/mol Å by use of the Polak–Ribiere conjugate gradient
method. After this search the found conformations were again
minimized to an energy convergence of 0.01 kJ/mol Å. In both
strategies, duplicate structures and those greater than 50 kJ/mol
above the global minimum were discarded. The generated struc-
tures were clustered in families with Xcluster 1.7. An RMSD value of
0.2 Å was used.
time the tetrapeptide model 11b adopted a
Methyl substitution at position 5 was not able to shift the ring
conformation from trans2 to trans1, and no -turn conformation was
b-turn conformation.
b
4.3. X-ray crystallography
observed. Whereas the 1(R)-phenyl-substituted analog 14 adopts
a trans1 conformation with a pseudo-axial phenyl substituent, 1(S)-
isomer 15 adopts a trans2 conformation. However, neither the
molecular modeling nor the solution conformational study was able
to indicate the formation of a turn structure. Therefore no correla-
tion between the ring conformation and the propensity to adopt
a turn conformation could be demonstrated. This study identified
Crystal of 11 suitable for single crystal X-ray diffraction was fixed
at glass fiber using epoxy resin. Diffraction data were collected at rt
on Nonius BV MACH3 diffractometer with graphite mono-
chromated Cu K
a
radiation (l¼1.54178 Å). The structure was solved
by direct methods using SHELXS9759 and refined on F2 by using
full-matrix least-squares methods with program SHELXL97.60
Software used to prepare material for publication was WinGX.61
Anisotropic thermal parameters were refined for non-H atoms.
Hydrogen atoms were localized from Dr maps and refined with
isotropic thermal parameters. CCDC-705574 contains the supple-
mentary crystallographic data for this paper. These data can be
obtained free of charge at ‘http://www.ccdc.cam.ac.uk/conts/
retrieving.html’ [or from the Cambridge Crystallographic Data
Centre, 12, Union Road, Cambridge CB2 1EZ, UK; E-mail:
deposit@ccdc.cam.sc.uk].
the
a-MeAba scaffold as a strong turn inducer, which is in agreement
with the finding that
a
-MePro is a stronger turn inducer than
Pro.57,58 An asymmetric synthesis of this scaffold and its application
in peptide mimetic design are currently in progress.
4. Experimental section
4.1. General methods
RP-HPLC was performed using an RP C-18 column (Supelco
Crystal
data
for
11:
a¼15.3280(3),
b¼26.3269(9),
Discovery ÒBIO Wide Pore C18, l¼25 cm, d¼0.45 cm, PS¼5
mm)
c¼7.7231(12) Å; V¼3116.6(5)Å3; F(000)¼1296; (Cu K
a
)¼0.740; or-
with a mobile phase consisting of water/acetonitrile containing
0.1% TFA. Products were eluted using the gradient: t¼0 min, 3%
thorhombic space group Pccn; 3077 reflections collected, 1706
unique (Rint¼0.0614); R1¼0.0594, and for all data wR2¼0.1928.

R. De Wachter et al. / Tetrahedron 65 (2009) 2266–2278
2273
4.4. Opioid receptor binding assay
134.86 (CH-Phth), 131.13 (Cq-Phth), 143.38 (Cq-arom), 167.34 (C]O-
Phth), 168.66 (COOCH3), 170.39 (CO-NH) ppm.
Opioid radioreceptor binding assays have been performed on
brain homogenates of male Wistar rats (250–300 g body weight) as
reported in detail elsewhere.65 Binding affinities of the compounds
Compound 22. Yield: 5.1 g (30%). Mp 120.1–123.2 ꢀC. HPLC
tR¼16.9 min. Rf 0.57 (CHCl3/EtOAc 1:1). MS 395 (MþHþ), 264
(MꢂCONHCH2COOCH2CH3), 292 (MꢂNHCH2COOCH2CH3), 321
(MꢂCOOCH2CH3), 349 (MꢂOCH2CH3). 1H NMR (DMSO, 250 MHz)
to the
m- and d-opioid receptors were measured against tritiated
compounds [3H]DAMGO and [3H][Ile5,6]deltorphin 2, respectively.
1.05–1.10 (6H, M, CH3-Etþ
b
-CH3), 3.59 (1H, dd, H
a
-Gly, 3J¼5.6 Hz,
2J¼17.3 Hz), 3.73 (1H, dd, H
a
0-Gly, 3J¼5.8 Hz, 2J¼17.2 Hz), 3.82–4.03
(3H, M, CH2-Etþ
b
-H), 4.88 (1H, d,
a
-H, 3J¼9.1 Hz), 7.08–7.35 (5H, M,
4.5. Phthaloyl-erythro-(2S
*
,3S
*
)-b-methyl phenylalanine 19
H-arom), 7.86–7.94 (4H, M, H-Phth), 8.35 (1H, t, NH,
and threo-(2S ,3R )- -methyl phenylalanine 20
*
*
b
3J¼5.6 Hz) ppm. 13C NMR (DMSO, 63 MHz) 13.86 (CH3 Et), 18.45 (
b-
CH3), 38.11 (b-CH), 40.85 (a-CH2-Gly), 57.27 (a-CH), 60.27 (CH2 Et),
To a solution of b-Me-Phe$HCl and Na2CO3 (6.0 equiv erythro;
123.31, 126.29, 127.42 (CH-arom), 128.27, 134.68 (CH-Phth), 131.26
(Cq-Phth), 144.11 (Cq-arom), 167.27 (C]O-Phth), 167.38 (C]O es-
ter), 169.39 (CO-NH) ppm.
4.0 equiv threo) in H2O/CH3CN (3:5), methyl-2-((succinimidooxy)
carbonyl)benzoate (MSB, 1.0 equiv) was added and the reaction
mixture was stirred until HPLC analysis indicated the disappear-
ance of the starting material (typically 24 h). The mixture was
acidified with a 2 N HCl solution to pH 1–2, diluted with EtOAc, and
extracted with a 1 N HCl solution (2ꢃ) and H2O (1ꢃ). The organic
layer was dried (MgSO4), filtered, and evaporated. The residue was
crystallized from hot EtOH to yield the phthaloyl-protected com-
pounds for the erythro derivative. The threo derivative was purified
by flash column chromatography (EtOAc/petroleum ether 3:7þ1%
AcOH).
4.7. Phthaloyl-erythro-(2S
*
,3S
*
)-
b-methyl phenyl-
alaninylglycine 23 and threo-(2S
* *
,3R )-b-methyl
phenylalaninylglycine 24
Phthaloyl-b-Me-Phe-Gly-OMe/OEt 21 or 22 was dissolved in
acetone. To this solution a 1 N HCl solution was added slowly. The
reaction mixture was refluxed in an oil bath at 90 ꢀC, until HPLC
analysis indicated the absence of the starting material (typically
16–20 h). The mixture was cooled down to rt and evaporated. The
obtained white solid was used in the next step without further
purification.
Compound 19. Yield: 3.7 g (65%). Mp 135.8–142.2 ꢀC. HPLC
tR¼17.0 min. Rf 0.61 (CHCl3/EtOH 3:1). MS 310 (MþHþ), 332
(MþNaþ), 348 (MþKþ), 641 (2MþNaþ). 1H NMR (DMSO, 250 MHz)
1.50 (3H, d,
b
-CH3, 3J¼6.8 Hz), 3.78 (1H, m,
b-H), 4.89 (1H, d, a-H,
3J¼10.8 Hz), 6.95–7.10 (5H, M, H-arom), 7.67–7.79 (4H, M, H-
Compound 23. Yield: 2.6 g (100%). Mp 187.4–196.2 ꢀC. HPLC
tR¼15.5 min. Rf 0.40 (CHCl3/CH3OH 1:1). MS 367 (MþHþ), 264
(MꢂCONHCH2COOH), 292 (MꢂNHCH2COOH), 349 (MꢂOH), 733
Phth) ppm. 13C NMR (DMSO, 63 MHz) 21.33 (
b-CH3), 39.44 (b-CH),
56.52 (a-CH), 123.52, 126.91, 127.62 (CH-arom), 128.42, 135.12 (CH-
Phth), 130.83 (Cq-Phth), 143.31 (Cq-arom), 167.02 (C]O-Phth),
170.22 (C]O COOH) ppm.
(2Mþ1). 1H NMR (DMSO, 250 MHz) 1.43 (3H, d,
b
-CH3, 3J¼6.9 Hz),
3.68 (1H, dd, H
a
-Gly, 3J¼5.6 Hz, 2J¼17.4 Hz), 3.88 (1H, dd, H
a
0-Gly,
Compound 20. Yield: 125 mg (72%). Mp 114.7–117.4 ꢀC. HPLC
tR¼16.2 min. Rf 0.59 (CHCl3/EtOH 3:1). MS 310 (MþHþ), 163
(MꢂPhth), 264 (MꢂCOOH), 292 (MꢂOH), 332 (MþNaþ), 348
3J¼6.0 Hz, 2J¼17.6 Hz), 3.97 (1H, m,
b-H), 4.90 (1H, d, a-H,
3J¼11.2 Hz), 6.95–7.11 (5H, M, H-arom), 7.64–7.74 (4H, M, H-Phth),
8.53 (1H, t, NH, 3J¼5.8 Hz) ppm. 13C NMR (DMSO, 63 MHz) 21.05 (
b-
(MþKþ). 1H NMR (DMSO, 500 MHz) 1.13 (3H, d,
b
-CH3, 3J¼7.2 Hz),
CH3), 38.70 (b-CH), 41.32 (a-CH2-Gly), 58.52 (a-CH), 123.31, 126.89,
3.80 (1H, m,
b
-H), 4.95 (1H, d,
a
-H, 3J¼8.9 Hz), 7.09–7.30 (5H, M, H-
127.61 (CH-arom), 128.45, 134.85 (CH-Phth), 131.14 (Cq-Phth),
143.46 (Cq-arom), 167.39 (C]O-Phth), 168.48 (COOH), 171.30 (CO-
NH) ppm.
arom), 7.89–7.95 (4H, M, H-Phth), 12.96 (1H, br s, COOH) ppm. 13C
NMR (DMSO, 63 MHz) 18.79 (b-CH3), 38.64 (b-CH), 56.44 (a-CH),
123.54, 126.36, 127.36 (CH-arom), 128.30, 135.00 (CH-Phth), 130.84
(Cq-Phth), 144.05 (Cq-arom), 167.32 (C]O-Phth), 169.10 (C]O
COOH) ppm.
Compound 24. Yield: 3.7 g (100%). Mp 55.8–58.3 ꢀC. HPLC
tR¼15.9 min. Rf 0.52 (CHCl3/CH3OH 1:1). MS 367 (MþHþ), 264
(MꢂCONHCH2COOH), 292 (MꢂNHCH2COOH), 389 (MþNaþ), 405
(MþKþ). 1H NMR (DMSO, 250 MHz) 1.07 (3H, d,
b
-CH3, 3J¼7.2 Hz),
3.48 (1H, dd, H
a
-Gly, 3J¼5.4 Hz, 2J¼17.4 Hz), 3.70 (1H, dd, H
a
0-Gly,
4.6. Phthaloyl-erythro-(2S
*
,3S )-b-methyl phenylalaninyl-
*
3J¼6.1 Hz, 2J¼17.4 Hz), 3.88 (1H, m,
b-H), 4.87 (1H, d, a-H,
glycine methyl ester 21 and phthaloyl-threo-(2S
*
,3R )-b-
*
3J¼9.0 Hz), 7.14–7.27 (5H, M, H-arom), 7.85–7.93 (4H, M, H-Phth),
methyl phenylalaninylglycine ethyl ester 22
8.23 (1H, t, NH, 3J¼5.8 Hz) ppm. 13C NMR (DMSO, 63 MHz) 18.41 (
b-
CH3), 38.23 (b-CH), 40.76 (a-CH2-Gly), 57.36 (a-CH), 123.29, 126.28,
In an oven dried flask, the phthaloyl-protected compound was
dissolved in dry CH2Cl2. The solution was basified by addition of
Et3N (3.0 equiv), after which the coupling reagent TBTU (1.1 equiv)
and the hydrochloric salt of Gly-OMe or Gly-OEt (1.1 equiv) were
added. The mixture was kept at pH 8 by means of Et3N and stirred
for 1 h at rt. Subsequently, the solution was washed with a 1 N HCl
solution (3ꢃ), a saturated NaHCO3 solution (3ꢃ) and brine (3ꢃ).
The organic layer was dried (MgSO4), filtered, and evaporated. The
residue was crystallized from a minimum amount of hot EtOH.
Compound 21. Yield: 2.8 g (64%). Mp 129.3–131.2 ꢀC. HPLC
tR¼17.0 min. Rf 0.51 (CHCl3/EtOAc 1:1). MS 381 (MþHþ), 264
(MꢂCONHCH2COOCH3), 292 (MꢂNHCH2COOCH3), 403 (MþNaþ),
127.43 (CH-arom), 128.27, 134.63 (CH-Phth), 131.29 (Cq-Phth),
144.14 (Cq-arom), 167.18 (C]O-Phth), 167.46 (COOH), 170.87 (CO-
NH) ppm.
4.8. Phthaloyl-erythro-(2S
glycine oxazolidinone 25 and threo-(2S
phenylalaninylglycine oxazolidinone 26
*
,3S
*
)-
b
-methyl phenylalaninyl-
*
*
,3R )-b-methyl
In an oven dried one necked flask, the hydrolyzed compound
was suspended in dry benzene (45 ml/g 23 or 24), followed by the
addition of (CH2O)n (15 equiv) and a catalytic amount of p-TosOH
(0.2 equiv). A Dean–Stark apparatus was mounted on the flask and
the mixture was brought to reflux in an oil bath at 90 ꢀC for 5 h (23)
or 12 h (24). After every 1.5 h, (CH2O)n (5 equiv) was added. The
reaction mixture was cooled down to rt and evaporated. The resi-
due was dissolved in CHCl3 and extracted with a 6% NaHCO3 so-
lution (2ꢃ) and H2O (1ꢃ). The CHCl3 layer was dried (MgSO4),
419 (MþKþ). 1H NMR (DMSO, 250 MHz) 1.43 (3H, d,
b-CH3,
3J¼6.9 Hz), 3.61 (3H, s, OCH3), 3.75–4.00 (3H, M,
b
-Hþ
a-CH2-Gly),
4.90 (1H, d,
a
-H, 3J¼11.2 Hz), 6.95–7.11 (5H, M, H-arom), 7.66–7.78
(4H, M, H-Phth), 8.63 (1H, t, NH, 3J¼5.8 Hz) ppm. 13C NMR (DMSO,
63 MHz) 21.00 (b-CH3), 38.69 (b-CH), 41.23 (a-CH2-Gly), 52.05
(OCH3), 58.36 (
a
-CH), 123.32, 126.90, 127.62 (CH-arom), 128.45,

2274
R. De Wachter et al. / Tetrahedron 65 (2009) 2266–2278
filtered, and evaporated to yield the corresponding oxazolidinones.
No further purification was required.
(b-CH3), 35.46 (b-CH), 49.35 (a-CH2-Gly), 50.97 (3-CH2), 58.23 (a-
CH), 123.36, 134.83 (CH-Phth), 126.30, 128.36, 128.75, 128.98 (CH-
Aba), 131.17 (Cq-Phth), 135.61, 141.15 (Cq-Aba), 166.93 (C]O-Phth),
167.79 (C]O-Aba), 170.10 (C]O Gly) ppm.
Compound 25. Yield: 2.3 g (89%). Mp 205.7–208.7 ꢀC. HPLC
tR¼16.1 min. Rf 0.40 (EtOAc/petroleum ether 3:2). MS 379 (MþHþ),
264 (MꢂCONCH2OCOCH2), 292 (MꢂNCH2OCOCH2). 1H NMR
(DMSO, 250 MHz) 1.34 (3H, d,
b
-CH3, 3J¼6.8 Hz), 3.99 (2H, m,
a
-
4.10. erythro-(4S
dihydro-1H-benzo[c]azepin-2(3H)-yl)-N-methylacetamide
[Ac-erythro-(4S ,5S )-5-Me-Aba-Gly-NH-Me] 12 and threo-
(4S ,5R )-2-4-(acetamido-5-methyl-3-oxo-4,5-dihydro-1H-
benzo[c]azepin-2(3H)-yl)-N-methylacetamide [Ac-threo-
(4R ,5S )-5-Me-Aba-Gly-NH-Me] 13
*
,5S )-2-(4-Acetamido-5-methyl-3-oxo-4,5-
*
CH2-Gly), 4.41 (1H, m,
b
-H), 5.02 (1H, d,
a
-H, 3J¼10.9 Hz), 5.27 (1H,
2
d, H1 N-CH2-O, J¼4.9 Hz), 5.59 (1H, d, H10 N-CH2-O, 2J¼4.7 Hz),
*
*
6.95–7.13 (5H, M, H-arom), 7.68–7.75 (4H, M, H-Phth) ppm. 13C
*
*
NMR (DMSO, 63 MHz) 20.09 (b-CH3), 38.40 (b-CH), 44.87 (a-CH2-
Gly), 56.05 ( -CH), 79.31 (N-CH2-O), 123.44, 127.04, 127.99 (CH-
a
*
*
arom), 128.39, 134.94 (CH-Phth), 130.88 (Cq-Phth), 142.32
(Cq-arom), 165.03 (C]O-Phth), 167.14 (C]O oxazolidinone), 170.98
(CO-NR2) ppm.
To a solution of the benzazepinone product in EtOH, hydrazine
hydrate (6.0 equiv) was added. The solution was refluxed for 1.5 h,
at an oil bath temperature of 95 ꢀC. After cooling the reaction
mixture to rt, the solvent was evaporated. The residue was re-dis-
solved in H2O and the pH was adjusted to 5 by means of careful
addition (pH-meter) of AcOH. This suspension was stirred at rt for
1 h, after which the formed precipitate was filtered and the filtrate
was evaporated. The residue was dissolved in H2O. The pH was
adjusted to 6 with Et3N and Ac2O (7.0 equiv) was added in three
portions. Meanwhile the pH was kept at 6 with Et3N. After stirring
2 h at rt the reaction mixture was evaporated. The crude product
was not purified at this stage and directly used in the next step. To
Compound 26. Yield: 3.3 g (89%). Mp 168.7–173.1 ꢀC. HPLC
tR¼17.7 min. Rf 0.37 (EtOAc/petroleum ether 3:2). MS (ESþ) 379
(MþHþ), 264 (MꢂCONCH2OCOCH2), 292 (MꢂNCH2OCOCH2), 401
(MþNaþ), 417 (MþKþ). 1H NMR (DMSO, 250 MHz) 0.87 (3H, d,
b
a
-
-
CH3, 3J¼6.7 Hz), 3.65–4.00 (3H, M,
a
-CH2-Glyþ
b-H), 4.84 (1H, d,
H, 3J¼10.4 Hz), 4.93 (1H, d, H1 N-CH2-O, 2J¼5.4 Hz), 5.07 (1H, d, H10
N-CH2-O, 2J¼4.9 Hz), 6.91–7.20 (5H, M, H-arom), 7.68–7.80 (4H, M,
H-Phth) ppm. 13C NMR (DMSO, 63 MHz) 18.62 (
b-CH3), 37.49 (b-
CH), 44.14 (a-CH2-Gly), 55.92 (a-CH), 78.54 (N-CH2-O), 123.60,
126.57, 127.69 (CH-arom), 128.33, 134.95 (CH-Phth), 130.80 (Cq-
Phth), 143.22 (Cq-arom), 164.33 (C]O-Phth), 167.30 (C]O oxazo-
lidinone), 170.23 (CO-NR2) ppm.
a solution of Ac-erythro- or threo-b-Me-Aba-Gly-OH in CH3CN/H2O
(3:1), pyridine was added until a pH of 8 was reached. The reaction
mixture was cooled down to 0 ꢀC, followed by the addition of N,N-
dicyclohexylcarbodiimide (1.4 equiv). After 10 min, CH3NH2$HCl
(4.0 equiv) and pyridine (5.0 equiv) were added. The coupling was
continued for 2.5 h. HPLC analysis did indicate that starting mate-
rial was still present. So 30–60 extra equivalents of CH3NH2$HCl
and 9–13 extra equivalents of N,N-dicyclohexylcarbodiimide were
added and the pH was kept at 8. The solution was evaporated and
the residue was re-dissolved in CH2Cl2 (10 mM solution). The pre-
cipitated DCU was filtered and the filtrate was evaporated. The
residue was dissolved again in CH2Cl2 (130 mM solution), and after
filtration of DCU and evaporation of the filtrate, a sample of the
product was purified by prep HPLC. After lyophilization a white
solid was obtained.
4.9. erythro-2-(4S
dihydro-1H-benzo[c]azepin-2(3H)-yl)acetic acid [Phth-
erythro-(4S ,5S )-5-Me-Aba-Gly-OH] 27 and threo-2-(4S
4-phthaloyl-5-methyl-3-oxo-4,5-dihydro-1H-benzo[c]azepin-
*
,5S
*
)-(4-Phthaloyl-5-methyl-3-oxo-4,5-
*
*
*
,5R )-
*
2(3H)-yl)acetic acid [Phth-threo-(4S
*
,5R )-5-Me-Aba-Gly-OH]
*
28
In an oven dried one necked flask the oxazolidinone was dis-
solved in dry CH2Cl2 and kept under Ar atmosphere. Then TFMSA
(10 equiv) was added dropwise, which turned the solution dark
brown. The flask was sealed with a septum and stirring was con-
tinued overnight under Ar atmosphere and at rt. The reaction
mixture was cooled down to 0 ꢀC by use of an ice bath and
quenched by careful addition of a cold mixture of CH2Cl2 and water.
The phases were separated and the aqueous layer was extracted
with CH2Cl2. The combined organic phases were washed with H2O
(1ꢃ) and diluted with acetone to obtain complete dissolution of all
product. This phase was dried (MgSO4), filtered, and evaporated to
obtain the benzazepinones.
Compound 12. Mp 145.6–149.8 ꢀC. tR¼11.5 min. Rf 0.50
(CH3CH2OH). MS 304 (MþHþ), 273 (MꢂNHCH3), 326 (MþNaþ), 342
(MþKþ). 1H NMR (DMSO, 500 MHz) 1.05 (3H, d,
b
-CH3, 3J¼7.2 Hz),
1.95 (3H, s, CH3 Ac), 2.60 (3H, d, CH3 NHMe, 3J¼4.5 Hz), 3.10 (1H, m,
b
-H), 3.75 (1H, d, H
a
-Gly, 2J¼16.3 Hz), 4.12 (1H, d,
3
0-H, 2J¼17.3 Hz),
4.24 (1H, d, H
(1H, m,
a
0-Gly, 2J¼16.3 Hz), 5.19 (1H, d,
3
-H, 2J¼17.2 Hz), 5.45
a
-H, 3Ja bz2.6 Hz), 7.14–7.25 (4H, M, H-Aba), 7.84 (1H, br s,
,
Compound 27. Yield: 1.5 g (100%). Mp 190.7–193.5 ꢀC. HPLC
tR¼15.6 min. Rf 0.63 (petroleum ether/CH3OH 3:2þ1% CH3COOH).
MS 379 (MþHþ), 333 (MꢂCOOH), 361 (MꢂOH), 401 (MþNaþ). 1H
NH-CH3), 8.04 (1H, d, NH-Ac, 3J¼6.7 Hz) ppm. 13C NMR (DMSO,
126 MHz) 19.03 (b-CH3), 22.50 (CH3-Ac), 25.39 (NH-CH3), 39.84 (b-
CH), 50.45 (
a-CH2-Gly), 51.04 (a-CH), 52.54 (3-CH2), 125.91, 127.38,
NMR (DMSO, 250 MHz) 1.33 (3H, d,
b
-CH3, 3J¼6.7 Hz), 3.97 (1H, m,
128.51, 130.50 (CH-Aba), 132.98, 141.87 (Cq-Aba), 168.11 (C]O-
NHMe), 168.60 (C]O-Ac), 170.51 (C]O Aba) ppm.
b
-H), 4.04 (1H, d, H
a
-Gly, 2J¼17.3 Hz), 4.38 (1H, d, H
a
0-Gly,
2J¼17.3 Hz), 4.40 (1H, br s,
3
0-H), 5.15 (1H, d,
3
-H, 2J¼15.7 Hz), 5.32
Compound 13. Mp 257.1–263.9 ꢀC. HPLC tR¼10.3 min. Rf 0.55
(CH3CH2OH). MS 304 (MþHþ), 245 (MꢂCONHCH3), 273
(MꢂNHCH3), 326 (MþNaþ), 342 (MþKþ). 1H NMR (DMSO,
(1H, d,
a
-H, 3J¼3.8 Hz), 7.10–7.34 (4H, M, H-Aba), 7.83 (4H, br s, H-
Phth) ppm. 13C NMR (DMSO, 63 MHz) 18.16 (
b-CH3), 36.22 (b-CH),
51.26 (
a
-CH2-Gly), 52.63 (
3
-CH2), 57.55 (a-CH), 123.55, 135.08 (CH-
500 MHz) 1.28 (3H, d,
b
-CH3, 3J¼6.8 Hz), 1.92 (3H, s, CH3 Ac), 2.57
Phth), 126.75, 127.05, 128.26, 128.75 (CH-Aba), 131.39 (Cq-Phth),
135.67, 142.18 (Cq-Aba), 167.74 (C]O-Phth), 168.09 (C]O-Aba),
170.85 (C]O Gly) ppm.
(3H, d, CH3 NHMe, 3J¼4.4 Hz), 2.91 (1H, m,
b
-H), 3.35 (1H, d, H
a
-Gly,
0-Gly,
-H,
2J¼16.2 Hz), 3.96 (1H, d,
3
0-H, 2J¼16.1 Hz), 4.18 (1H, d, H
a
2J¼16.2 Hz), 5.08 (1H, d,
3
-H, 2J¼15.9 Hz), 5.17 (1H, m,
a
Compound 28. Yield: 3.18 g (100%). Mp 85.3–87.6 ꢀC. HPLC
tR¼16.3 min. Rf 0.65 (petroleum ether/CH3OH 3:2þ1% CH3COOH).
MS 379 (MþHþ), 333 (MꢂCOOH), 361 (MꢂOH), 401 (MþNaþ). 1H
3Ja b z11.0 Hz), 7.09–7.25 (4H, M, H-Aba), 7.75 (1H, br s, NH-CH3),
0
,
8.19 (1H, d, NH-Ac, 3J¼8.5 Hz) ppm. 1H NMR (CDCl3, 500 MHz) 1.38
(3H, d,
b
-CH3, 3J¼6.8 Hz), 2.06 (3H, s, CH3 Ac), 2.60 (3H, d, CH3
NMR (DMSO, 250 MHz) 1.32 (3H, d,
b
b
-CH3, 3J¼6.8 Hz), 3.86 (1H, d,
NHMe, 3J¼4.5 Hz), 2.90 (1H, m,
b
-H), 3.73 (1H, d,
H
a
a
-Gly,
H
a
-Gly, 2J¼17.3 Hz), 4.13 (1H, m,
-H), 4.20 (1H, d, H
a
0-Gly,
2J¼16.1 Hz), 3.88 (1H, d,
2J¼16.0 Hz), 5.08 (1H, d,
3
0-H, 2J¼16.1 Hz), 4.16 (1H, d, H
0-Gly,
2J¼17.3 Hz), 4.45 (1H, d,
3
0-H, 2J¼15.7 Hz), 4.95 (1H, d,
3-H,
3
-H, 2J¼15.9 Hz), 5.24 (1H, m,
a-H), 5.95
2J¼15.6 Hz), 5.14 (1H, d,
a
-H, 3J¼10.7 Hz), 7.17–7.36 (4H, M, H-Aba),
(1H, br s, NH-CH3), 6.40 (1H, d, NH-Ac, 3J¼7.8 Hz), 7.04–7.23 (4H, M,
7.87–7.96 (4H, M, H-Phth) ppm. 13C NMR (DMSO, 63 MHz) 21.72
H-Aba) ppm. 13C NMR (DMSO, 126 MHz) 22.58 (CH3-Ac), 23.67

R. De Wachter et al. / Tetrahedron 65 (2009) 2266–2278
2275
(b
-CH3), 25.45 (NH-CH3), 39.53 (
b
-CH), 48.62 (
a
-CH2-Gly), 51.03 (
3
-
4.14. (R,S)-2-Amino-3-(2-cyanophenyl)-2-methylpropanoic
CH2), 54.34 ( -CH), 125.75, 128.08, 128.94, 130.68 (CH-Aba), 134.51,
a
acid hydrochloride 33
141.69 (Cq-Aba), 168.15 (C]O-NHMe), 169.22 (C]O-Ac), 170.47
(C]O Aba) ppm.
A solution of ethyl (R,S)-2-amino-3-(2-cyanophenyl)-2-meth-
ylpropanoate hydrochloride 32 (5.0 g, 18.7 mmol, 1.0 equiv) in 6 N
HCl (70 mL) was stirred for 50 h at 65 ꢀC and was subsequently
cooled down to rt, which induced crystallization. The white crystals
were filtered and rinsed with a minimum amount of cold 6 N HCl.
Yield: 4.5 g (81%). Mp 139–144 ꢀC. HPLC tR¼9.6 min. Rf 0.40
(CH3CN/CH3OH/H2O 4:1:1). MS 205 (MþHþ), 159 (MꢂCOOH). 1H
4.11. Ethyl (R,S)-2-(benzylideneamino)propanoate 29
(R,S)-Ala-OEt$HCl (8.85 g, 58.0 mmol,1.0 equiv) was dissolved in
dry CH2Cl2 (120 mL) to which benzaldehyde (6.15 g, 58.0 mmol,
1.0 equiv), Et3N (16 mL, 116 mol, 2.0 equiv), and 4.8 g MgSO4 were
added. After 24 h stirring at rt, MgSO4 was filtered and the filtrate
was evaporated. The residue was dissolved in Et2O and washed
with brine, after which the organic layer was dried (MgSO4), fil-
tered, and evaporated. A light yellow oil was obtained and was used
without further purification.
NMR (DMSO, 250 MHz) 1.47 (3H, s,
a-CH3), 3.34 (1H, d, b-H,
2J¼14.2 Hz), 3.41 (1H, d,
b
-H0, 2J¼14.2 Hz), 7.47–7.84 (4H, M, H-
arom), 8.87 (3H, br s, NH3þ) ppm. 13C NMR (DMSO, 63 MHz) 21.50
(a-CH3), 40.40 (b-CH2), 59.52 (a-Cq), 113.70 (arom Cq-CN), 118.30
(CN), 128.80, 132.20, 133.50, 133.50 (CH-arom), 137.80 (Cq-arom),
171.80 (COO–) ppm.
Yield: 11.1 g (93%). Rf 0.7 (EtOAc/cyclohexane 2:1). MS 206
(MþHþ), 132 (MꢂCOOEt), 118 (MꢂPhꢂCH]N), 91 (tropylium ion).
1H NMR (CDCl3, 250 MHz) 1.27 (3H, t, CH3 ethyl, 3J¼7.1 Hz), 1.53
4.15. (R,S)-3-(2-Cyanophenyl)-2-(methoxycarbonylamino)-2-
methylpropanoic acid 34
(3H, d,
a
-CH3, 3J¼6.8 Hz), 4.10–4.29 (3H, M, OCH2þ
a-H), 7.38–7.80
(5H, M, H-arom), 8.32 (1H, s, imine H) ppm. 13C NMR (CDCl3,
To a solution of (R,S)-2-amino-3-(2-cyanophenyl)-2-methyl-
propanoic acid hydrochloride 33 (2.0 g, 8.43 mmol, 1.0 equiv) in
a 1 N NaOH solution (60 mL), methyl chloroformate (2.6 mL,
33.7 mmol, 4.0 equiv) was added. After 45 min stirring, the reaction
mixture was cooled down by use of an ice bath and acidified to pH
2–3 with a 6 N HCl solution. The aqueous phase was extracted with
cold CH2Cl2 (4ꢃ100 mL). The combined organic layers were washed
with water (pH 2,1ꢃ100 mL). The organic phase was dried (MgSO4),
filtered, and evaporated. A white solid was obtained and used in the
next step without further purification.
63 MHz) 14.19 (CH3 ethyl), 19.39 (a-CH3), 61.01 (OCH2), 67.99 (a-
CH), 128.50, 128.60, 131.00 (CH-arom), 135.80 (Cq-arom), 162.80
(CH]N), 172.50 (COO–) ppm.
4.12. Ethyl (R,S)-2-(benzylideneamino)-3-(2-cyanophenyl)-2-
methylpropanoate 30
To a solution of ethyl (R,S)-2-(benzylideneamino)propanoate 29
(10.0 g, 48.7 mmol, 1.0 equiv) in CH2Cl2 (300 mL), o-cyanobenzyl
bromide (11.5 g, 57.4 mmol, 1.2 equiv), KOH (4.1 g, 73.1 mmol,
1.5 equiv), K2CO3 (20.2 g, 146.0 mmol, 3.0 equiv), and (Et)3BnNCl
(1.11 g, 4.87 mmol, 0.1 equiv) were added. After overnight stirring,
the mixture was filtered and the precipitate was washed with
CH2Cl2 (2ꢃ15 mL). The filtrate was washed with water until neutral.
The organic phase was dried (MgSO4), filtered, and evaporated to
yield a pale yellow oil.
Yield: 1.5 g (69%). Mp 145.0–147.5 ꢀC. HPLC tR¼15.3 min. Rf 0.70
(CH3CN/CH3OH/H2O 4:1:1). MS 263 (MþHþ), 203 (MꢂCH3OCO),
525 (2Mþ1). 1H NMR (DMSO, 250 MHz) 1.14 (3H, s,
a-CH3), 3.25
(1H, d,
b
-H, 2J¼13.7 Hz), 3.50 (1H, d,
b
-H0, 2J¼13.7 Hz), 3.57 (3H, s,
MocCH3), 7.26–7.81 (4H, M, H-arom), 12.70 (1H, br s, COOH) ppm.
13C NMR (DMSO, 63 MHz) 22.30 (
Moc), 58.67 ( -Cq), 113.60 (arom Cq-CN),118.40 (CN), 127.60, 132.00,
a-CH3), 38.51 (b-CH2), 51.37 (CH3-
a
Yield: 15.3 g (98%). MS 321 (MþHþ), 233 (hydrolyzed imine). 1H
132.70, 132.90 (CH-arom), 140.50 (Cq-arom), 155.60 (C]O-Moc),
174.90 (COO–) ppm.
NMR (CDCl3, 250 MHz) 1.27 (3H, t, CH3 ethyl, 3J¼7.1 Hz), 1.52 (3H, s,
a
-CH3), 3.48 (1H, d,
b
-H, 2J¼13.7 Hz), 3.62 (1H, d,
b
-H0, 2J¼13.7 Hz),
4.23 (2H, q, OCH2, 3J¼7.1 Hz), 7.26–7.78 (9H, M, H-arom), 8.16 (1H, s,
4.16. (R,S)-3-(2-(Aminomethyl)phenyl)-2-(methoxy-
carbonylamino)-2-methylpropanoic acid 35
imine H) ppm. 13C NMR (CDCl3, 63 MHz) 14.56 (CH3 ethyl), 22.97
(a-CH3), 43.88 (b-CH2), 61.83 (OCH2), 69.33 (a-Cq), 115.00 (arom Cq-
CN), 119.10 (CN), 127.50–135.40 (CH-arom), 136.60 (arom Cq-
To
a
suspension of (R,S)-3-(2-cyanophenyl)-2-(methoxy
carbonylamino)-2-methylpropanoic acid 34 (1.86 g, 7.1 mmol,
1.0 equiv) in EtOH/H2O (3:1, 60 mL), 10% Pd/C (40w%, 0.744 g) and
a 10% aqueous AcOH solution (5.5 mL, 10.6 mmol, 1.5 equiv) were
added. The suspension was hydrogenated in a Parr apparatus
(50 psi, rt, 2 days). The mixture was filtered over dicalite and rinsed
with water. After evaporation of the solvent, the residue was
crystallized from a minimum amount of hot EtOH.
CH]N), 160.60 (CH]N), 173.50 (COO–) ppm.
4.13. Ethyl (R,S)-2-amino-3-(2-cyanophenyl)-2-
methylpropanoate hydrochloride 32
Ethyl (R,S)-2-(benzylideneamino)-3-(2-cyanophenyl)-2-meth-
ylpropanoate 30 (17.5 g, 54.6 mmol, 1.0 equiv) was suspended in an
Et2O/1.5 N HCl (200 mL/140 mL) mixture. After 3 h stirring, the
layers were separated and the aqueous phase was evaporated. The
residue was dissolved in a minimum amount of EtOH and dropwise
addition of EtOAc/Et2O (1:1) led to the formation of white crystals.
Yield: 14.7 g (63%) (four steps). Mp 103–108 ꢀC. HPLC
tR¼13.2 min. Rf 0.50 (CH3CN/CH3OH/H2O 4:1:1). MS 233
(MþHþ), 159 (MꢂCOOEt). 1H NMR (DMSO, 250 MHz) 1.14 (3H, t,
Yield: 1.4 g (74%). Mp 218.4–219.2 ꢀC. HPLC tR¼9.8 min. Rf 0.50
(CH3CN/CH3OH/H2O 4:1:1). MS 267 (MþHþ), 533 (2Mþ1). 1H NMR
(DMSO, 250 MHz) 1.41 (3H, s,
a
-CH3), 3.12 (1H, d,
b
-H, 2J¼14.0 Hz),
3.25 (1H, d,
b
-H0, 2J¼14.0 Hz), 3.58 (3H, s, MocCH3), 3.87 (1H, d, CH-
2
NH2, J¼13.4 Hz), 4.03 (1H, d, CH0-NH2, 2J¼13.4 Hz) 7.09–7.39 (4H,
M, H-arom) ppm. 13C NMR (DMSO, 63 MHz) 24.52 (
a
a
-CH3), 36.92
-Cq), 126.80,
(b
-CH2), 40.06 (CH2-NH2), 51.56 (CH3-Moc), 60.38 (
128.40, 131.10, 131.40 (CH-arom), 133.40, 137.80 (Cq-arom), 155.20
(C]O-Moc), 175.50 (COO–) ppm.
CH3 ethyl, 3J¼7.1 Hz), 1.53 (3H, s,
a
-CH3), 3.37 (1H, d,
b
-H,
2J¼13.9 Hz), 3.44 (1H, d,
b
-H0, 2J¼13.9 Hz), 4.13 (2H, m, OCH2),
7.49–7.86 (4H, M, H-arom), 9.07 (3H, br s, NHþ3 ) ppm. 13C NMR
4.17. (R,S)-4-Methyloxycarbonylamino-4-methyl-1,2,4,5-
tetrahydro-2-benzazepin-3-one [Moc-(R,S)-4-Me-Aba] 36
(DMSO, 63 MHz) 13.90 (CH3 ethyl), 21.00 (
a-CH3), 40.64 (b-
CH2), 59.60 ( -Cq), 62.80 (OCH2), 113.50 (arom Cq-CN), 118.10
a
(CN), 128.90, 132.40, 133.60, 133.70 (CH-arom), 137.60 (Cq-
arom), 170.10 (COO–) ppm.
A
solution of (R,S)-3-(2-(aminomethyl)phenyl)-2-(methoxy
carbonylamino)-2-methylpropanoic acid 35 (0.5 g, 1.88 mmol,

2276
R. De Wachter et al. / Tetrahedron 65 (2009) 2266–2278
1.0 equiv) and pyridine (303
mL, 3.76 mmol, 2.0 equiv) in CH3CN/
NMR (DMSO, 63 MHz) 21.39 (
a-CH3), 39.02 (b-CH2), 52.17–52.32 (a-
H2O (3:1, 170 mL) was cooled in an ice bath for 10 min, followed by
the addition of 1-ethyl-3-(30-dimethylaminopropyl)carbodiimide
(0.469 g, 2.4 mmol, 1.3 equiv, EDC). After 30 min the ice bath was
removed and the reaction mixture was stirred overnight. After
evaporation of the solvent, a white solid was obtained. This residue
was re-dissolved in water (100 mL) and extracted with CH2Cl2
(4ꢃ100 mL). The organic layer was washed with H2O (2ꢃ80 mL),
dried (MgSO4), filtered, and evaporated (yield: 60%). The combined
aqueous phases were evaporated again, re-dissolved in H2O
(100 mL), and extracted with CH2Cl2 (3ꢃ100 mL). The organic layer
was washed with H2O (50 mL), dried (MgSO4), filtered, and evap-
orated to yield a white solid.
CH2-Glyþ
3
-CH2), 59.57 ( -Cq), 127.90, 128.90, 130.10 (CH-Aba),
a
135.60, 137.30 (Cq-Aba), 170.40þ171.20 (C]O-AbaþC]O Gly) ppm.
4.20. (R,S)-2-(4-Acetamido-4-methyl-3-oxo-4,5-dihydro-1H-
benzo[c]azepin-2(3H)-yl)-N-methylacetamide [(R,S)-Ac-4-Me-
Aba-Gly-NHMe] 11
HBr$(R,S)-4-Me-Aba-Gly-OH 38 (185 mg, 0.745 mmol, 1.0 equiv)
was dissolved in H2O (9 mL). The pH was adjusted to 6 with Et3N
and Ac2O (490 mL, 5.22 mmol, 7.0 equiv) was added in five portions
over 5 min. Meanwhile the pH was kept at 6 with Et3N. After
30 min stirring at rt the reaction mixture was evaporated. The
residue was dissolved in a minimum amount 2 N HCl and extracted
with CH2Cl2 (4ꢃ40 mL). The organic layer was again extracted with
2 N HCl (5 mL), dried (MgSO4), filtered, and evaporated. A trans-
parent oil was obtained and used without further purification. To
a solution of (R,S)-Ac-4-Me-Aba-Gly-OH (177 mg, 0.584 mmol,
Yield: 0.45 g (97%). Mp 231.4–233.2 ꢀC. HPLC tR¼14.8 min. Rf
0.40 (Et2O/hexane 1:1). MS 249 (MþHþ), 217 (MꢂOCH3), 174
(MꢂCH3OCOꢂNH), 497 (2Mþ1). 1H NMR (DMSO, 250 MHz) 1.00
(3H, s,
3.79 (1H, m,
a
-CH3), 2.51 (1H, d,
b
-H, 2J¼13.7 Hz), 3.53 (3H, s, MocCH3),
3
-H), 3.98 (1H, d,
b
-H0, 2J¼13.7 Hz), 4.57 (1H, d,
3
-H0,
2J¼14.3 Hz), 7.20–7.29 (4H, M, H-Aba), 7.64 (1H, s, NHMoc), 7.80
1.0 equiv) in CH3CN/H2O (3:1, 30 mL), pyridine (96.2 mL, 1.17 mmol,
(1H, d, NH-Aba, 3J¼6.2 Hz) ppm. 13C NMR (DMSO, 63 MHz) 25.84
2.0 equiv) was added. The reaction mixture was cooled down to
0 ꢀC, followed by the addition of N,N-dicyclohexylcarbodiimide
(176 mg, 0.817 mmol,1.4 equiv). After 10 min CH3NH2$HCl (164 mg,
(a-CH3), 41.25 (3-CH2), 44.30 (b-CH2), 51.50 (CH3Moc), 58.61 (a-Cq),
127.10, 127.50, 128.10, 130.50 (CH-Aba), 137.90, 139.00 (Cq-Aba),
156.20 (C]O-Moc), 174.70 (C]O-Aba) ppm.
2.33 mmol, 4.0 equiv) and pyridine (481 mL, 2.92 mmol, 5.0 equiv)
were added. The coupling was continued for 2.5 h. The reaction
mixture was evaporated and the product was purified by prep
HPLC. After lyophilization a white solid was obtained.
4.18. (R,S)-tert-Butyl-2-(4-(methoxycarbonylamino)-4-
methyl-3-oxo-4,5-dihydro-1H-benzo[c]azepin-2(3H)-yl)-
acetate [Moc-(R,S)-4-Me-Aba-Gly-OtBu] 37
Yield: 106 mg (60%). HPLC tR¼13.4 min. MS 304 (MþHþ), 273
(MꢂCH3NH), 245 (MꢂCH3CONH of –CH3NHCO). 1H NMR (DMSO,
Pentane was added four times to 20 g 60% NaH in oil and dec-
anted into EtOH. The residual pentane was each time evaporated by
use of a dry rotavap. A white powder was obtained (11.4 g, 95%). To
a cooled solution of (R,S)-4-methyloxycarbonylamino-4-methyl-
1,2,4,5-tetrahydro-2-benzazepin-3-one 36 (79.5 mg, 0.320 mmol,
1.0 equiv) in N,N-dimethylformamide (6 mL), NaH (11.1 mg,
0.480 mmol, 1.5 equiv) was added, followed by the addition after
250 MHz) 1.11 (3H, s,
a
-CH3), 1.90 (3H, s, CH3Ac), 2.55 (1H, d,
b
-H,
-Gly,
-H,
-H0,
2J¼14.0 Hz), 2.63 (3H, s, CH3 NHCH3), 3.63 (1H, d,
Ha
2J¼16.6 Hz), 3.91 (1H, d,
b
a
-H0, 2J¼14.0 Hz), 4.10 (1H, d,
3
3
2J¼14.6 Hz), 4.40 (1H, d, H0
-Gly, 2J¼16.6 Hz), 5.05 (1H, d,
2J¼14.5 Hz), 7.21–7.39 (4H, M, H-Aba), 7.68 (1H, s, NH-CH3), 8.77
(1H, s, NH-Ac) ppm. 13C NMR (DMSO, 63 MHz) 22.36 (CH3-Ac),
25.47 (
a
-CH3), 25.60 (NH-CH3), 41.37 (b-CH2), 50.95 (3-CH2), 53.89
20 min of tert-butyl-bromoacetate (103
m
L, 0.640 mmol, 2.0 equiv).
(a-CH2-Gly) 58.91 (a
-Cq), 126.90, 127.80, 128.10, 129.80 (CH-Aba),
After 20 min the ice bath was removed and the mixture was stirred
for 1 h. The mixture was diluted with EtOAc (20 mL) and the or-
ganic phase was subsequently extracted with a saturated NaHCO3
solution (3ꢃ15 mL) and H2O (2ꢃ20 mL), dried (MgSO4), filtered,
and evaporated. The product was purified by flash column chro-
matography (EtOAc/hexane 12:20) to yield a white solid.
136.90, 137.70 (Cq-Aba), 168.50 (C]O-NHMe), 170.20 (C]O-Ac),
172.60 (C]O-Aba) ppm.
4.21. (1R,4S)-2-(4-Acetamido-3-oxo-1-phenyl-4,5-dihydro-
1H-benzo[c]azepin-2(3H)-yl)-N-methylacetamide [(1R,4S)-Ac-
1-phenyl-Aba-Gly-NHMe] 14 and (1S,4S)-2-(4-acetamido-3-
oxo-1-phenyl-4,5-dihydro-1H-benzo[c]azepin-2(3H)-yl)-N-
methylacetamide [(1S,4S)-Ac-1-phenyl-Aba-Gly-NHMe] 15
Yield: 91.6 mg (79%). Mp 103.1–104.6 ꢀC. HPLC tR¼22.4 min. Rf
0.40 (EtOAc). MS 363 (MþHþ), 385 (MþNaþ), 289 (MꢂOtBu). 1H
NMR (DMSO, 250 MHz) 1.04 (3H, s, a
-CH3), 1.26 (9H, s, H-tBu), 2.56
(1H, d,
b
-H, 2J¼14.1 Hz), 3.53 (3H, s, MocCH3), 3.90 (1H, d, H
a
-Gly,
(1S,4S)-Phth-1-phenyl-Aba-Gly-OEt and (1R,4S)-Phth-1-phe-
nyl-Aba-Gly-OEt were prepared as previously described by us.37
They were transformed into (1R,4S)-Ac-1-phenyl-Aba-Gly-NHMe
14 and (1S,4S)-Ac-1-phenyl-Aba-Gly-NHMe 15 by the reaction se-
quence described above for the 5-methyl analogs. Final purification
was performed by prep HPLC.
2J¼16.6 Hz), 3.97–4.15 (2H, M,
b
-H0þ
3-H), 4.25 (1H, d, Ha
0-Gly,
2J¼16.6 Hz), 5.03 (1H, d,
3
-H0, 2J¼14.8 Hz), 7.21–7.30 (4H, M, H-Aba),
7.73 (1H, s, NHMoc) ppm. 13C NMR (DMSO, 63 MHz) 26.54 (
a
-CH3),
-CH2-Gly),
a
-Cq), 80.62 (Cq-tBu), 127.00, 128.20, 130.20
27.86 (CH3 tBu), 41.90 (
53.67 ( -CH2), 59.30 (
b-CH2), 51.50 (CH3Moc), 52.28 (a
3
(H-Aba), 137.70, 138.00 (Cq-Aba), 156.20 (C]O-Moc), 168.70
(COOtBu), 173.70 (C]O-Aba) ppm.
4.22. (1R,4S)-2-(4-Acetamido-3-oxo-1-phenyl-4,5-dihydro-
1H-benzo[c]azepin-2(3H)-yl)-N-methylacetamide [(1R,4S)-Ac-
1-phenyl-Aba-Gly-NHMe] 14
4.19. (R,S)-2-(4-Amino-4-methyl-3-oxo-4,5-dihydro-1H-
benzo[c]azepin-2(3H)-yl)acetic acid hydrobromide ((R,S)-4-
Me-Aba-Gly-OH) [HBr$(R,S)-4-Me-Aba-Gly-OH] 38
Yield: 25.4 mg (49%). HPLC tR¼13.5 min. Rf 0.75 (EtOAc/BuOH/
AcOH/H2O 1:1:1:1). MS 366 (MþHþ). 1H NMR (CDCl3, 250 MHz):
(R,S)-Moc-4-Me-Aba-Gly-OtBu
37
(453 mg,
1.25 mmol,
2.08 (3H, s, CH3Ac), 2.77 (3H, br s, CH3 NHMe), 2.86 (1H, dd, b-H,
1.0 equiv) was dissolved in 33% HBr/AcOH (24 mL) and stirred at
55 ꢀC for 1.5 h. The reaction mixture was evaporated and repeatedly
re-dissolved in AcOH and evaporated to yield an orange solid.
Yield: >100%. HPLC tR¼10.8 min. Rf 0.20 (CH3CN/CH3OH/H2O).
MS 249 (MþHþ), 232 (MꢂNH2), 497 (2Mþ1). 1H NMR (DMSO,
2J¼16.0 Hz, 3J¼4.0 Hz), 2.98 (1H, pseudo t,
b
0-H,2Jz3J¼8.5 Hz), 3.63
(1H, H
a
-Gly, 2J¼17 Hz), 4.45 (1H, m,
a
-H), 4.99 (1H, H
a
0-Gly,
2J¼17 Hz), 5.80 (1H, s,
3-H), 6.28 (1H, br s, NH-CH3), 6.60 (1H, br s,
NH-Ac) 7.07 (2H, m, H-arom), 7.22–7.46 (7H, M, H-arom) ppm. 13C
NMR (DMSO-d6, 63 MHz) 22.0 (NH-CH3) (HMBC), 25.3 (CH3-Ac)
250 MHz) 1.25 (3H, s,
Glyþ
a
-CH3), 3.00–4.72 (6H, M,
b
-CH2þ
a
-CH2-
(HMBC), 35.3 (b-CH2), 53.4 (a-CH2-Gly), 54.0 (Ca Aba), 68.7 (3-CH),
3
-CH2), 7.29–7.41 (4H, M, H-Aba), 8.45 (3H, s, NHþ3 ) ppm. 13C
126.7 (CH-arom), 127.3 (CH-arom), 128.6 (CH-arom), 129.3

R. De Wachter et al. / Tetrahedron 65 (2009) 2266–2278
2277
7. Zhang, J.; Xiong, C.; Ying, J.; Wang, W.; Hruby, V. J. Org. Lett. 2003, 5, 3115–3118.
8. Richardson, J. S. Adv. Protein Chem. 1981, 34, 167–339.
9. Rose, G. D.; Gierasch, L. M.; Smith, J. A. Adv. Protein Chem. 1985, 37, 1–109.
10. Lewis, P. N.; Momany, F. A.; Scheraga, H. A. Biochem. Biophys. Acta 1973, 303,
211–229.
(CH-arom), 130.0 (CH-arom), 130.7 (CH-arom), 137.7 (Cq-arom),
139.9 (Cq-arom), 140.6 (Cq-arom), 168.2 (C]O-NHMe), 168.3 (C]O-
Ac), 171.4 (C]O-Aba) ppm.
11. Freidinger, R. M.; Veber, D. F.; Perlow, D. S. Science 1980, 210, 656–658.
12. Freidinger, R. M.; Perlow, D. S.; Veber, D. F. J. Org. Chem. 1982, 47, 104–109.
13. Valle, G.; Crisma, M.; Toniolo, C.; Yu, K. L.; Johnson, R. L. J. Chem. Soc., Perkin
Trans. 2 1998, 83–87.
4.23. (1S,4S)-2-(4-Acetamido-3-oxo-1-phenyl-4,5-dihydro-
1H-benzo[c]azepin-2(3H)-yl)-N-methylacetamide [(1S,4S)-Ac-
1-phenyl-Aba-Gly-NHMe] 15
14. Piscopio, A. D.; Miller, J. F.; Koch, K. Tetrahedron 1999, 55, 8189–8198.
15. Hoffmann, M.; Waibel, R.; Gmeiner, P. J. Org. Chem. 2003, 68, 62–69.
16. Perdih, A.; Kikelj, D. Curr. Med. Chem. 2006, 13, 1525–1556.
17. Tourwe´, D.; Verschueren, K.; Frycia, A.; Davis, P.; Porreca, F.; Hruby, V. J.; Toth,
G.; Jaspers, H.; Verheyden, P.; Van Binst, G. Biopolymers 1996, 38, 1–12.
18. Tourwe´, D.; Verschueren, K.; Van Binst, G.; Davis, P.; Porreca, F.; Hruby, V. J.
Bioorg. Med. Chem. Lett. 1992, 2, 1305–1308.
19. Van den Eynde, I.; Laus, G.; Schiller, P. W.; Kosson, P.; Chung, N. N.; Lipkowksi,
A. W.; Tourwe´, D. J. Med. Chem. 2005, 48, 3644–3648.
20. Warshawsky, A. M.; Flynn, G. A.; Koehl, J. R.; Mehdi, S.; Vaz, R. J. Bioorg. Med.
Chem. Lett. 1996, 6, 957–962.
Yield: 22.6 mg (44%). HPLC tR¼13.0 min. Rf 0.75 (EtOAc/BuOH/
AcOH/H2O 1:1:1:1). MS 366 (MþHþ). 1H NMR (CDCl3, 250 MHz)
1.99 (3H, s, CH3Ac), 2.76 (3H, br s, CH3 NHMe), 2.91 (1H, dd, b-H,
2J¼14.0 Hz, 3J¼10 Hz), 3.40 (1H, dd,
b
0-H, 2J¼14 Hz, 3J¼4.5 Hz), 4.35
(1H, H
a
-Gly, 2J¼17 Hz), 4.55 (1H, H
a
0-Gly, 2J¼17 Hz), 4.87 (1H, m,
a-
H), 5.78 (1H, s, 3-H), 6.38 (1H, br s, NH-CH3), 6.87 (1H, br s, NH-Ac)
7.00 (2H, m, H-arom), 7.05–7.34 (7H, M, H-arom) ppm. 13C NMR
(DMSO-d6, 63 MHz) 22.1 (NH-CH3) (HMBC), 26.0 (CH3-Ac) (HMBC),
´
21. Le Diguarher, T.; Ortuno, J.-C.; Shanks, D.; Guilbaud, N.; Pierre, A.; Raimbaud, E.;
Fauche`re, J.-L.; Hickman, J.; Tucker, G. C.; Casara, P. J. Bioorg. Med. Chem. Lett.
2004, 14, 767–771.
36.0 (b-CH2), 48.3 (Ca Aba), 53.5 (a-CH2-Gly), 67.3 (3-CH), 125.3
(CH-arom), 126.0 (CH-arom), 127.3 (CH-arom), 128.0 (CH-arom),
128.7 (CH-arom), 131.8 (CH-arom), 132.0 (CH-arom), 134.7 (Cq-
arom), 135.3 (Cq-arom), 142.7 (Cq-arom), 168.1 (C]O-Ac), 168.4
(C]O-NHMe), 171.9 (C]O-Aba) ppm.
22. Flynn, G. A.; Giroux, E. L.; Dage, R. C. J. Am. Chem. Soc. 1987, 109, 7914–7915.
23. Robl, J. A.; Simpkins, L. M.; Sulsky, R.; Sieber-McMaster, E.; Stevenson, J.; Kelly,
Y. F.; Sun, C.-Q.; Misra, R. N.; Ryono, D. E.; Asaad, M. M.; Bird, J. E.; Trippodo,
N. C.; Karanewsky, D. S. Bioorg. Med. Chem. Lett. 1994, 4, 1795–1800.
24. Casimir, J. R.; Itterbeke, K.; Van Den Nest, W.; Trescol-Bie´mont, M.-C.; Du-
mortier, H.; Muller, S.; Gerlier, D.; Rabourdin-Combe, C.; Tourwe´, D.; Paris, J.
J. Pept. Res. 2000, 56, 398–408.
4.24. Protocol for peptide synthesis
25. Van Rompaey, K.; Ballet, S.; To¨mbo¨ly, C.; De Wachter, R.; Vanommeslaeghe, K.;
Biesemans, M.; Willem, R.; Tourwe´, D. Eur. J. Org. Chem. 2006, 2899–2911.
26. Kataoka, Y.; Seto, Y.; Yamamoto, M.; Yamada, T.; Kuwata, S.; Watanabe, H. Bull.
Chem. Soc. Jpn. 1976, 49, 1081–1084.
Compounds 42 to 44 were prepared on a 0.4 mmol scale by
manualsolid phase synthesisusingMBHAresin (loading0.95 mmol/
g) as a solid support and following standard Boc amino protection
procedures. The Boc-deprotection was performed in a mixture of
TFA/CH2Cl2/2% anisole (5 minþ20 min). After filtration of the TFA
mixture and neutralization with 20% DIPEA/CH2Cl2, the couplings
were performed by using a threefold excess of the amino acids and
activating agent (TBTU), and a ninefold excess of N-methyl-
morpholine. The completeness of the couplings was checked with
the Kaiser or NF 31 color tests.62,63 Cleavage of the peptide from the
resin and side chain deprotection was accomplished by treatment
with HFliq for 1 h at 0 ꢀC. The crude peptide was purified by pre-
parative HPLC and their structure was confirmed by mass spec-
trometry. (S)-Aba-Gly56 and (S)-spiroAba-Gly55 were obtained
following the literature procedures, whereas (4S)-Me-Aba-Gly-OH
waspreparedasdescribed forracemate38, startingfrom(S)-33.64 H-
(S)-Dmt-(R)-Ala-(S)-Aba-Gly-NH2 42. HPLC tR¼11.5 min. Rf 0.66
(EtOAc/BuOH/AcOH/H2O 1:1:1:1). MS 496 (MþHþ). H-(S)-Dmt-(R)-
Ala-(4S)-Me-(S)-Aba-Gly-NH2 43. HPLC tR¼10.3 min. Rf 0.71 (EtOAc/
BuOH/AcOH/H2O 1:1:1:1). MS 510 (MþHþ). H-(S)-Dmt-(R)-Ala-(S)-
spiroAba-Gly-NH2 44. HPLC tR¼10.8 min. Rf 0.69 (EtOAc/BuOH/
AcOH/H2O 1:1:1:1). MS 536 (MþHþ).
27. Tourwe´, D.; Mannekens, E.; Diem, T. N. T.; Verheyden, P.; Jaspers, H.; To´th, G.;
´
´
¨ ¨
Peter, A.; Kertesz, I.; Torok, G.; Chung, N. N.; Schiller, P. W. J. Med. Chem. 1998, 41,
5167–5176.
´
´
28. Alıas, M.; Lopez, M. P.; Cativiela, C. Tetrahedron 2004, 60, 885–891.
29. Casimir, R.; Guichard, G.; Briand, J.-P. J. Org. Chem. 2002, 67, 3764–3768.
30. de Laszlo, S. E.; Bush, B. L.; Doyle, J. J.; Greenlee, W. J.; Hangauer, D. G.; Halgren,
T. A.; Lynch, R. J.; Schorn, T. W.; Siegl, P. K. S. J. Med. Chem. 1992, 35, 833–846.
31. Flynn, G.; Burkholder, T. P.; Huber, E. W.; Bey, P. Bioorg. Med. Chem. Lett. 1991, 1,
309–312.
32. Murkin, A. S.; Tanner, M. E. J. Org. Chem. 2002, 67, 8389–8394.
33. Stork, G.; Leong, A. Y. W.; Touzin, A. M. J. Org. Chem. 1976, 41, 3491–3493.
34. O’Donnell, M. J.; Bennett, W. D.; Bruder, W. A.; Jacobson, W. N.; Knuth, K.;
LeClef, B.; Polt, R. L.; Bordwell, F. G.; Mrozack, S. R.; Cripe, T. A. J. Am. Chem. Soc.
1988, 110, 8520–8525.
35. Yaozhong, J.; Changyou, Z.; Shengde, W.; Daimo, C.; Youan, M.; Guilan, L. Tet-
rahedron 1988, 44, 5343–5353.
36. Ghosez, L.; Antoine, J.-P.; Deffense, E.; Navarro, M.; Libert, V. Tetrahedron Lett.
1982, 23, 4255–4258.
37. Ballet, S.; Urbanczyl-Lipkowska, Z.; Tourwe´, D. Synlett 2005, 2791–2795.
38. Kolossva´ry, I.; Guida, W. C. J. Am. Chem. Soc. 1996, 118, 5011–5019.
39. Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.; Caufield,
C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11, 440–467.
40. Allinger, N. L.; Yuh, Y. H.; Lii, J.-H. J. Am. Chem. Soc. 1989, 111, 8551–8566.
41. Goodman, J. M.; Still, W. C. J. Comput. Chem. 1991, 12, 1110–1117.
42. Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T. J. Am. Chem. Soc. 1990,
112, 6127–6129.
43. Belvisi, L.; Bernardi, A.; Manzoni, L.; Potenza, D.; Scolastico, C. Eur. J. Org. Chem.
2000, 14, 2563–2569.
44. Ball, J. B.;Hughes, R. A.; Alewood, P. F.; Andrews, P. R. Tetrahedron 1993,49, 3467–3478.
45. Bax, A.; Subramanian, S. J. Magn. Reson. 1986, 67, 565–569.
46. Bax, A.; Summers, M. F. J. Am. Chem. Soc. 1986, 108, 2093–2094.
47. Macura, S.; Ernst, R. R. Mol. Phys. 1980, 41, 95–117.
Acknowledgements
This work was supported by the Fund for Scientific
ResearchdFlanders (Belgium, grant G.0008.08) and European
grant ‘Normolife’ (LSHC-CT-2006-037733). D.F. is a Research As-
sistant and S.B. is a postdoctoral researcher of the Fund for Scientific
Research Flanders (FWO-Vlaanderen). L.B. thanks the Institute for
the Promotion of Innovation through Science and Technology in
Flanders (IWT-Vlaanderen) for the financial support.
48. Montecucchi, P. C.; Decastiglione, R.; Piani, S.; Gozzini, L.; Erspamer, V. Int. J.
Pept. Protein Res. 1981, 17, 275–283.
49. Amiche, M.; Sagan, S.; Mor, A.; Delfour, A.; Nicolas, P. Int. J. Pept. Protein Res.
1988, 32, 506–511.
50. Melchiorri, P.; Negri, L. Gen. Pharmacol. 1996, 27, 1099–1107.
51. Broccardo, M.; Erspamer, V.; Falconierierspamer, G.; Improta, G.; Linari, G.;
Melchiorri, P.; Montecucchi, P. C. Br. J. Pharmacol. 1981, 73, 625–631.
52. Bryant, S. D.; Jinsmaa, Y.; Salvadori, S.; Okada, Y.; Lazarus, L. H. Biopolymers
2003, 71, 86–102.
53. Schiller, P. W.; Fundytus, M. E.; Merovitz, L.; Weltrowska, G.; Nguyen, T. M. D.;
Lemieux, C.; Chung, N. N.; Coderre, T. J. J. Med. Chem. 1999, 42, 3520–3526.
54. Okada, Y.; Tsuda, Y.; Fujita, Y.; Yokoi, T.; Sasaki, Y.; Ambo, A.; Konishi, R.; Nagata,
M.; Salvadori, S.; Jinsmaa, Y.; Bryant, S. D.; Lazarus, L. H. J. Med. Chem. 2003, 46,
3201–3209.
References and notes
1. Tyndall, J. D. A.; Pfeiffer, B.; Abbenante, G.; Fairlie, D. P. Chem. Rev. 2005, 105,
793–826.
2. Chou, K.-C. Anal. Biochem. 2000, 286, 1–16.
3. Souers, A. J.; Ellman, J. A. Tetrahedron 2001, 57, 7431–7448.
4. Cluzeau, J.; Lubell, W. D. Biopolymers 2005, 80, 98–150.
5. De Borggraeve, W. D.; Verbist, B. M. P.; Rombouts, F. J. R.; Pawar, V. G.; Smets,
W. J.; Kamoue, L.; Alen, J.; Van der Eycken, E. V.; Compernolle, F.; Hoornaert, G.
J. Tetrahedron 2004, 60, 11597–11612.
55. To¨mbo¨ly, Cs.; Ballet, S.; Feytens, D.; Ko¨ve´r, K. E.; Borics, A.; Lovas, S.; Al-Khrasani,
M.; Fu¨rst, Zs.; Toth, G.; Benyhe, S.; Tourwe´, D. J. Med. Chem. 2008, 51, 173–177.
56. Ballet, S.; Frycia, A.; Piron, J.; Chung, N. N.; Schiller, P. W.; Kosson, P.; Lipkowski,
A. W.; Tourwe´, D. J. Pept. Res. 2005, 66, 222–230.
57. Bisang, C.; Weber, C.; Inglis, J.; Schiffer, C. A.; van Gunsteren, W. F.; Jelesarov, I.;
Bosshard, H. R.; Robinson, J. A. J. Am. Chem. Soc. 1995, 117, 7904–7915.
6. Wang, W.; Xiong, C.; Hruby, V. J. Tetrahedron Lett. 2001, 42, 3159–3161.

2278
R. De Wachter et al. / Tetrahedron 65 (2009) 2266–2278
58. Flores-Ortega, A.; Jime´nez, A. I.; Cativiela, C.; Nussinov, R.; Alema´n, C.; Casa-
novas, J. J. Org. Chem. 2008, 73, 3418–3427.
62. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal. Biochem. 1970, 34,
595–598.
59. Sheldrick, G. M. SHELS-97, Program for Crystal Structure Solution; Univerity of
Go¨ttingen: Go¨ttingen, Germany, 1997.
63. Madder, A.; Farcy, N.; Hosten, N. G. C.; De Muynck, H.; De Clerq, P. J.; Barry, J.;
Davis, A. P. Eur. J. Org. Chem. 1999, 2787–2791.
60. Sheldrick, G. M. SHELXL97, Program for Crystal Structure Refinement; University
of Go¨ttingen: Go¨ttingen, Germany, 1997.
61. Farrugia, L. J. J. Appl. Crystallogr. 1999, 32, 837–838.
64. De Wachter, R.; Tourwe´, D. Unpublished results.
65. Toth, G.; Ioja, E.; To¨mbo¨ly, Cs; Ballet, S.; Tourwe´, D.; Pe´ter, A.; Martinek, T.; Chung,
N. N.; Schiller, P. W.; Benyhe, S.; Borsodi, A. J. Med. Chem. 2007, 50, 328–333.