Synthesis of 4-amino-3-oxo-tetrahydroazepino[3,4-b]indoles: new conformationally constrained Trp analogs

Article abstract of DOI:10.1016/j.tet.2006.11.069

The synthesis of tryptophan analogs is reported in which the conformation has been constrained by formation of a seven-membered lactam. Boc-protected 2′-formyl tryptophan was obtained by SeO₂ oxidation of Boc-tetrahydro-β-carboline-3-carboxylic

Full text of DOI:10.1016/j.tet.2006.11.069

Tetrahedron 63 (2007) 1459–1466
Synthesis of 4-amino-3-oxo-tetrahydroazepino[3,4-b]indoles: new
conformationally constrained Trp analogs
Karolina Pulka,a Debby Feytens,b Isabelle Van den Eynde,b Rien De Wachter,b
Piotr Kosson,c Aleksandra Misicka,a,c Andrzej Lipkowski,c Nga N. Chung,d
d
Peter W. Schiller and Dirk Tourwe
ꢀb,
*
aDepartment of Chemistry, Warsaw University, Pasteura 1, 02-093 Warsaw, Poland
bOrganic Chemistry Department, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
cMedical Research Centre, Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, Poland
dLaboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, Montreal, PQ H2W 1R7, Canada
Received 20 September 2006; revised 22 November 2006; accepted 24 November 2006
Available online 15 December 2006
Abstract—The synthesis of tryptophan analogs is reported in which the conformation has been constrained by formation of a seven-
membered lactam. Boc-protected 20-formyl tryptophan was obtained by SeO2 oxidation of Boc-tetrahydro-b-carboline-3-carboxylic acid.
Reductive amination was performed with a variety of amines and amino acid esters using sodium cyanoborohydride, followed by ring closure
to the target compounds. The constrained Trp derivative has been incorporated into the endomorphin-1 opioid peptide sequence to probe the
bioactive conformation.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
(R0¼OH)) skeleton.3,5 This type of Phe or Tyr analog has
been applied successfully in the design of peptides with
improved pharmacological properties and of selective
protease inhibitors.5,7,18–21 We have also published a solid-
supported synthesis allowing a parallel synthesis of 2,4-di-
substituted analogs of this drug-like molecule.22 We now
report an efficient synthetic route toward the Trp analog
4-amino-3-oxo-3,4,5,10-tetrahydro-1H-azepino[3,4-b]indol-
2-yl acetic acid (5, Aia). Only one report of this heterocycle
has been found in the literature, where it is part of a polycy-
clic structure having ACE inhibition properties (6).23,24 We
also report the use of the Aia residue to evaluate the proposed
Aromatic amino acids (Phe, Tyr, Trp, and His) are often a key
element of bioactivepeptides. The conformational restriction
of such residues has been a successful strategy to increase the
potency, selectivity, and metabolic stability of peptides, and
to influence their agonist/antagonist properties.1–8 The con-
cept of topographical control or the control of c-space has
been used to design many constrained analogs of these natu-
ral amino acids.1–23 One of the most successfully applied
analogs is that in which the a-nitrogen has been linked to
the aromatic ring through a methylene unit, resulting in the
six-membered ring derivatives tetrahydroisoquinoline-3-car-
boxylic acid (1, Tic), tetrahydro-b-carboline-3-carboxylic
acid (2, Tcc) or spinacine (3, Spi), which limit the side chain
conformation to a c1 of ꢀ60ꢁ or +60ꢁ.9 These have found
many applications in peptides1,4,5,7,9–11 as well as in peptide
mimetics.12–17 This widespread use was certainly helped by
the easy availability of the homochiral compounds (Fig. 1).
NH
HN
N
χ1
N
H
COOH
N
H
COOH
N
H
COOH
3
2
1
R'
N
An alternative conformational constraint involves the fixa-
tion of the side chain conformation to a c1 of +60ꢁ or 180ꢁ
by linking the aromatic ring to the nitrogen atom of the
succeeding amino acid, resulting in the 4-amino-1,2,4,
5-tetrahydro-2-benzazepin-3-one (4, Aba (R0¼H), Hba
NH
NH
H
N
N
R''
R
R
H2N
H2N
N
H
O
O
4
O
5
COOH
6
* Corresponding author. Tel.: +32 2 629 32 95; fax: +32 2 629 33 04; e-mail:
datourwe@vub.ac.be
Figure 1. Conformationally constrained analogs of Phe, Trp, and His.
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.11.069

1460
K. Pulka et al. / Tetrahedron 63 (2007) 1459–1466
bioactive conformation of the opioid peptide endomorphin-
1. Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) 7 is a highly po-
tent and selective endogenous agonist of the m-receptor. A
model for its bioactive conformation with the Trp side chain
in the gauche (+) or trans conformation was proposed using
NMR spectroscopy and molecular modeling.25 Since these
are the c1 values allowed by the Aia structure, the bioactivity
of [Aia]endomorphin-1 can either confirm or disprove the
proposal.
an N-acyliminium ion, which is generated from an
enamide23,24 or from an oxazolidinone precursor,8,28,29 the
other proceeds through lactam formation after reductive
amination of N-protected o-formyl phenylalanine.30,31 In
Scheme 1, these synthetic routes are shown for the Trp
derivatives. However, all our attempts to prepare the oxazo-
lidinone of phthaloyl-protected Trp-Gly 11 failed. We there-
fore focused our attention on the preparation of N-protected
20-formyl tryptophan 14.
2. Results and discussion
The conformation of Ac-Aia-Phe-NH2 8 was studied by
molecular modeling using MacroModel, as a model for
the C-terminus of endomorphin-1. The results confirmed
that the low energy conformations for 8 have a trans c1 con-
formation for the Aia ring. All low energy conformations
are extended structures, which are in agreement with our
previous studies on the benzazepine analog Ac-Aba-Gly-
NHMe.26 These also indicated that despite the fact that
similar dehydro-lactams were reported as b-turn mimics,27
the benzazepinones prefer extended conformations. Most
of the conformations of 8 have a gauche (ꢀ) orientation of
the Phe side chain. An extended conformation with the
3Trp side chain in the gauche (+) and the 4Phe side chain in
the trans conformation has been proposed as the bioactive
conformation of endomorphin-1.25 The conformation of 8
having a trans conformation of the Phe side chain has an
energy of 13.9 kJ/mol above the minimum and showed the
best backbone similarity with the proposed bioactive confor-
mation of endomorphin-1. A superimposition of both confor-
mations is shown in Figure 2.
NH
5
1
2N
COOH
4
3
RHN
O
10
NH
NH
H
N
COOR
O
O
N
RHN
RHN
COOH
12
O
11
NH
NH
CHO
H
N
COOH
RHN
COOH
RHN
14
O
13
Scheme 1. Retrosynthetic pathways for 4-amino-azepino[3,4-b]indoles.
Although the formation of the 20-formyl derivative by
direct formylation of N,N-dimethyl-homotryptamine under
Vilsmeier conditions was reported,32 the formylation of
Pht-Trp-OMe resulted in only 15% of the desired 20-formyl
compound, the main product being the 10-formyl isomer.
The SeO2 oxidation of N-acetyl-1,2,3,4-tetrahydro-b-carbo-
line-3-carboxylic acid methyl ester was reported to give
N-acetyl-20-formyl Trp-OMe in 52% yield.33 We have found
that this oxidation reaction does not require the ester protec-
tive group, and that the yield was better with Boc-nitrogen
protection (76%, Scheme 2). As a result, the oxidation of
Boc-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid 1534
resulted in Boc-20-formyl tryptophan 16, which could be
isolated by precipitation, and has an optimal protection for
further transformation.
Figure 2. Superimposition of the low energy trans-conformation for the
C-terminus of endomorphin-1 as reconstructed from Podlogar et al.25 and
Ac-Aia-Phe-NH2 8.
A maintained or an increased binding affinity of [Aia]endo-
morphin-1 9 is not in agreement with the bioactive model,
while a decreased binding affinity can be ascribed to a differ-
ent orientation of the indole ring, since the conformation of
the remaining part of the molecule is very similar, including
the positioning of the carboxamide, which is claimed to be
responsible for the m-selectivity.25
The reductive amination of 16 was performed with various
amines or amino acid esters using NaCNBH3. Intermediates
17 were not isolated,31 but the crude mixtures were used in
the following cyclization reaction using DCC as the coupling
agent. Afterovernight stirring, the cyclizationwas completed.
Two general synthetic routes have been described for
the synthesis of 4-amino-tetrahydro-2-benzazepin-3-ones
derived from Phe and Tyr: one involves the cyclization of
The crude products 18 were purified by flash chromato-
graphy. The isolated yields were calculated over two steps

K. Pulka et al. / Tetrahedron 63 (2007) 1459–1466
1461
The receptor affinities of the peptide analogs for the m- and
d-opioid receptors were measured in rat brain membranes36
and are summarized in Table 2.
NH
HN
dioxane, SeO2
reflux, 3h
CHO
COOH
Table 2. Receptor binding affinities of the endomorphin-1 analogs
N
BocHN
COOH
Boc
16
Peptide sequence
IC50 mꢂSEM (nM)a IC50 dꢂSEM (nM)b
15
Tyr-Pro-Trp-Phe-NH2
7
9.7ꢂ2.21
1600ꢂ7.2
1300ꢂ6.8
>104
RNH2, NaCNBH3,
MgSO4, CH2Cl2, pH6
Tyr-Pro-Trp-Phe-OH 20 1023.8ꢂ3.03
Tyr-Pro-Aia-Phe-NH2 9
223.8ꢂ3.64
Tyr-Pro-Aia-Phe-OH 21 >104
>104
m-Ligand:[3H]naloxone.
a
NH
N
d-Ligand:[3H][5,6Ile]deltorphin.
b
NH
DCC, pyridine,
CH3CN,
overnight, rt
BocHN
17
NHR
COOH
Both analogs showed a substantial loss in m-affinity com-
pared to the native m-selective endomorphin-1 sequence 7
or to the non-selective C-terminal acid analog 20. No affinity
for the d-opioid receptor was observed. The most active
analog 9 was tested in bioassays based on inhibition of
electrically evoked contractions of the guinea pig ileum
(GPI) and mouse vas deferens (MVD) as reported in detail
elsewhere.37 This [Aia]endomorphin-1 analog showed to
be a full agonist in both assays with IC50¼299ꢂ22 nM
and IC50¼678ꢂ83 nM. The GPI contains m- and k-recep-
tors, whereas the MVD contains mainly d-receptors, al-
though m- and k-receptors are also present in this tissue.37
This may explain the agonist activity of 9 in the GPI assay,
despite the low d-affinity of the compound.
BocHN
R
O
18
Scheme 2. Synthesis of 4-amino-azepino[3,4-b]indoles.
from 16 and are reported in Table 1. The optical purity of the
final compounds was studied by preparing (S)- and (R)-18b,
starting from L- and D-Trp, respectively. After Boc-deprotec-
tion, theenantiomerswerederivatizedwithMarfey’sreagent35
and separated by RP-HPLC. The individual enantiomers
showed less than 1% racemization.
Table 1. Yields of amino-indoloazepinones 18 after flash chromatography
Entry
Yield (%)
The conformation of Ac-Aia-Phe-NH2 8 was studied as
a model for the C-terminus of endomorphin-1 by NMR spec-
troscopy in DMSO-d6 solution. The large temperature depen-
dence of the NH2 signals indicates that there is no formation
of a turn conformation having an intramolecular hydrogen
bond (Table 3). The Ha and Hb coupling constants are consis-
tent with a trans c1 conformation for the Aia ring. This is in
complete agreement with the molecular modeling results.
18a R¼CH2C6H5
48
42
45
61
53
54
31
22
38
28
24
18b R¼CH2COOCH2CH3
18c R¼CH2COOCH2C6H5
18d R¼(S)CH(CH3)COOCH3
18e R¼(S)CH(CH3)COOCH2C6H5
18f R¼(S)(CH2)5COOCH3
18g R¼(S)CH(CH2C6H5)COOCH3
18h R¼CH(CH3)2
18i R¼CH2CH(CH3)2
18j R¼CH2CH2CH2CH3
18k R¼cyclohexyl
Table 3. NMR study of Ac-Aia-Phe-NH2 8
d (CDCl3, ppm) d (DMSO, ppm) Dd (ppm) Dd/DT (ppb/K)
In our previous studies on the preparation of 4-amino-tetra-
hydro-2-benzazepin-3-ones, we used solid-supported cyano-
borohydride for the reductive amination and supported
carbodiimide for the cyclization, in order to facilitate isola-
tion of the target compounds.22 Reductive aminations of 16
with several amines were carried out in the presence of solid-
supported cyanoborohydride. However, the reaction times
required for the completion of these reactions were exceed-
ingly long (between 8 and 13 days). The resulting secondary
amines 17h–k were cyclized using DCC in solution, and the
obtained yields and purities of the cyclic compounds 18h–k
were lower than those of 18a–g, which were obtained from
the solution phase synthesis.
Ac-NH 7.0
8.1
7.1
6.9
1.1
1.0
1.0
ꢀ4.7
ꢀ4.3
ꢀ4.3
NH2
NH2
6.1
5.9
These results indicate that the conformational constraints
imposed by this type of fixation of the Trp residue are not
3
well tolerated by the m-opioid receptor. Figure 2 indicates
that the decreased binding affinity of the [Aia]endomor-
phin-1 9 can be ascribed to an orientation of the indole
ring that is not well accepted by the receptor. Nevertheless,
analog 9 was able to fully activate the m-receptor.
3. Conclusions
We have incorporated the constrained dipeptide 18g into the
endomorphin-1 sequence. The synthesis of the tetrapeptide
was performed in solution. Boc-Aia-Phe-OMe 18g was
Boc-deprotected using 1 M HCl–EtOAc and coupled to
Boc-Tyr-Pro using TBTU as the coupling reagent. The re-
sulting peptide (Boc-Tyr-Pro-Aia-Phe-OMe 19) was treated
with saturated ammonia in MeOH or 0.58 M LiOH (5 equiv)
to give 9 and 21, respectively. These peptides were purified
by semi-preparative HPLC.
We have developed a suitable synthetic procedure giving an
easy access to the hitherto largely unexplored homochiral 4-
amino-3-oxo-tetrahydroazepino[3,4-b]indole skeleton. The
procedure allows the introduction of a variety of substituents
at the ring nitrogen. After Boc-deprotection, further derivati-
zation of 4-amino group is possible. This will be very impor-
tant to generate new leads in medicinal chemistry.22
Moreover we have demonstrated that the dipeptide analog

1462
K. Pulka et al. / Tetrahedron 63 (2007) 1459–1466
can be introduced into the bioactive peptides using standard
methodology and that these peptide analogs can provide in-
teresting information about the receptor-bound conformation
of Trp-containing peptides. We are currently further explor-
ing the potential of this building block in peptide mimicry.
182.5 (C); HRMS (ESI+) exact mass calcd for
C17H20N2O5Na [M+H]+: 355.1270, found: 355.1279.
4.2. General procedure for the synthesis of 4-amino-3-
oxo-tetrahydroazepino[3,4-b]indoles 18a–g
Aldehyde 16 (0.332 g, 1 mmol) was suspended in CH2Cl2
(puriss. p.a. 15 mL) and amino acid ester (hydrochloric or
p-toluenesulfonic acid salt, 1.05 mmol) was added. The
pH was adjusted to 6 with acetic acid or N-methyl-morpho-
line, followed by the addition of MgSO4 (20 wt %) and
NaCNBH3 (2.5 mmol). The reaction was monitored with
HPLC or TLC. When the reaction was completed (typically
3 h) the solvent was evaporated and the residue was redis-
solved in acetonitrile (80 mL). Pyridine (2 mmol) was added
and the reaction mixture was cooled to 0 ꢁC for 10 min and
DCC (1.1 mmol) was added. After 1 h of stirring at 0 ꢁC
the reaction was continued overnight at room temperature.
Oxalic acid (5 M solution in DMF, 2.5 mL) was added and
after 30 min of stirring the precipitate was filtered off. The
filtrate was evaporated and the redissolved in EtOAc
(100 mL) then washed with 1 M HCl (3ꢃ30 mL), saturated
aqueous NaHCO3 (3ꢃ30 mL), and brine (3ꢃ20 mL). The
organic layer was dried over MgSO4. After evaporation there
was a small amount of remaining N,N’-dicyclohexylurea
(DCU). The crude product was dissolved in small amount
of CH2Cl2 and DCU was filtered off. The solvent was
evaporated and the product was purified by flash column
chromatography (CH2Cl2–EtOAc).
4. Experimental section
4.1. General
RP-HPLC was performed using an Agilent 1100 Series
system (Waldbronn, Germany) with a Discovery BIO
Wide Pore C18 column (ID¼0.46 cm, L¼25 cm, PS¼5 mm,
Bellefonte, PA, USA). Gradient 1 or 2 was used (grad. 1:
t¼0 min, 97% A, 3% B; t¼30 min, 3% A, 97% B. Grad.
2: t¼0 min, 97% A, 3% B; t¼20 min, 3% A, 97% B), flow
rate: 1 mL/min, l¼215 nm. The mobile phases (water and
acetonitrile) contained 0.1% TFA. Semi-preparative HPLC
was performed using a Merck Hitachi with DAD detector
and RP C-18 column (Discovery BIO Wide Pore C18,
ID¼1.0 cm, L¼25 cm, PS¼5 mm), gradient: t¼0 min, 97%
A, 3% B; t¼15 min, 70% A, 30% B; t¼30 min, 60% A,
40% B, flow rate: 3 mL/min, l¼215 nm. The mobile phases
(water and acetonitrile) contained 0.05% TFA. TLC analysis
was performed on a plastic sheet precoated with silica gel
60F254 (Merck). Silica gel 60 (0.04–0.063 mm) from Merck
was used for flash chromatography. 1H and 13C NMR spectra
were recorded on an AC 250 Bruker spectrometer at 250 and
63 MHz or on a Varian Unity Plus spectrometer at 200 and
50 MHz in CDCl3 or DMSO-d6. Mass spectra were recorded
on a VG Quattro II spectrometer using electrospray ioniza-
tion (positive ion mode). High-resolution mass spectra were
recorded on a LCT TOF spectrometer. Optical rotations were
measured on a Perkin–Elmer 241 polarimeter. Infrared spec-
tral data were obtained using an Avatar 370 FTIR. Receptor
binding and functional assays were performed as described
previously.36,37 1,2,3,4-Tetrahydro-b-carboline-3-carbox-
ylic acid was synthesized from L-Trp and formaldehyde
using the Pictet–Spengler reaction.34 The Boc-protected
compound was obtained according to the standard proce-
dure38 in a mixture of dioxane–water.
4.2.1. 2(S)-tert-Butyl 2-(benzyl)-3-oxo-1,2,3,4,5,10-hexa-
hydroazepino[3,4-b]indol-4-yl carbamate 18a. Yield: 48%;
[a]2D0 +54.1 (c 1, CHCl3); IR (neat, cmꢀ1): 3293, 2974, 1694,
1641, 1161, 731; HPLC (grad. 1): tR¼23.5 min; Rf (CH2Cl2–
EtOAc 1:1): 0.82; MS: 306 [MꢀBoc]+, 350 [MꢀtBu]+, 406
1
[M+H]+, 428 [M+Na]+, 444 [M+K]+; H NMR (250 MHz,
CDCl3): d 1.51 (s, 9H), 2.86 (pseudo-t, 1H), 3.29 (pseudo-d,
1H), 3.88 (d, J¼17.25 Hz, 1H), 4.48 (d, J¼15.25 Hz, 1H),
4.83 (d, J¼18.25 Hz, 1H), 4.84 (d, J¼15 Hz, 1H), 5.02–5.12
(m, 1H), 6.02 (d, J¼7.25 Hz), 7.00–7.33 (m, 9H), 8.09 (s,
1H); 13C NMR (63 MHz, CDCl3): d 28.9 (CH3), 29.5 (CH2),
44.7 (CH2), 51.4 (CH), 51.9 (CH2), 80.2 (C), 109.8–141.7
(C+CH), 155.7 (C), 173.2 (C); HRMS (ESI+) exact mass
calculated for C24H27N3O3Na [M+Na]+: 428.1950, found:
428.1960.
4.1.1. 2(S)-N-(tert-Butoxycarbonyl)-20-formyl trypto-
phan 16. To a stirred solution of Boc-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid (1.00 g, 3.16 mmol) in dioxane
(25 mL), selenium dioxide (0.457 g, 4.11 mmol) was added.
The reaction mixture was refluxed for 3 h (monitored with
TLC, sprayed with 2,4-dinitrophenylhydrazine to give
orange spots). After filtration of the hot mixture through
dicalite and the removal of the solvent, the residue was redis-
solved in methanol and filtered through filter paper. The fil-
trate was evaporated and the product was precipitated from
CHCl3 to give a yellow solid (0.80 g, 76%). [a]2D0 +21.9
(c 1, CH3OH); IR (neat): 3328, 2979, 1683, 1649, 1164,
741; HPLC (grad. 1): tR¼17.3 min; Rf (CHCl3–MeOH–
AcOH 9:1:0.1): 0.44; MS: 215 [MꢀBocꢀH2O]+, 233
[MꢀBoc]+, 277 [MꢀtBu]+, 333 [M+H]+, 665 [2M+H]+;
1H NMR (250 MHz, DMSO-d6): d 1.26 (s, 9H), 3.16–3.74
(m, 2H), 4.19 (br s, 1H), 7.09–7.64 (m, 4H), 7.76 (s, 1H),
9.96 (s, 1H), 11.68 (s, 1H); 13C NMR (63 MHz, DMSO-
d6): d 25.8 (CH2), 28.4 (CH3), 44.7 (CH2), 55.0 (CH),
78.50 (C), 113.1–137.9 (C+CH), 155.6 (C), 173.4 (C),
4.2.2. 2(S)-Ethyl [4-[(tert-butoxycarbonyl)amino]-3-oxo-
3,4,5,10-tetrahydroazepino[3,4-b]indol-2(1H)-yl]acetate
18b. Yield 42%; [a]D20 +106.7 (c 1, CHCl3); IR (neat, cmꢀ1):
3325, 2978, 1710, 1649, 1162, 738; HPLC (grad. 1):
tR¼20.5 min; Rf (CH2Cl2–EtOAc 1:1): 0.55; MS: 302
[MꢀBoc]+, 346 [MꢀtBu]+, 402 [M+H]+, 424 [M+Na]+,
440 [M+K]+; 1H NMR (250 MHz, CDCl3): d 1.11 (t,
J¼7.13 Hz, 3H), 1.50 (s, 9H), 2.77 (pseudo-t, 1H), 3.22
(pseudo-d, 1H), 3.98 (d, J¼17.25 Hz, 1H), 4.02 (d,
J¼17.75 Hz, 1H), 4.07 (k, J¼7 Hz, 2H), 4.45 (d, J¼17.5 Hz,
1H), 5.00–5.07 (m, 1H), 5.14 (d, J¼17.5 Hz, 1H), 5.94 (d,
J¼7 Hz, 1H), 6.97–7.26 (m, 4H), 8.45 (s, 1H); 13C NMR
(63 MHz, CDCl3): d 14.3 (CH3), 28.8 (CH3), 29.4 (CH2),
46.9 (CH2), 50.7 (CH2), 51.4 (CH), 61.9 (CH2), 80.3 (C),
109.5–135.1 (C+CH), 155.6 (C), 169.4 (C), 173.4 (C);
HRMS (ESI+) exact mass calculated for C21H27N3O5Na
[M+Na]+: 424.1848, found: 424.1832.

K. Pulka et al. / Tetrahedron 63 (2007) 1459–1466
1463
4.2.3. 2(S)-Benzyl [4-[(tert-butoxycarbonyl)amino]-
3-oxo-3,4,5,10-tetrahydroazepino[3,4-b]indol-2(1H)-yl]-
acetate 18c. Yield 45%; [a]2D0 +74.0 (c 1, CHCl3); IR (neat,
cmꢀ1): 3315, 2975, 1707, 1649, 1164, 738; HPLC (grad. 1):
tR¼24.36 min; Rf (CH2Cl2–EtOAc 1:1): 0.7; MS: 364
[MꢀBoc]+, 408 [MꢀtBu]+, 464 [M+H]+, 486 [M+Na]+,
34.1 (CH2), 45.6 (CH2), 48.9 (CH2), 51.3 (CH), 51.9
(CH3), 80.1 (C), 109.7–135.1 (C+CH), 155.6 (C), 172.6
(C), 174.6 (C); HRMS (ESI+) exact mass calculated for
C24H33N3O5Na [M+Na]+: 466.2318, found: 466.2328.
4.2.7. 2(S)-Methyl 2-[4-[(tert-butoxycarbonyl)amino]-
3-oxo-3,4,5,10-tetrahydroazepino[3,4-b]indol-2(1H)-yl]-
3-phenylpropanoate 18g. Yield 31%; [a]2D0 +36.4 (c 1,
CHCl3); IR (neat, cmꢀ1): 3332, 2976, 1708, 1646, 1163,
735; HPLC (grad. 1): tR¼24.51 min; Rf (CH2Cl2–EtOAc
1:1): 0.72; MS: 378 [MꢀBoc]+, 422 [MꢀtBu]+, 478
1
502 [M+K]+; H NMR (250 MHz, CDCl3): d 1.49 (s, 9H),
2.75 (m, 1H), 3.23 (pseudo-d, 1H), 3.96 (d, J¼17 Hz, 1H),
4.09 (d, J¼17.5 Hz, 1H), 4.47 (d, J¼17.5 Hz, 1H), 4.99
(m, 1H), 5.05 (s, 2H), 5.13 (d, J¼17.5 Hz, 1H), 5.92 (d,
J¼7 Hz, 1H), 6.99–7.27 (m, 9H), 8.30 (s, 1H); 13C NMR
(63 MHz, CDCl3): d 29.3 (CH3), 30.1 (CH2), 46.9 (CH2),
50.7 (CH2), 51.4 (CH2), 67.6 (CH2), 80.2 (C), 118.7–135.4
(C+CH), 155.6 (C), 169.2 (C), 173.5 (C); HRMS (ESI+)
exact mass calculated for C26H29N3O5Na [M+Na]+: 486.2005,
found: 486.1981.
1
[M+H]+, 500 [M+Na]+, 516 [M+K]+; H NMR (250 MHz,
CDCl3): d 1.47 (s, 9H), 2.80 (pseudo-t, 1H), 3.06 (dd,
1H), 3.30 (d, 2H), 3.34 (s, 3H), 4.1 (d, J¼17.75 Hz, 1H),
4.92 (d, J¼17 Hz, 1H), 4.98–5.06 (m, 1H), 5.28 (k,
J¼6.25 Hz, 1H), 5.87 (d, J¼7.25 Hz, 1H), 6.93–7.35 (m,
9H), 7.85 (s, 1H); 13C NMR (63 MHz, CDCl3): d 28.8
(CH3), 29.4 (CH2), 35.8 (CH2), 43.2 (CH2), 51.3 (CH),
52.6 (CH3), 60.1 (CH), 80.1 (C), 109.6–136.7 (C+CH),
155.5 (C), 171.2 (C), 173 (C); HRMS (ESI+) exact mass
calculated for C27H31N3O5Na [M+Na]+: 500.2161, found:
500.2139.
4.2.4. 2(S)-Methyl 2-[4-[(tert-butoxycarbonyl)amino]-3-
oxo-3,4,5,10-tetrahydroazepino[3,4-b]indol-2(1H)-yl]-
propanoate 18d. Yield 61%; [a]2D0 +57.0 (c 1, CHCl3); IR
(neat, cmꢀ1): 3341, 2979, 1701, 1646, 1165, 735; HPLC
(grad. 1): tR¼21.02 min; Rf (CH2Cl2–EtOAc 1:1): 0.65;
MS: 302 [MꢀBoc]+, 346 [MꢀtBu]+, 402 [M+H]+; 1H
NMR (250 MHz, CDCl3): d 1.41 (d, J¼7 Hz, 3H), 1.49 (s,
9H), 2.86 (pseudo-t, 1H), 3.27 (s, 3H), 3.33 (pseudo-d,
1H), 4.17 (d, J¼17.25 Hz, 1H), 4.94 (d, J¼16.75 Hz, 1H),
5.08–5.17 (m, 1H), 5.33 (k, J¼7.25 Hz, 1H), 5.95 (d,
J¼7.25 Hz, 1H), 7.02–7.37 (m, 4H), 8.32 (s, 1H); 13C
NMR (63 MHz, CDCl3): 15.3 (CH3), 28.8 (CH3), 29.5
(CH2), 41.4 (CH2), 51.4 (CH), 52.5 (CH3), 52.7 (CH), 80.2
(C), 109.5–135.1 (C+CH), 155.6 (C), 172.3 (C), 173.1 (C);
HRMS (ESI+) exact mass calculated for C21H27N3O5Na
[M+Na]+: 424.1848, found: 424.1849.
4.3. General procedure for the reductive amination with
solid-supported cyanoborohydride 18h–k
Aldehyde 16 (0.332 g, 1 mmol) and amine (1.2 mmol) were
dissolved in CH2Cl2 (10 mL). Pivalic acid (10 mmol) and
polymer-bound cyanoborohydride (0.581 g, 2.5 mmol,
loading¼4.3 mmol/g) were added. When the reaction was
finished (between 8 and 13 days), polymer-bound benzalde-
hyde (0.167 g, 0.5 mmol, loading¼3 mmol/g) was added to
scavenge the excess of primary amine. Scavenging took 12
days. The resin was filtered off and the filtrate was evapo-
rated. Cyclization of products 17h–k was performed accord-
ing to the general procedure that was used for compounds
18a–g.
4.2.5. 2(S)-Benzyl 2-[4-[(tert-butoxycarbonyl)amino]-3-
oxo-3,4,5,10-tetrahydroazepino[3,4-b]indol-2(1H)-yl]-
propanoate 18e. Yield 53%; [a]2D0 +62.0 (c 1, CHCl3); IR
(neat, cmꢀ1): 3259, 2978, 1712, 1647, 1150, 734; HPLC
(grad. 1): tR¼26.27 min; Rf (CHCl3–EtOAc 3:1): 0.4; MS:
378 [MꢀBoc]+, 422 [MꢀtBu]+, 478 [M+H]+; 1H NMR
(250 MHz, CDCl3): d 1.42 (d, J¼7.18 Hz, 3H), 1.47 (s, 9H),
2.77 (pseudo-t, 1H), 3.23–3.31 (m, 1H), 4.14 (d, J¼16.9 Hz,
1H), 4.58 (d, J¼12.13 Hz, 1H), 4.83 (d, J¼12.13 Hz, 1H),
4.88 (d, J¼16.9 Hz, 1H), 5.03–5.12 (m, 1H), 5.37–5.45 (m,
1H), 5.93 (d, J¼7.28 Hz, 1H), 7.01–7.37 (m, 9H), 8.13 (s,
1H); 13C NMR (63 MHz, CDCl3): d 15.2 (CH3), 28.5
(CH3), 29.4 (CH2), 41.4 (CH2), 51.5 (CH), 52.8 (CH), 67.5,
80.2 (C), 111.3–135.1 (C+CH), 155.6 (C), 171.7 (C), 173.0
(C); HRMS (ESI+) exact mass calculated for C27H31N3O5Na
[M+Na]+: 500.2161, found: 500.2153.
4.3.1. 2(S)-tert-Butyl 2-isopropyl-3-oxo-1,2,3,4,5,10-hexa-
hydroazepino[3,4-b]indol-4-yl carbamate 18h. Yield:
22%; [a]2D0 +143.2 (c 1, CHCl3); IR (neat, cmꢀ1): 3315,
2977, 1689, 1631, 1164, 738; HPLC (grad. 1): tR¼23.40 min;
Rf (CH2Cl2–EtOAc 1:1): 0.62; MS: 358 [M+H]+, 380
1
[M+Na]+, 737 [2M+Na]+; H NMR (200 MHz, CDCl3):
d 0.94 (d, J¼6.8 Hz, 3H), 1.16 (d, J¼6.8 Hz, 3H), 1.49 (s,
9H), 2.81 (pseudo-t, 1H), 3.27 (pseudo-d, 1H), 4.08 (d,
J¼17.4 Hz, 1H), 4.78 (d, J¼17.4 Hz, 1H), 4.87–5.11 (m,
2H), 6.05 (d, J¼7.2 Hz, 1H), 7.01–7.36 (m, 4H), 8.22 (s, 1H);
13C NMR (50 MHz, CDCl3): d 19.9 (CH3), 20.2 (CH3), 28.4
(CH3), 29.1 (CH2), 38.1 (CH2), 45.2 (CH), 50.9 (CH), 79.7
(C), 109.7–134.7 (C+CH), 155.2 (C), 171.8 (C); HRMS
(ESI+) exact mass calculated for C20H27N3O3Na [M+Na]+:
380.1950, found: 380.1945.
4.2.6. 2(S)-Methyl 6-[4-[(tert-butoxycarbonyl)amino]-3-
oxo-3,4,5,10-tetrahydroazepino[3,4-b]indol-2(1H)-yl]-
hexanoate 18f. Yield 54%; [a]2D0 +91.1 (c 1, CHCl3); IR
(neat, cmꢀ1): 3315, 2931, 1711, 1639, 1156, 736; HPLC
(grad. 1): tR¼20.86 min; Rf (CH2Cl2–EtOAc 1:1): 0.58;
MS: 344 [MꢀBoc]+, 388 [MꢀtBu]+, 444 [M+H]+; 1H
NMR (250 MHz, CDCl3): d 1.05–1.49 (m, 15H), 2.18 (t,
J¼7 Hz, 2H), 2.78 (pseudo-t, 1H), 3.22–3.37 (m, 2H),
3.48–3.62 (m, 4H), 3.98 (d, J¼17.25 Hz, 1H), 4.97–5.04
(m, J¼16.25 Hz, 2H), 5.98 (d, J¼7 Hz, 1H), 7.00–7.33 (m,
4H), 8.62 (s, 1H); 13C NMR (63 MHz, CDCl3): d 24.7
(CH2), 26.5 (CH2), 28.1 (CH2), 28.8 (CH3), 29.4 (CH2),
4.3.2. 2(S)-tert-Butyl 2-isobutyl-3-oxo-1,2,3,4,5,10-hexa-
hydroazepino[3,4-b]indol-4-yl carbamate 18i. Yield:
38%; [a]2D0 +136.8 (c 1, CHCl3); IR (neat, cmꢀ1): 3313,
2964, 1691, 1638, 1155, 736; HPLC (grad. 1): tR¼24.72 min;
Rf (CH2Cl2–EtOAc 1:1): 0.66; MS: 394 [M+Na]+, 765
[2M+Na]+; 1H NMR (200 MHz, CDCl3): d 0.73 (d,
J¼6.6 Hz, 3H), 0.84 (d, J¼6.6 Hz, 3H), 1.49 (s, 9H), 1.72–
1.93 (m, 1H), 2.80 (pseudo-t, 1H), 3.14–3.48 (m, 2H), 3.95
(d, J¼17.0 Hz, 1H), 4.98–5.06 (m, J¼16.6 Hz, 2H), 6.03

1464
K. Pulka et al. / Tetrahedron 63 (2007) 1459–1466
(d, J¼7.2 Hz, 1H), 7.00–7.33 (m, 4H), 8.37 (s, 1H); 13C
NMR (50 MHz, CDCl3): d 19.8 (CH3), 20.0 (CH3), 27.6,
28.4 (CH3), 29.0 (CH2), 45.8 (CH2), 50.9 (CH), 56.3
(CH2), 79.7 (C), 109.5–134.7 (C+CH), 155.3 (C), 172.5
(C); HRMS (ESI+) exact mass calculated for C21H29N3O3Na
[M+Na]+: 394.2107, found: 394.2102.
sealed tightly with a cap and the reaction was stirred during
2 days. Thirty percent of racemization occurred. The solu-
tion was evaporated and the crude product was used in the
following step. The Boc protecting group was removed as
described for compound 18b. The resulting TFA salt
(0.42 mmol) was dissolved in water (4 mL). The pH was
adjusted to 6 with NEt3 and Ac2O (0.2 mL, 2.1 mmol) was
added in three portions. Meanwhile the pH was kept at 6
with NEt3. After stirring for 2 h at room temperature,
HPLC analysis showed completion of the reaction. The re-
action mixture was evaporated and the residue was purified
by preparative HPLC (gradient 2).
4.3.3. 2(S)-tert-Butyl 2-butyl-3-oxo-1,2,3,4,5,10-hexa-
hydroazepino[3,4-b]indol-4-yl carbamate 18j. Yield: 28%;
[a]2D0 +131.1 (c 1, CHCl3); IR (neat, cmꢀ1): 3315, 2960,
1690, 1636, 1155, 736; HPLC (grad. 1): tR¼24.79 min;
Rf (CH2Cl2–EtOAc 1:1): 0.65; MS: 294 [MꢀBoc+Na]+,
394 [M+Na]+; 1H NMR (200 MHz, CDCl3): d 0.83
(t, J¼7.2 Hz, 3H), 1.15–1.42 (m, 4H), 1.49 (s, 9H), 2.78
(pseudo-t, 1H), 3.24 (pseudo-d, 1H), 3.32–3.58 (m, 2H),
3.96 (d, J¼17.2 Hz, 1H), 4.98–5.06 (m, J¼17.0 Hz,
2H), 6.02 (d, J¼7.0 Hz, 1H), 7.00–7.33 (m, 4H), 8.34 (s,
1H); 13C NMR (50 MHz, CDCl3): d 19.9 (CH3), 27.1
(CH2), 28.4 (CH3), 29.0 (CH2), 30.1 (CH2), 45.2 (CH2),
48.7 (CH2), 50.9 (CH), 79.7 (C), 109.5–134.7 (C+CH),
155.3 (C), 172.2 (C); HRMS (ESI+) exact mass calculated
for C21H29N3O3Na [M+Na]+: 394.2107, found: 394.2115.
Yield: 43% over three steps; [a]2D0 ꢀ17.2 (c 1.25, MeOH); IR
(neat, cmꢀ1): 3328, 2978, 3361, 2330, 1638, 1182; HPLC
(grad. 2): tR¼11.39 min; Rf (EtOAc): 0.16; MS 404 [M]+;
1H NMR (500 MHz, DMSO): d 10.82 (s, 1H), 8.09 (d,
J¼6.5 Hz, 1H), 7.31–6.82 (m, 11H), 5.14 (t, J¼6.9 Hz,
1H), 5.07–5.05 (m, 1H), 4.94 (d, J¼16.8 Hz, 1H), 4.59 (d,
J¼16.8 Hz, 1H), 3.27–3.23 (m, 1H), 3.00 (d, J¼14.0 Hz,
1H), 2.95–2.92 (m, 1H), 2.7 (t, J¼14.0 Hz, 1H), 1.92 (s,
3H); 13C NMR (126 MHz, DMSO): d 172.1 (C), 171.0
(C), 168.7 (C), 137.7–106.8 (C+CH), 58.9 (CH), 49.9
(CH), 41.2 (CH2), 34.7 (CH2), 27.5 (CH2), 22.6 (CH3);
HRMS (ESI+) exact mass calculated for C23H24N4O3
[M+H]+: 405.1927, found: 405.1868.
4.3.4. 2(S)-tert-Butyl 2-cyclohexyl-3-oxo-1,2,3,4,5,10-hexa-
hydroazepino[3,4-b]indol-4-yl carbamate 18k. Yield:
24%; [a]2D0 +88.3 (c 1, CHCl3); IR (neat, cmꢀ1): 3289,
2930, 1698, 1621, 1698, 1620, 1154, 735; HPLC (grad. 1):
tR¼25.99 min; Rf (CH2Cl2–EtOAc 1:1): 0.75; MS 320
[MꢀBoc+Na]+, 420 [M+Na]+; 1H NMR (200 MHz,
CDCl3): d 1.19–1.23 (m, 10H), 1.49 (s, 9H), 2.82 (pseudo-
t, 1H), 3.26 (pseudo-d, 1H), 4.15 (d, J¼17.2 Hz, 1H), 4.52
(m, 1H), 4.82 (d, J¼16.8 Hz, 1H), 5.02–5.14 (m, 1H),
6.09 (d, J¼7.6 Hz, 1H), 7.00–7.36 (m, 4H), 8.25 (s, 1H);
13C NMR (50 MHz, CDCl3): d 24.8–30.8 (CH2), 28.4
(CH3), 29.2 (CH2), 39.1 (CH2), 50.9 (CH), 53.5 (CH), 79.9
(C), 109.7–134.7 (C+CH), 155.3 (C), 172.1 (C); HRMS
(ESI+) exact mass calculated for C23H31N3O3Na [M+Na]+:
420.2263, found: 420.2249.
4.6. Synthesis of Tyr-Pro-Aia-Phe-NH2 9
Boc-Tyr-Pro-Aia-Phe-OMe 19 (0.1 g, 0.136 mmol) was
dissolved in MeOH saturated with ammonia (5 mL). After
72 h, the solvent was evaporated to give a yellow solid.
The peptide amide was treated with 1 M HCl–EtOAc
(2 mL). After 2 h, cold Et2O was added to precipitate the
product. Analytical HPLC indicated two epimeric forms of
the peptide (ratio 66:34). Semi-preparative HPLC yielded
31% of 9 (purity>99%). HPLC (grad. 2): tR¼14.23 min;
MS: 606 [MꢀNH2]+, 623 [M+H]+, 645 [M+Na]+.
4.4. Boc-Tyr-Pro-Aia-Phe-OMe 19
4.7. Synthesis of Tyr-Pro-Aia-Phe-OH 21
Boc-Aia-Phe-OMe 18g (0.150, 0.31 mmol) was treated with
1 M HCl–EtOAc (2 mL). After 3 h, cold Et2O was added
to precipitate the product. Aia-Phe-OMe$HCl (0.119 g,
0.288 mmol) was suspended in CH2Cl2 (6 mL). Diisopropyl-
ethylamine (0.126 mL, 0.72 mmol), Boc-Tyr-Pro-OH
(0.109 g, 0.288 mmol), HOBt (0.039 g, 0.288 mmol), and
TBTU (0.102 g, 0.317 mmol) were added. The reaction
mixture was stirred at 0 ꢁC for 1 and 3 h at room temperature
until TLC indicated the absence of the substrates. The sol-
vent was evaporated and the residue was redissolved in
EtOAc (15 mL) and washed with 1 M HCl (3ꢃ6 mL), satu-
rated NaHCO3 (3ꢃ6 mL), and brine (3ꢃ6 mL). The organic
layer was dried over MgSO4. After evaporation of the
solvent, the crude product was obtained (0.197 g, 93%).
HPLC (grad. 1): tR¼15.5 min; MS: 738 [M+H]+.
To a solution of Boc-Tyr-Pro-Aia-Phe-OMe 19 (0.0858 g,
0.116 mmol) in MeOH (1.5 mL) at 0 ꢁC, LiOH (0.58 M,
1 mL) was added and the reaction mixture was stirred for
5 h at this temperature. MeOH was evaporated, water was
added (2 mL) and acidified to pH 3.0 with 10% citric acid.
The aqueous phase was extracted with EtOAc (4ꢃ3 mL),
combined organic layers were washed with brine (3ꢃ2 mL),
and dried over MgSO4 to give a yellow foam after evapora-
tion. The peptide acid was treated with 1 M HCl–EtOAc
(2 mL). After 2 h, cold Et2O was added to precipitate the
product. Semi-preparative HPLC yielded 44% of 21 (purity-
>99%). HPLC (gradient 2): tR¼14.60 min; MS: 624
[M+H]+, 646 [M+Na]+.
4.8. Procedure for the Boc-deprotection of 18c and its
derivatization with Marfey’s reagent (FDAA)
4.5. Synthesis and characterization of 2-[4-(acetyl-
amino)-3-oxo-3,4,5,10-tetrahydroazepino[3,4-b]indol-
2(1H)-yl]-3-phenylpropanamide 8
Analog 18c (0.013 g, 0.028 mmol) was dissolved in a mix-
ture of TFA–water 95:5 (231 mL) and CH2Cl2 (100 mL)
was added. The mixture was stirred for 1 h at room tem-
perature. The solution was evaporated to give the crude
TFA-salt.
Compound 18g (0.20 g, 0.42 mmol) was dissolved in
a solution of NH3 in MeOH (25%, 20 mL). The vessel was

K. Pulka et al. / Tetrahedron 63 (2007) 1459–1466
1465
Table 4. The 10 lowest-energy conformers of Ac-Aia-Phe-NH2 8 after clustering into families
c1 (Trp,
)
c1 (Phe,
ꢁ
ꢁ
)
Conformer number
Potential energy (kJ/mol)
Relative energy (kJ/mol)
42 (ꢁ)
j2 (ꢁ)
43 (ꢁ)
j3 (ꢁ)
1
2
3
4
5
6
7
8
9
10
118.21
118.72
126.50
127.76
130.51
131.30
132.11
133.77
134.06
134.50
0.00
0.51
8.29
9.55
12.30
13.09
13.90
15.56
15.85
16.29
ꢀ161
ꢀ161
ꢀ67
ꢀ162
ꢀ161
180
159
158
ꢀ52
160
161
ꢀ59
160
153
153
159
68
ꢀ123
ꢀ121
76
ꢀ134
ꢀ125
ꢀ119
ꢀ123
67
19
37
33
123
38
37
107
35
20
ꢀ107
ꢀ175
ꢀ174
60
ꢀ177
ꢀ172
58
ꢀ175
ꢀ166
ꢀ167
ꢀ175
ꢀ53
ꢀ53
ꢀ52
ꢀ55
55
ꢀ53
ꢀ173
ꢀ53
ꢀ53
ꢀ59
ꢀ161
85
85
ꢀ161
ꢀ125
A stock solution of the TFA-salt of the analyte (0.005 g,
0.009 mmol) in acetone–water 1:1 (1 mL) was prepared
and triethylamine was added to this solution (0.13 mL).
The superimposition of all the backbone atoms of Ac-Trp-
Phe-NH2 with those of conformer 7 (Table 3) was carried
out with MacroModel 5.0.39
A stock solution of FDAA (0.005 g, 0.183 mmol) in acetone
(1 mL) was prepared.
Acknowledgements
FDAA-solution (40 mL, 0.007 mmol) was added to the ana-
lyte stock solution (28 mL, 0.0003 mmol) and the mixture
was incubated at 40 ꢁC overnight. The mixture was then
quenched with aqueous HCl (1M, 28 mL) and the solution
was diluted to 1 mL with acetonitrile–water 1:1.
Supported by the Flemish–Polish Bilateral Agreement grant
AWI/BILA 03/03. D.F. is a Research Assistant of the Fund
for Scientific Research—Flanders (Belgium). We would like
to thank Ir. Ingrid Verbruggen for taking the NMR spectra.
References and notes
HPLC analysis was performed using gradient 1 (l¼340 nm).
Retention times were 24.01 min (S) and 25.35 min (R).
1. Hruby, V. J.; Li, G.; Haskell-Luevano, C.; Shenderovich, M.
Biopolymers 1997, 43, 219.
4.9. Molecular modeling of Ac-Aia-Phe-NH2 8
2. Gibson, S. E.; Guillo, N.; Tozer, M. J. Tetrahedron 1999,
5, 585.
3. Flynn, G. A.; Akeson, A. L.; Dharanipragada, R.; Genin, M. J.;
Malikayil, J. A.; Pottorf, R.; Sabol, J. S.; Schreuder, H.;
Tomlinson, R.; Waid, P.; Barrett, R.; Jacobs, J.; Yanofsky, S.
Lett. Pept. Sci. 1998, 5, 93.
4. Schiller, P. W.; Weltrowska, G.; Berezowska, I.; Nguyen,
T. M. D.; Wilkes, B. C.; Lemieux, C.; Chung, N. N.
Biopolymers 1999, 51, 411.
The calculations were carried out using MacroModel 5.039
with Maestro 8.0 as a graphic interface. The MM3* force
field40 was used for energy minimization in combination
with the GB/SA solvation model of Still et al.,41 using
MacroModel’s default parameters for an aqueous medium.
The conformational analysis of Ac-Aia-Phe-NH2 8 structure
was carried out with the Pure Low Mode search.42 Structures
(130,000) were generated and minimized by means of the
ꢁ
Polak–Ribiere conjugate gradient method as implemented
in MacroModel, using a gradient convergence criterion of
0.1 kJ/mol A. After this search the found conformations
were again minimized to an energy convergence of
˚
0.01 kJ/mol A. Duplicate structures and those greater than
50 kJ/mol above the global minimum were discarded. The
generated structures were clustered in families with Xcluster
˚
1.7. A RMSD value of 0.2 A was used.
ꢀ
5. 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.
6. Ruzza, P.; Cesaro, L.; Tourwe, D.; Calderan, A.; Biondi, B.;
ꢀ
Maes, V.; Menegazzo, I.; Osler, A.; Rubini, C.; Guiotto, A.;
Pinna, L. A.; Borin, G.; Donella-Deana, A. J. Med. Chem.
2006, 49, 1916.
7. 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.
8. 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.
This revealed 10 low energy conformations of Ac-Aia-Phe-
NH2 8 in a range of 16.74 kJ/mol above the global minimum
(E¼118.21 kJ/mol) (Table 4).
4.10. Superimposition of Ac-Aia-Phe-NH2 8 with the C-
terminus of the trans-endomorphin-1 NMR structure
9. Kazmierski, W. M.; Yamamura, H. I.; Hruby, V. J. J. Am. Chem.
Soc. 1991, 113, 2275.
10. Lembeck, F.; Griesbacher, T.; Eckhardt, M.; Henke, St.;
Breipohl, G.; Knolle, J. Br. J. Pharmacol. 1991, 102, 297.
11. Ye, Z. X.; MacNeil, T.; Weinberg, D. H.; Kalyani, R. N.; Tang,
R.; Strack, A. M.; Murphy, B. A.; Mosley, R. T.; MacIntyre,
D. E.; Van der Ploeg, L. H. T.; Patchett, A. A.; Wyvratt,
M. J.; Nargund, R. R. Peptides 2005, 26, 2017.
Ac-Trp-Phe-NH2 has been built using the torsional angles of
the trans-endomorphin-1 NMR structure:25 (4,j)i+1¼(ꢀ106,
ꢀ179), (4,j)i+2¼(ꢀ50,112) and with c1 (Trp)¼ꢀ39ꢁ and c1
(Phe)¼ꢀ179ꢁ. The MM3* force field40 was used in vacuo
for the energy minimization of this structure. The minimiza-
ꢁ
tion with constraints was carried out with the Polak–Ribiere
conjugate gradient method as implemented in MacroModel,
using a gradient convergence criterion of 0.02 kJ/mol A.
12. Wexler, R. R.; Greenlee, W. J.; Irvin, J. D.; Goldberg, M. R.;
Prendergast, K.; Smith, R. D.; Timmermans, P. B. M. W. M.
J. Med. Chem. 1996, 39, 625.
˚

1466
K. Pulka et al. / Tetrahedron 63 (2007) 1459–1466
13. Sawa, M.; Kiyoi, T.; Kurokawa, K.; Kumihara, H.; Yamamoto,
M.; Miyasaka, T.; Ito, Y.; Hirayama, R.; Inoue, T.; Kirii, Y.;
Nishiwaki, E.; Ohmoto, H.; Maeda, Y.; Ishibushi, E.; Inoue,
Y.; Yoshino, K.; Kondo, H. J. Med. Chem. 2002, 45, 919.
14. Grunewald, G. L.; Romero, F. A.; Criscione, K. R. J. Med.
Chem. 2005, 48, 134.
25. Podlogar, B. L.; Paterlini, M. G.; Ferguson, D. M.; Leo, G. C.;
Demeter, D. A.; Brown, F. K.; Reitz, A. B. FEBS Lett. 1998,
439, 13.
€
26. Van Rompaey, K.; Ballet, S.; Tomboly, C.; De Wachter, R.;
Vanommeslaeghe, K.; Biesemans, M.; Willem, R.; Tourwe,
€
ꢀ
D. Eur. J. Org. Chem. 2006, 13, 2899.
27. Hoffmann, T.; Waibel, R.; Gmeiner, P. J. Org. Chem. 2003,
68, 62.
15. (a) Matter, H.; Schwab, W.; Barbier, D.; Billen, G.; Haase, B.;
Neises, B.; Schudok, M.; Thorwart, W.; Schreuder, H.;
€
Brachvogel, V.; Lonze, P.; Weithmann, K. U. J. Med. Chem.
28. Flynn, G. A.; Burkholder, T. P.; Huber, E. W.; Bey, P. Bioorg.
Med. Chem. Lett. 1991, 1, 309.
1999, 42, 1908; (b) Matter, H.; Schwaben, W. J. Med. Chem.
1999, 42, 4506.
16. Thomas, J. B.; Atkinson, R. N.; Rothman, R. B.; Fix, S. E.;
Mascarella, S. W.; Vinson, N. A.; Xu, H.; Dersch, C. M.; Lu,
Y.-F.; Cantrell, B. E.; Zimmerman, D. M.; Carroll, F. I.
J. Med. Chem. 2001, 44, 2687.
17. Chen, K. V.; Njoroge, F. G.; Pichardo, J.; Prongay, A.;
Butkiewicz, N.; Yao, N.; Madison, V.; Girijavallabhan, V.
J. Med. Chem. 2006, 49, 567.
18. Ruzza, P.; Calderan, A.; Donella-Deana, A.; Biondi, B.;
Cesaro, L.; Osler, A.; Elardo, S.; Guiotto, A.; Pinna, L. A.;
Borin, G. Biopolymers (Pept. Sci.) 2003, 71, 478.
19. Flynn, G. A.; Beight, D. W.; Mehdi, S.; Koehl, J. R.; Giroux,
E. L.; French, J. F.; Hake, P. W.; Dage, R. C. J. Med. Chem.
1993, 36, 2420.
ꢀ
29. Casimir, J. R.; Tourwe, D.; Iterbeke, K.; Guichard, G.; Braind,
J.-P. J. Org. Chem. 2000, 65, 6487.
30. Warshawsky, A. M.; Flynn, G. A.; Koehl, J. R.; Mehdi, S.; Vaz,
R. J. Bioorg. Med. Chem. Lett. 1996, 6, 957.
ꢀ
31. Van Rompaey, K.; Van den Eynde, I.; De Kimpe, N.; Tourwe,
D. Tetrahedron 2003, 59, 4421.
32. Guan, H.; Laird, A. D.; Blake, R. A.; Tang, Ch.; Liang, Ch.
Bioorg. Med. Chem. Lett. 2004, 14, 187.
33. Gatta, F.; Misiti, D. J. Heterocycl. Chem. 1987, 24, 1183.
ꢀ
34. Iterbeke, K.; Laus, G.; Verheyden, P.; Tourwe, D. Lett. Pept.
Sci. 1998, 5, 121.
35. Marfey, P. Carlsberg Res. Commun. 1984, 49, 591.
36. Olma, A.; Lachwa, M.; Lipkowski, A. W. J. Pept. Res. 2003,
62, 45.
ꢀ
20. Le Diguarher, T.; Ortuno, J.-C.; Shanks, D.; Guilbaud, N.; Pierre,
A.; Raimbaud, E.; Fauchere, J.-L.;Hickman, J. A.; Tucker, G. C.;
Casara, P. J. Bioorg. Med. Chem. Lett. 2004, 14, 767.
21. Casimir, J. R.; Iterbeke, K.; Van den Nest, W.; Trescol-
Biemont, M. C.; Dumortier, H.; Muller, S.; Gerlier, D.;
37. DiMaio, J.; Nguyen, T. M. D.; Lemieux, C.; Schiller, P. W.
J. Med. Chem. 1982, 25, 1432.
38. Millet, R.; Goossens, J.-F.; Bertrand-Caumont, K.; Chavatte,
ꢁ
ꢀ
P.; Houssin, R.; Henichart, J.-P. Lett. Pept. Sci. 1999, 6, 221.
39. Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Hendrickson, T.; Still, W. C.
J. Comput. Chem. 1990, 11, 440.
40. Allinger, N. L.; Yuh, Y. H.; Lii, J.-H. J. Am. Chem. Soc. 1989,
111, 8551.
ꢀ
Rabourdin-Combe, C.; Tourwe, D.; Paris, J. J. Pept. Res.
2000, 56, 398.
22. Van den Eynde, I.; Van Rompaey, K.; Lazzaro, F.; Tourwe, D.
ꢀ
J. Comb. Chem. 2004, 6, 468.
23. Robl, J. A. EP Patent 0 657 453 A1, 1995; Chem. Abstr. 1995,
123, 285985.
41. Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T.
J. Am. Chem. Soc. 2005, 112, 6127.
ꢀ
42. Kolossvary, I.; Guida, W. C. J. Am. Chem. Soc. 1996, 118,
5011.
24. Flynn, G. A.; Giroux, E. L.; Dage, R. C. J. Am. Chem. Soc.
1987, 109, 7914.