Structure based design of simplified analogues of insect kinins

Article abstract of DOI:10.1016/j.tet.2005.07.085

Based on a receptor interaction model, simplified analogues of insect kinins were prepared. The compounds were templated on a conformationally restricted amino piperidinone carboxylate scaffold. The conformation of the analogues was studied by NMR and the

Full text of DOI:10.1016/j.tet.2005.07.085

Tetrahedron 61 (2005) 9555–9562
Structure based design of simplified analogues of insect kinins
a
b
Laila Kamoune,a Wim M. De Borggraeve,a, Bie M. P. Verbist, Jozef Vanden Broeck,
*
Geoffrey M. Coast,c Frans Compernollea and Georges Hoornaerta
aLaboratory for Organic Synthesis, Chemistry Department, K.U. Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium
bLaboratory for Developmental Physiology, Genomics and Proteomics, Department of Animal Physiology and Neurobiology,
K.U. Leuven, Naamsestraat 59, B-3000 Leuven, Belgium
cBiology Department, Birkbeck College, Malet Street, London WC1E 7HX, UK
Received 5 May 2005; revised 19 July 2005; accepted 26 July 2005
Available online 11 August 2005
Abstract—Based on a receptor interaction model, simplified analogues of insect kinins were prepared. The compounds were templated on a
conformationally restricted amino piperidinone carboxylate scaffold. The conformation of the analogues was studied by NMR and the
biological activity of the compounds was tested in a bio-assay.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
which are known to mimic cis-peptide bonds. Similar,
conclusions were obtained using a 4-aminopyroglutamate as
a cis-peptide bond inducer.3
Insect neuropeptides control essential physiological pro-
cesses such as maturation, water balance, pheromone
production and metamorphosis.1 Natural insect neuro-
peptides are readily degraded by peptidases; however,
neuropeptide analogues, which could resist peptidase
degradation would be attractive leads in the development
of new insect control agents.
A receptor interaction model was presented in which it was
assumed that the type VI b-turn over residues 1–4 places the
side-chains of Phe and Trp on the same side of the structure
where they can interact as a continuous aromatic surface
with the Malpighian tubule receptor. On the other hand, the
side-chains of Phe and Pro lie on the opposite face away
from the receptor explaining why these positions are
tolerant of considerable modification in the unconstrained
structures described above.
Design of such analogues requires knowledge of the
chemical and conformational features of the neuropeptides.
Earlier structure-activity studies have shown that the
generalised pentapeptide sequence Phe-Xaa-Xbb-Trp-Gly-
NH2 (XaaZTyr, Phe, His, Ser or Asn, and XbbZAla, Ser or
Pro) is present at the C-terminus of a number of insect
neuropeptides, which are controlling diuretic and myotropic
activity in cockroaches and crickets.2 Within this active core
pentapeptide, the aromatic residues Phe1 and Trp4 are both
critical for myotropic and diuretic activity as was revealed
by an alanine replacement scan. Moreover, by means of
NMR and molecular dynamics analysis, a cis-Pro type VI
b-turn has been shown to be involved in an active cyclo(Ala-
Phe-Phe-Pro-Trp-Gly) insect kinin peptide analog centered
around the Phe-Phe-Pro-Trp moiety. The possible presence
of this turn conformation in the receptor bound state was
further corroborated via the synthesis and biological
screening of tetrazole containing analogues of these kinins
Based on this structural information, a small library of
‘pseudotetrapeptides’ was designed containing the basic
features assumed to be important for receptor binding
(minimalistic approach): these are the cis-peptide bonds of
the type VI b-turn and the side-chains of the Phe and Trp
amino acids important for myotropic and/or diuretic activity
(Fig. 1), the N-terminal amine function and the C-terminal
carboxylate (as well as the glycine amide) were omitted in
this approach. We assumed the beta turn properties of the
systems could be imposed by means of a turn mimicking
amino piperidinone carboxylate system (APC).4 The side-
chains of this APC system (which replaces the residues 2
and 3 in the parent peptide) are of no major importance since
according to the receptor model these are diverted away
from the receptor. Hence we chose one of the most simple
APC systems available (Fig. 1). Phe and Trp side-chains
were introduced as simple phenylalkylamides and indolyl-
alkyl amides. Different tether lengths were chosen and also
Keywords: cis-peptide bond; Insect kinin analogues.
*
Corresponding author. Tel.: C32 16 327464; fax: C32 16 327990;
e-mail: wim.deborggraeve@chem.kuleuven.be
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.07.085

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L. Kamoune et al. / Tetrahedron 61 (2005) 9555–9562
Scheme 2. Generation of bislactam 4. Reagents and conditions: (i) (CH3)4Sn,
toluene, 110 8C, tetrakis; (ii) ethene (35 atm), 145 8C, toluene, 4 h; (iii) air
moisture.
established procedure via cyclisation of 2-[N-(4-methoxy-
benzyl) amino] acetonitrile (in the form of an HCl salt) with
oxalyl chloride.5 The PMB group by now has become the
standard protective group for the lactam function in this
type of precursors. According to our design approach, the
substituent R3 is believed to be of minor importance for
activity, so we have chosen to introduce the easily
accessible methyl group at this position.
The 3-methyl analogue 2 was obtained through catalytic
cross-coupling of 3,5-dichloro-2(1H)-pyrazinone 1 with
tetramethyltin using the procedure described before.6
Figure 1. Design approach towards simplified insect kinin analogues.
The 2(1H)-pyrazinone 2 underwent Diels–Alder addition
with ethene7 to produce the imidoyl chloride 3, which was
converted into the bis (lactam) 4 via hydrolysis (Scheme 2).
In this reaction step, both the conformational restriction and
the cis-relationship of the amine and the carboxylate of the
target APC system are imposed.
the position of the side-chains (N- or C-terminus) was
varied. Compounds of this type, incorporating a b-turn
mimic, are of course also interesting candidates for more
general broad screening, especially in view of the major
importance of the Phe and Trp amino acids in biological
processes.
2.1.2. Conversion of the APC precursor into tetrapeptide
analogues.
2. Results and discussion
2.1.2.1. Methanolysis of the bicyclic lactam systems.
Introduction of the side-chains of both Phe and Trp occurs in
the next steps in the synthesis. Different combinations of
alkylphenyl and alkylindolyl amides were envisaged both
for the N- and the C-terminus of the APC scaffold. To do so,
the bislactam system 4 firstly was selectively converted into
the HCl salt 5 using a selective methanolysis reaction via
treatment with an HCl–saturated methanol solution for 12 h
(Scheme 3).8 The ammonium chloride salt was trapped as
the corresponding amide by dissolving the evaporated
residue 5 from the methanolysis in dichloromethane and
adding an acyl chloride R0COCl (3 equiv) and Et3N
(4 equiv) under an inert atmosphere. After stirring for 16 h
at room temperature, the reaction mixture was purified by
column chromatography (silica gel, 10% MeOH/CH2Cl2 as
eluent) to yield the N-acylated products 6a–c (Scheme 3).
In the following subsections, we will first describe our
synthetic approach towards the target analogues I–V and
discuss structural features of models I and IV by analysis of
the NMR spectra. Finally, a bioassay performed on some of
the analogues will be presented.
2.1. Insect kinin analogue synthesis
The target compounds I–V can be brought back to a 3,5-
dichloropyrazinone 1 using chemistry previously developed
in our laboratory (Scheme 1).4 Key steps in the synthesis are
a Diels–Alder reaction and a selective methanolysis as will
be discussed further.
Scheme 1. Pyrazinone 1 as a precursor for compounds I–V.
2.1.1. Synthesis of the APC precursor.
2.1.1.1. Diels–Alder reaction and hydrolysis of the
adduct: introduction of the conformational restriction of
the APC system. The initially required N-(p-methoxy-
benzyl) pyrazinone 1 (Scheme 2) was generated using our
Scheme 3. Reagents and conditions: (i) MeOH/HCl, rt, 16 h; (ii) R0COCl
(3 equiv), Et3N (4 equiv), DCM, rt, 16 h or R0CO2H (1 equiv), DIEA
(5 equiv), CH3CN, TBTU (1.2 equiv), rt, 4 h.

L. Kamoune et al. / Tetrahedron 61 (2005) 9555–9562
9557
For the synthesis of compound 6d with the Trp side-chain
mimick, we first activated indol-3-yl propionic acid as its
OBt ester by treating it with TBTU and DIEA in CH3CN. To
this solution, product 5 was added. Compound 6d was
isolated in a moderate yield of 43%. The lower yield is due
to the recyclisation of the ammonium chloride salt 5 to the
bicyclic 4 because the OBt ester in this case reacts slower
than the corresponding acyl chlorides used for the
preparation of 6a–c. Using the same conditions as for 6d,
product 6e was obtained in 37% yield.
conformation of pseudotetrapeptides I and IV in CDCl3.
The analysis was performed using one dimensional NOE-
diff experiments and two-dimensional COSY- and NOESY-
techniques.
An APC system can in theory adopt two conformations A or
B (Fig. 2). The actual conformation in solution depends on
the substitution pattern of the APC system.
2.1.2.2. Hydrolysis of the ester function and subse-
quent coupling reaction with amines: addition of the
fourth amino acid mimicking residue. Direct conversion
of the methyl ester 6a to the target compound I with amines
proved to be impossible (we tried to do the substitution with
different amines under reflux conditions in methanol or
pyridine). In order to further functionalise the C-terminus of
these compounds, we first needed to convert them into the
corresponding acids.
The conversion of 6 into the corresponding acids 7 was
successfully effected using LiI in dry pyridine for 16 h at
115 8C (the more standard ester hydrolysis methods using
KOH or NaOH in water/methanol mixtures failed in this
case). Upon completion of the reaction, the pH of the
reaction mixture was adjusted to 2 by adding a dilute HCl
solution. After extraction, the acids 7 were further purified
by chromatography on silica gel.
Figure 2. Observed through-space NOE’s for pseudopeptide I.
In pseudopeptide I, the proton H2 can be identified as the
doublet at d 3.87, which couples with both H3 protons. One
3J coupling constant is 4.7 Hz; the other H3 coupling
constant is not resolved in the 1D spectrum though it can be
observed in the COSY spectrum. The axial protons H3 and
H4 can be distinguished from the equatorial ones because
they show broader multiplets due to the larger axial–axial
coupling constants present in their spectrum. The axial H3
proton shows a coupling in the NOESY spectrum with the
methyl group and with H2.
The obtained derivatives 7 were activated with TBTU in dry
DMF and reacted with different amines again representing
side-chains of Phe or Trp. After stirring these mixtures for
16 h at room temperature, the products were extracted
several times with dichloromethane and purified by column
chromatography to yield the final target compounds I–V
(Scheme 4).
Based on the NOE-effect between the NH–amide proton at d
8.51 and the axial proton H4, it can be concluded that the
conformation A of this pseudotetrapeptide prevails in
solution and not conformation B (Fig. 2). The other NOE-
effects in Figure 2 (as seen in the NOESY spectrum) are in
agreement with the proposed conformation.
2.2. NMR analysis of model systems I and IV
NMR techniques were used to study the solution
The chemical shift of the NHCH2 amide proton shows a
temperature dependence of K4.6 ppb/K indicating that it is
not completely solvent exposed. This partial solvent
shielding could be explained by the fact that a hydrogen
bond is present in this structure as would be expected with a
turn inducing APC scaffold.4 However, the shielding is not
complete and probably this hydrogen bonded conformation
is in equilibrium with other non-hydrogen bonded
conformations.
A similar analysis was performed for peptide IV. According
to our NMR data the peptide IV adopts the same
conformation as in the case of pseudopeptide I. Also in
this case the temperature dependence of the chemical shift
of the NHCH2 amide proton of K4.7 ppb/K shows partial
solvent shielding of this proton.
2.3. Fluid secretion assay
In order to check whether the systems were suitable
analogues (in structure and function) of the insect kinins,
Scheme 4. Reagents and conditions: (i) LiI, dry pyridine, 115 8C, 16 h;
(ii) TBTU, amine, dry DMF, rt.

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L. Kamoune et al. / Tetrahedron 61 (2005) 9555–9562
biological testing of the products was performed. The exact
details of the assay are discussed elsewhere.1
meeting the minimal requirements for receptor-binding,
which was the initial goal of this project.10 Hence the APC
system is an appropriate scaffold for stabilising the cis-
peptide bond and for directing the side-chains of Phe and
Trp towards the receptor surface as was stated in the
receptor binding model. However, oversimplification of the
parent system results in poor bioactivity.
The most common way to analyse the rate of fluid secretion
by Malpighian tubules (i.e., insect excretory organs) is the
‘Ramsay assay’. Tubules are dissected and incubated
in vitro in a physiological Ringer solution and the secretory
tubule terminal is inserted in a drop of paraffin oil. A small
droplet of liquid secretion is then easily visualised
(appearing in the oil) and measured under a microscope
(diameter can be measured and droplet volume can be
calculated) after a given time interval (Schematic represen-
tation in Fig. 3).
3. Conclusion
In this paper, design and synthesis of simplified insect kinin
analogues incorporating APC scaffolds was described.
In the target pseudopeptides I–V the side-chains important
for receptor binding were retained as well as the global beta
turn conformation of the peptide backbone. We have shown
that these simplified analogues are recognised by the
receptor, for which they were developed. A similar
minimalistic approach can be applied to other peptides, of
which the bioactive conformation is established.
Figure 3. Schematic representation of the diuretic assay system.
All analogues were tested in the assay using 10 mM
solutions (Fig. 4).9 At first sight, all compounds seem to
slightly increase the fluid secretion by the tubules compared
to the secretion in the absence of analogue (basal level).
A paired t-test however, reveals that the observed increase is
only significant in the case of compounds I, II and III. In
compound IV the side-chains of the N- and C-terminus were
exchanged and, therefore, it shows less resemblance to the
natural peptide than the other compounds.
4. Experimental
4.1. General
All melting points are uncorrected, and were measured on
an Electrothermal IA 9000 Digital melting point apparatus.
Infrared spectra were recorded on a Perkin-Elmer 1720
Fourier transform spectrometer. Mass spectra were run
using a Hewlett Packard MS-Engine 5989A apparatus for EI
and CI spectra and a Kratos MS 50TC instrument using
DS90 data system for exact mass measurements performed
in the EI mode at a resolution of 10.000. APCI and ESI
spectra were recorded on a Thermo Finnigan LCQ
Advantage mass spectrometer. For NMR spectra (d, ppm)
a Bruker AMX 400 and a Bruker Avance 300 spectrometer
were used. Analytical and preparative thin-layer chroma-
tography; TLC plates were performed on Alugram Sil G/
UV254. Column chromatography was carried out using
70–230 mesh silica gel 60 (E. M. Merck) as the stationary
phase.
4.2. Synthesis
4.2.1. Synthesis of 2-(1H)-pyrazinones 1 and 2. For the
preparation of 3,5-dichloro-2(1H)-pyrazinone 1 and its
functionalisation to pyrazinone 2, see Refs. 4 and 7.
5-Chloro-1-(4-methoxybenzyl)-3-methyl-2(1H)-pyrazinone
2. Yield: 92%; mp 92 8C (EtOH); IR (KBr) cmK1: 1652 (s,
1
CO), 1599 (s, C]N); H NMR (CDCl3, 400 MHz, ppm):
7.26 (d, JZ8 Hz, 2H, H-20CH-60), 7.06 (s, 1H, H-6), 6.90
(d, JZ8 Hz, 2H, H-30CH-50), 4.98 (s, 2H, N–CH2 of PMB),
3.82 (s, 3H, O–CH3), 2.49 (s, 3H, 3-CH3); 13C NMR
(CDCl3, 100 MHz, ppm):160.0 (CO), 158.7 (C–OCH3
(PMB)), 155.4 (C-3), 130.7 (C-20CC-60), 126.6 (C-ipso
PMB), 126.0 (C-5), 124.3 (C-6), 115.0 (C-30CC-50),
55.7 (O–CH3), 52.3 (N–CH2 of PMB), 21.3 (CH3); EIMS
m/z (%): 264 (MC, 34), 121 (CH3OC6H4CHC2 , 100);
HRMS: Calcd for C13H13ClN2O2: 264.0664; found:
264.0665.
Figure 4. Diuretic assay: tubular fluid secretion in the absence (control;
basal level) or presence of the analogues. Values marked with ** are
significantly different from the basal level of secretion according to a paired
t-test. As a positive test Achdo-K-II was added to the control sample.
The stimulation caused by compounds I, II and III should
be considered very modest, this can be shown by adding a
positive control (Achdo-K-II), which almost doubles the
secretion. The fact however, that some stimulation is
observed with the analogues is most probably due to

L. Kamoune et al. / Tetrahedron 61 (2005) 9555–9562
9559
4.2.2. Diels–Alder reaction and hydrolysis of the adduct:
introduction of the conformational restriction in the
APC system.
H-50), 6.57 (s, 1H, NH), 5.37 (d, JZ15 Hz, 1H, N–CH2 of
PMB), 3.94 (br d, JZ4 Hz, 1H, H-2), 3.79 (s, 3H, O–CH3),
3.74 (s, 3H, CH3 of ester), 3.63 (d, JZ15 Hz, 1H, N–CH2 of
PMB), 2.94 (t, JZ7 Hz, 2H, CH2–CO), 2.68–2.64 (m, 1H,
CH2CH2), 2.49–2.45 (m, 2H, CH2-Ph), 2.08–2.02 (m, 3H,
CH2CH2), 1.55 (s, 3H, CH3); 13C NMR (CDCl3, 100 MHz,
ppm): 173.7 (CO), 172.1 (CO), 172.0 (CO), 159.6 (C–OCH3
(PMB)), 141.2 (C-ipso Ph), 129.9 (C-20CC-60), 128.5,
128.3, 126.5 (CH-Ph), 128.2 (C-ipso PMB), 114.5 (C-30C
C-50), 58.4 (C-2), 56.8 (C-5), 55.2 (O–CH3), 53.1 (CH3 of
ester), 49.8 (N–CH2 of PMB), 39.3 (CH2–CO), 32.0
(CH2-Ph), 30.2 (C-4), 25.1 (CH3), 23.4 (C-3); EIMS m/z
(%): 438 (MC%, 30), 289 (MC%KNHCOCH2CH2Ph, 17),
261 (MC%KCH3–CO–NCH2PhOCH3, 100); HRMS: Calcd
for C25H30N2O5: 438.2147; found: 438.2154.
4.2.2.1. Synthesis of adduct 4. Pyrazinone 2 (1 mmol) is
dissolved in 20 mL of toluene and the solution is transferred
to a stainless steel bomb. The mixture is heated under ethene
pressure (35 atm) at 145 8C for 4 h. Upon cooling and
removal of ethene, the solvent is evaporated under reduced
pressure. The imidoyl chloride intermediate is further
hydrolised to the desired compound 4 by stirring it for
16 h in toluene open to air moisture. This compound is
further purified by column chromatography (silica gel,
MeOH–CH2Cl2 5/95).
5-(4-Methoxybenzyl)-1-methyl-2,5-diazabicyclo [2.2.2]
octane-3,6-dione 4. Yield: 83%; mp 156 8C (EtOH); IR
(KBr) cmK1: 3270 (w, NH), 1685 (s, CO); 1H NMR
(CDCl3, 400 MHz, ppm): 7.18 (d, JZ8 Hz, 2H, H-20C
H-60), 7.17 (s, 1H, NH), 6.85 (d, JZ8 Hz, 2H, H-30CH-50),
4.72 (d, JZ14 Hz, 1H, N–CH2 of PMB), 4.29 (d, JZ14 Hz,
1H, N–CH2 of PMB), 3.79 (s, 3H, O–CH3), 3.87 (br s, 1H,
H-4), 1.83–1.75 (m, 4H, CH2CH2), 1.51 (s, 3H, CH3); 13C
NMR (CDCl3, 100 MHz, ppm): 172.2 (CO), 171.2 (CO),
159.4 (C–OCH3 (PMB)), 129.5 (C-20CC-60), 128.3 (C-ipso
PMB), 114.2 (C-30CC-50), 59.0 (C-4), 58.1 (C-1),
55.2 (O–CH3), 47.9 (N–CH2 of PMB), 32.4 (CH2–C1),
(2S*,5S*) Methyl 1-(4-methoxybenzyl)-5-methyl-6-oxo-5-
(phenacylamino)-2-piperidinecarboxylate 6b. Yield: 91%;
Method A; mp oil; IR (NaCl) cmK1: 3350 (w, NH), 1739 (s,
1
CO), 1647 (s, CO); H NMR (CDCl3, 400 MHz, ppm):
7.36–7.25 (m, 5H, Ph-H), 7.070 (d, JZ8 Hz, 2H, H-20C
H-60), 6.83 (d, JZ8 Hz, 2H, H-3 CH-50), 6.59 (s, 1H, NH),
5.36 (d, JZ14 Hz, 1H, N–CH2 of PMB), 3.93 (br d, JZ
5 Hz, 1H, H-2), 3.78 (s, 3H, O–CH3), 3.74 (s, 3H, CH3 of
ester), 3.61 (d, JZ14 Hz, 1H, N–CH2 of PMB), 3.52 (s, 2H,
CH2-Ph), 2.64 (br d, JZ14 Hz, 1H, H-4eq), 2.16–1.99 (m,
3H, CH2CH2), 1.54 (s, 3H, CH3); 13C NMR (CDCl3,
100 MHz, ppm):173.1 (CO), 171.6 (CO), 170.4 (CO), 1590.2
(C–OCH3 (PMB)), 135.0 (C-ipso Ph), 129.5 (C-20CC-6 ),
129.2, 128.8, 127.0 (CH-Ph), 128.1 (C-ipso PMB), 114.1
(C-30CC-50), 58.0 (C-2), 56.8 (C-5), 55.2 (O–CH3), 52.6
(CH3 of ester), 49.4 (N–CH2 of PMB), 44.4 (CH2-Ph), 29.8
24.7 (CH2–C4), 18.1 (CH3); EIMS m/z (%): 274 (MC%
,
92), 153 (MC%KCH3OPhCH2, 42), 121 (CH3OC6H4CHC2 ,
100); HRMS: Calcd for C15H18N2O3: 274.1317; found:
274.1319.
4.2.2.2. Conversion of the bislactam into APC
analogues. a. Methanolysis reaction. The bislactam 4
(0.5 mmol) was stirred in 10 mL of HCl–saturated methanol
under an inert atmosphere for 16 h. Then methanol and HCl
were evaporated to afford the amino piperidinone as the
hydrochloride salt 5. This crude product is used in step b
without further purification.
(C-4), 24.7 (CH3), 23.0 (C-3); EIMS m/z (%): 424 (MC%
,
35), 289 (MC%KCH3OC6H4CH2–CH3, 30), 261 (MC%K
CH3OPhCH2–CO2CH3, 100); HRMS: Calcd for
C24H28N2O5: 424.1994; found: 424.1998.
(2S*,5S*) Methyl 5-benzoylamino-1-(4-methoxybenzyl)-5-
methyl-6-oxo-2-piperidinecarboxylate 6c. Yield: 77%;
Method A; mp oil; IR (NaCl) cmK1: 3393 (w, NH), 1739
b. Trapping the amine (Method A) (6a–c). The hydro-
chloride salt of the amino piperidinone 5 (0.7 mmol) was
dissolved in CH2Cl2 and the acid chloride (2.1 mmol) was
added followed by Et3N (2.8 mmol). The reaction mixture
was stirred for 4 h at room temperature. After evaporation of
the solvent, the residue was subjected to column chroma-
tography on silica gel using 5% EtOAc–CH2Cl2 as eluent to
afford 6a–c.
1
(s, CO), 1646 (s, CO); H NMR (CDCl3, 400 MHz, ppm):
7.94 (s, 1H, NH), 7.75 (d, JZ7 Hz, 2H, ortho Ph), 7.43 (t,
JZ7 Hz, 1H, para Ph), 7.330(t, JZ7 Hz, 2H, meta Ph), 7.09
(d, JZ8 Hz, 2H, H-20CH-6 ), 6.81 (d, JZ8 Hz, 2H, H-30C
H-50), 5.39 (d, JZ14 Hz, 1H, N–CH2 of PMB), 3.97 (br d,
JZ4 Hz, 1H, H-2), 3.72 (s, 3H, O–CH3), 3.70 (s, H, CH3 of
ester), 3.66 (d, JZ14 Hz, 1H, N–CH2 of PMB), 2.77 (br d,
JZ13 Hz, 1H, CH2CH2), 2.23 (td, JZ13, 5 Hz, 1H,
CH2CH2), 2.08–1.86 (m, 2H, CH2CH2), 1.68 (s, 3H,
CH3); 13C NMR (CDCl3, 100 MHz, ppm): 173.5 (CO),
171.5 (CO), 166.7 (CO), 159.3 (C–OCH3 (PMB)), 134.9
(C-ipso Ph), 131.3, 128.5, 127.0 (CH-Ph), 129.5 (C-20C
C-60), 127.9 (C-ipso PMB), 114.2 (C-30CC-50), 58.2 (C-2),
57.1 (C-5), 55.2 (O–CH3), 52.6 (CH3 of ester), 49.5 (CH2 of
PMB), 29.8 (C-4), 24.7 (CH3), 23.0 (C-3); EIMS m/z (%):
410 (MC%, 25), 289 (MC%KCH3OPhCH2, 23), 121
(CH3OC6H4CHC2 , 86); HRMS: Calcd for C23H26N2O5:
410.1827; found: 410.1841.
(Method B) (6d–e). A solution of indol-3-yl propionic acid
(or indole-3-acetic acid) (0.48 mmol) in CH3CN (3 mL) was
treated with DIEA (0.72 mmol), ammonium chloride salt 5
(0.58 mmol) and TBTU (0.58 mmol). The reaction mixture
was stirred at room temperature for 18 h. After filtration, the
filtrate was evaporated and purified by preparative thin-
layer chromatography (silica gel) using EtOAc as eluent to
give 6d–e.
(2S*,5S*) Methyl 1-(4-methoxybenzyl)-5-methyl-6-oxo-
5[(3-phenylpropanoyl) amino]-2-piperidinecarboxylate 6a.
Yield: 84%; Method A; mp 103 8C (EtOH); IR (KBr) cmK1
:
(2S*,5S*) Methyl 5-(3-1H-indol-3-yl-propionylamino)-1-
(4-methoxybenzyl)-5-methyl-6-oxo-2-piperidine-carboxyl-
ate 6d. Yield: 43%; Method B; mp 90 8C (EtOH); IR (KBr)
cmK1: 3301 (w, NH), 1740 (s, CO), 1640 (s, CO); 1H NMR
3310 (w, NH), 1740 (s, CO), 1623 (s, CO); 1H NMR
(CDCl3, 400 MHz, ppm): 7.29–7.16 (m, 5H, Ph-H), 7.09
(d, JZ8 Hz, 2H, H-20CH-60), 6.84 (d, JZ8 Hz, 2H, H-30C

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L. Kamoune et al. / Tetrahedron 61 (2005) 9555–9562
(CDCl3, 400 MHz, ppm): 8.54 (br s, 1H, NH-ind), 7.58 (d,
00
72%; mp 78 8C (EtOH); IR (KBr) cmK1: 3288 (w, NH),
1648 (s, CO); H NMR (CDCl3, 400 MHz, ppm, 55 8C):
1
JZ7 Hz, 1H, H-4 i), 7.32 (d, JZ7 Hz, 1H, H-700i), 7.15 (t,
JZ7 Hz, 1H, H-6 i), 7.08 (d, JZ8 Hz, 2H, H-20CH-60),
8.6611 (br s, 1H, OH), 7.14–7.06 (m, 7H, Ph-HC (H-20C
H-60)), 6.70 (d, JZ7 Hz, 2H, H-30CH-50), 6.20 (br s, 1H,
NH), 5.50 (d, JZ14 Hz, 1H, N–CH2 of PMB), 3.85 (br d,
JZ4 Hz, 1H, H-2), 3.69 (s, 3H, O–CH3), 3.60 (d, JZ14 Hz,
1H, N–CH2 of PMB), 2.89–2.88 (m, 2H, CH2-Ph), 2.55–
2.30 (m, 2H, CH2–CO), 2.42–2.32 (m, 1H, CH2CH2), 2.19–
2.15 (m, 1H, CH2CH2), 1.99–1.89 (m, 1H, CH2CH2), 1.81–
1.76 (m, 1H, CH2CH2), 1.36 (s, 3H, CH3); 13C NMR
(CDCl3, 100 MHz, ppm, 55 8C): 172.5 (CO), 171.7 (CO),
158.7 (C–OCH3 (PMB)), 140.7 (C-ipso Ph), 129.2 (C-20C
C-60), 128.03 (C-ipso PMB), 129.2, 128.3, 126.0 (CH-Ph),
113.8 (C-3 CC-50), 59.5 (C-2), 56.0 (C-5), 55.1 (O–CH3),
49.3 (N–CH2 of PMB), 38.0 (CH2–CO), 31.3 (CH2-Ph),
30.3 (C-4), 25.3 (CH3), 23.3 (C-3); EIMS m/z (%): 424
(MC%, 3), 406 (MC%KH2O, 6), 380 (MC%KCO2H, 2);
HRMS: Calcd for C24H28N2O5: 424.1993; found: 424.1998.
00
7.07 (t, JZ7 Hz, 1H, H-500i), 6.98 (s, 1H, H-200i), 6.83 (d,
JZ8 Hz, 2H, H-30CH-50), 6.56 (s, 1H, NH), 5.39 (d, JZ
15 Hz, 1H, N–CH2 of PMB), 3.95 (br d, JZ4 Hz, 1H, H-2),
3.77 (s, 3H, O–CH3), 3.73 (s, 3H, CH3 of ester), 3.66 (d, JZ
15 Hz, 1H, N–CH2 of PMB), 3.07 (t, JZ7 Hz, 2H,
CH2-ind), 2.57–2.55 (m, 3H, H-4eqCCH2–CO), 2.12–
2.00 (m, 3H, CH2CH2), 1.53 (s, 3H, CH3); 13C NMR
(CDCl3, 100 MHz, ppm): 173.1 (CO), 172.2 (CO), 171.5
(CO), 159.1 (C–OCH3 (0P0 MB)), 136.3 (C-7a00i), 129.4
(C-20CC-60), 128.1 (C-3a i), 127.1 (C-ipso PMB), 121.7,
121.6 (C-200iCC-6000i), 118.9 (C-500i), 1010 8.6 (C-400i), 114.7
(C-300i), 114.1 (C-3 CC-50), 111.0 (C-7 i), 57.9 (C-2), 56.5
(C-5), 55.1 (O–CH3), 52.5 (CH3 of ester), 49.3 (N–CH2 of
PMB), 37.7 (CH2–CO), 29.9 (C-4), 24.7 (CH3), 22.9 (C-3),
21.0 (CH2-ind); EIMS m/z (%): 477 (MC%, 57), 445 (MC%K
OCH3, 16), 261 (MC%KOCH3–NHCH2CH2indole, 58);
HRMS: Calcd for C27H31N3O5: 477.2244; found: 477.2263.
(2S*,5S*) 1-(4-Methoxybenzyl)-5-methyl-6-oxo-5-(phen-
acylamino) piperidine-2-carboxylic acid 7b. Yield: 82%;
mp 113 8C (EtOH); IR (KBr) cmK1: 3259 (w, NH), 1725 (s,
(2S*,5S*) Methyl 5-(2-1H-indol-3-yl-acetylamino)-1-(4-
methoxybenzyl)-5-methyl-6-oxo-2-piperidine carboxylate
6e. Yield: 37%; Method B; mp oil; IR (NaCl) cmK1: 3311
1
CO), 1649 (s, CO); H NMR (CDCl3, 400 MHz, ppm):
7.32–7.19 (m, 5H, Ph-H), 7.14 (d, JZ8 Hz, 2H, H-200C
H-600), 7.06 (br s, 1H, NH), 6.83 (d, JZ8 Hz, 2H, H-3 C
H-5 ), 5.52 (d, JZ14 Hz, 1H, N–CH2 of PMB), 3.98 (br d,
JZ5 Hz, 1H, H-2), 3.82 (d, JZ14 Hz, 1H, N–CH2 of PMB),
3.78 (s, 3H, O–CH3), 3.50 (s, 2H, CH2-Ph), 2.45 (td(ddd),
JZ14 Hz, 1H, CH2CH2), 2.24 (br d, JZ14 Hz, 1H,
CH2CH2), 1.87 (tdd(dddd), JZ14, 4 Hz, 1H, CH2CH2),
1
(w, NH), 1741 (s, CO), 1642 (s, CO); H NMR (C6D6,
400 MH0z0 , ppm): 9.27 (s, 1H, NH-ind), 7.74 (dd, JZ6, 2 Hz,
1H, H-4 i), 7.3800(dd, JZ6, 2 Hz, 1H, H-700i), 7.21–7.17 (m,
2H, 0H-500iCH-6 i), 6.99 (s, 1H, NH), 6.97 (d, JZ8 Hz, 2H,
H-2 CH-60), 6.81 (br0 d, JZ2 Hz, 1H, H-200i), 6.70 (d, JZ
8 Hz, 2H, H-30CH-5 ), 5.55 (d, JZ14 Hz, 1H, N–CH2 of
PMB), 3.75 (br d, JZ5 Hz, 1H, H-2), 3.67 (d, JZ14 Hz,
1H, N–CH2 of PMB), 3.62 (s, 2H, CH2–CO), 3.37 (s, 3H,
CH3 of ester), 3.30 (s, 3H, O–CH3), 2.44 (td(ddd), JZ
12 Hz, 1H, H-4ax), 2.36 (br d, JZ14 Hz, 1H, H-4eq), 1.82
(br d, JZ14 Hz, 1H, H-3eq), 1.53–1.51 (m, 1H, H-3ax),
1.40 (s, 3H, CH3); 13C NMR (C6D6, 100 MHz, ppm): 173.9
(CO), 172.0 (CO), 171.9 (CO), 160.2 (C–OCH3 (PMB)),
137.7 (C-7a00i), 130.2 (C-3a00i), 129.5 (C-20CC-60), 128.3
(C-ipso PMB), 125.0 (C-200i), 122.5, 120.1 (C-600iCC-500i),
119.6 (C-400i), 114.9 (C-30CC-50), 112.5 (C-700i), 109.5
(C-300i), 58.9 (C-2), 57.1 (C-5), 55.3 (O–CH3), 52.6 (CH3 of
ester), 50.4 (N–CH2 of PMB), 34.9 (CH2–CO), 31.1 (C-4),
25.4 (CH3), 23.7 (C-3); EIMS m/z (%): 463 (MC%, 34), 307
(MC%KCOCH2indol, 18); 261 (MC%KNHCOCH2indol–
OCH3, 18); HRMS: Calcd for C26H29N3O5: 463.2096;
found: 463.2107.
1.65 (br d, JZ14 Hz, 1H, CH2CH2), 1.35 (s, 3H, CH3); 13
C
NMR (CDCl3, 100 MHz, ppm): 172.9 (CO), 172.2 (CO),
171.8 (CO), 159.3 (C–OCH3 (PMB)), 134.6 (C-ipso Ph),
129.7 (C-20CC-60), 129.3, 128.8, 127.1 (CH-Ph), 128.2
(C-ipso PMB), 114.6 (C-30CC-50), 60.3 (C-2), 58.7 (C-5),
55.2 (O–CH3), 49.6 (N–CH2 of PMB), 42.7 (CH2-Ph), 30.8
(C-4), 25.1 (CH3), 23.0 (C-3); EIMS m/z (%): 410 (MC%),
396 (MC%KCH3, 18), 121 (CH3OC6H4CHC2 , 100); HRMS:
Calcd for C23H26N2O5: 410.1841; found: 410.1841.
(2S*,5S*) 5-Benzoylamino-1-(4-methoxybenzyl)-5-methyl-
6-oxo-piperidine-2-carboxylic acid 7c. Yield: 69%; mp
84 8C (EtOH); IR (KBr) cmK1: 1730 (s, CO), 1635 (s, CO);
1H NMR (CDCl3, 400 MHz, ppm): 7.80 (d, JZ7 Hz, 2H,
ortho Ph), 7.50 (t, JZ7 Hz, 1H, para Ph), 7.430(t, JZ7 Hz,
2H, meta Ph), 7.17 (d, JZ8 Hz, 2H, H-20CH-6 ), 7.14 (br s,
1H, NH), 6.86 (d, JZ8 Hz, 2H, H-30CH-50), 5.46 (d, JZ
14 Hz, 1H, N–CH2 of PMB), 4.10 (br d, JZ5 Hz, 1H, H-2),
3.79 (s, 3H, O–CH3), 3.72 (d, JZ14 Hz, 1H, N–CH2 of
PMB), 2.55 (br t(dd), JZ16 Hz,1H, H-4ax), 2.22 (br d, JZ
16 Hz,1H, H-3eq), 2.06–1.94 (m, 2H, H-4eqCH-3ax), 1.65
(s, 3H, CH3); 13C NMR (CDCl3, 100 MHz, ppm):172.9
(CO), 171.3 (CO), 167.8 (CO–NH), 159.4 (C–OCH3
(PMB)), 132.9 (C-ipso Ph), 132.3, 128.6, 127.3 (CH-Ph),
129.8 (C-20CC-60), 127.9 (C-ipso PMB), 114.2 (C-30C
C-50), 59.6 (C-2), 56.2 (C-5), 55.2 (O–CH3), 49.5 (N–CH2
of PMB), 30.9 (C-4), 25.5 (CH3), 23.1 (C-3); EIMS m/z (%):
396 (MC%, 4), 352 (MC%KCO2H, 2), 121 (CH3OC6H4CHC2 ,
100); HRMS: Calcd for C22H24N2O5: 396.1685; found:
396.1700.
4.3. General procedure for the deprotection of the ester
compounds
Ester compound 6 (0.63 mmol) and anhydrous lithium
iodide (1.9 mmol) in 10 mL of dry pyridine was refluxed for
16 h under argon. After cooling to room temperature the
solution was adjusted to pHZ2 by slowly adding dilute HCl
solution. The residue was extracted with EtOAc (3!). The
combined organics layers were dried over magnesium
sulphate and evaporated under reduced pressure. The crude
product was subjected to column chromatography on silica
gel using 5% MeOH/CH2Cl2 as eluent to give the
corresponding acid compound 7.
(2S*,5S*) 1-(4-Methoxybenzyl)-5-methyl-6-oxo 5 [(3-phenyl-
propanoyl) amino]- piperidine-2-carboxylic acid 7a. Yield:
(2S*,5S*) 5-(3-1H-Indol-3-yl-propionylamino)-1-(4-meth-
oxybenzyl)-5-methyl-6-oxo-piperidine-2-carboxylic acid

L. Kamoune et al. / Tetrahedron 61 (2005) 9555–9562
9561
7d. Yield: 85%; mp 64 8C; IR (KBr) cmK1: 3350 (w, NH),
1634 (s, CO); 1H NMR (CDCl3, 400 MHz, ppm): 8.04 (br s,
1H, NH-ind), 007.57 (d, JZ7 Hz, 1H, H-400i), 7.3400(d, JZ
7 Hz, 1H, H-7 i), 7.19 (t, JZ7 Hz, 2H, H-500iCH-6 i), 7.13
(d, JZ8 Hz, 2H, H-20CH-60), 7.06 (s, 1H, H-200i), 6.83 (d,
JZ8 Hz, 2H, H-30CH-50), 6.18 (s, 1H, NH), 5.39 (d, JZ
14 Hz, 1H, N–CH2 of PMB), 4.07 (br d, JZ5 Hz, 1H, H-2),
3.78 (s, 3H, O–CH3), 3.73 (d, JZ14 Hz, 1H, N–CH2 of
PMB), 3.12–3.08 (m, 2H, CH2-ind), 2.64–2.58 (m, 2H,
CH2–CO), 2.34–1.61 (m, 4H, CH2CH2), 1.36 (s, 3H, CH3);
13C NMR (CDCl3, 100 MHz, ppm): 173.1 (CO), 172.2
(CO), 171.5 (CO),0159.1 (C–OCH3 (PMB)), 134.6 (C-7a00i),
129.4 (C-20CC-600), 127.8, 127.1 (C-3a00iCC-ipso PMB),
121.7, 121.6 (C-6 0iCC-200i), 119.1 (C-500i), 118.6 (C-400i),
114.2 (C-30CC-5 ), 111.2 (C-700i), 109.1 (C-300i), 57.9
(C-2), 56.5 (C-5), 55.2 (O–CH3), 49.4 (N–CH2 of PMB),
37.7 (CH2–CO), 29.9 (C-4), 24.7 (CH3), 22.9 (C-3), 21.0
(CH2-ind); EIMS m/z (%): 463 (MC%, 10), 445 (MC%K
H2O, 11), 130 (indolCHC2 , 49); HRMS: Calcd for
C26H29N3O5: 463.2107; found: 463.2116.
NMR (CDCl3, 400 MHz, ppm): 8.52 (t, JZ5 Hz, 1H, NH–
CO–C2), 7.95 (s, 1H, NH-ind), 7.69 (d, JZ8 Hz, 1H,
H-400i), 7.32 (d, JZ8 Hz, 1H, H-700i), 7.24–7.18 (m, 5H,
Ph-H), 70.014 (t, JZ7 Hz, 10H0 , H-600i or H-500i), 7.10–7.06 (m,
2H, 0H-2 iCH-600i or H-5 i), 7.05 (d, JZ80Hz, 2H, H-20C
H-6 ), 6.79 (d, JZ8 Hz, 2H, H-30CH-5 ), 5.68 (s, 1H,
NH-C5), 5.29 (d, JZ15 Hz, 1H, N–CH2 of PMB), 3.86 (br
d, JZ6 Hz, 1H, H-2), 3.76 (s, 3H, O–CH3), 3.71–3.64 (m,
2H, CH2–NH), 3.30 (d, JZ15 Hz, 1H, N–CH2 of PMB),
3.11 (t, JZ2 Hz, 2H, CH2-ind), 2.96 (t, JZ2 Hz, 2H,
CH2-Ph), 2.62–2.54 (m, 1H, CH2–CO), 2.48–2.41 (m, 1H,
CH2–CO), 2.35 (td(ddd), JZ14, 4 Hz, 1H, H-4ax), 2.03 (br
d, JZ14 Hz, 1H, H-3eq), 1.87 (tdd(dddd), JZ14, 6, 4 Hz,
1H, H-3ax), 1.44 (br d, JZ14 Hz, 1H, H-4eq), 1.38 (s, 3H,
CH3); 13C NMR (CDCl3, 100 MHz, ppm): 171.7 (CO),
171.1 (CO), 170.6 (CO), 159.1 (C–OCH3 (PMB)), 140.5
(C-ipso Ph), 136.2 (C-7a00i), 129.7 (C-20CC-60), 128.6
(C-3a00i), 12070 .6 (C-ipso PMB), 128.5, 128.3, 126.3 (CH-Ph),
122.0 (C-6i ), 1210.0 (C-200i), 119.3, 119.0 (C-4i00CC-5i00),
114.8 (C-30CC-5 ), 113.5 (C-700i), 110.9 (C-300i), 60.9
(C-2), 55.3 (C-5), 55.2 (O–CH3), 48.9 (N–CH2 of PMB),
40.0 (CH2–NH), 37.8 (CH2–CO), 31.3 (CH2-Ph), 30.4
(C-4), 25.4 (CH3), 24.8 (CH2-ind), 23.1 (C-3); EIMS m/z
(%): 566 (MC%, 20), 424 (MC%KCH2CH2indol, 58), 143
(CH2CH2indole, 55); HRMS: Calcd for C34H38N4O4:
566.2893; found: 566.2910.
(2S*,5S*) 5-(2-1H-Indol-3-yl-acetylamino)-1-(4-methoxy-
benzyl)-5-methyl-6-oxo-piperidine-2-carboxylic acid 7e.
Yield: 75%; mp 104 8C (EtOH); IR (KBr) cmK1: 3389 (w,
NH), 1730 (s, CO), 1638 (s, CO); 1H NMR (CDCl3,
300 MHz, ppm): 9.60 (br s, 1H, OH), 8.73 (s, 1H, NH-ind),
00
7.56 (d, JZ7 Hz, 1H, H-4 i), 7.49 (d, JZ7 Hz, 1H, H-700i),
00
7.29 (d, JZ7 Hz, 2H, H-5 iCH-600i), 7.09 (d, JZ8 Hz, 1H,
(2S*,5S*) 1-(4-Methoxybenzyl)-5-methyl-6-oxo-5-(phen-
acylamino) piperidine-2-carboxylic acid {2-(1H-indol-3-
yl)-ethyl}-amide II. Yield: 44%; mp 213 8C (EtOH); IR
(KBr, cmK1): 3439 (w, NH), 1634 (s, CO); 1H NMR
(CDCl3, 400 MHz, ppm): 8.45 (t, JZ5 Hz, 1H, NH–CH2),
8.10 (s, 1H, NH-ind), 7.62 (d, JZ7 Hz, 1H, H-400i), 7.31 (d,
JZ7 Hz, 1H, H-700i), 7.28–7.24 (m, 5H, Ph-H), 7.13 (t, JZ
7 Hz, 1H, H-6000i), 7.09 (t, JZ7 Hz, 1H, H-500i), 7.01 (d, JZ
9 Hz, 2H, H-2 0CH-600), 7.00 (br s, 1H, H-200i), 6.76 (d, JZ
9 Hz, 2H, H-3 CH-5 ), 5.82 (s, 1H, NH-C5), 5.26 (d, JZ
14 Hz, 1H, N–CH2 of PMB), 3.85 (br d, JZ6 Hz, 1H, H-2),
3.73 (s, 3H, O–CH3), 3.65 (m, 2H, CH2–NH), 3.52 (s, 2H,
CH2-Ph), 3.30 (d, JZ14 Hz, 1H, N–CH2 of PMB), 3.04 (t,
JZ8 Hz, 2H, CH2-ind), 2.31 (td(ddd), JZ14, 3 Hz, 1H,
H-4ax), 1.99 (br d, JZ14 Hz, 1H, H-3eq), 1.83 (tdd(dddd),
JZ14, 6, 3 Hz, 1H, H-3ax), 1.41 (br d, JZ14 Hz, 1H,
H-4eq), 1.31 (s, 3H, CH3); 13C NMR (CDCl3, 100 MHz,
ppm): 171.0 (CO), 170.7 (CO), 170.6 (CO), 159.1 (C–OCH3
(PMB)), 136.3 (C-ipso Ph), 134.5 (C-7a00i), 129.5 (C-20C
C-60), 129.4, 128.9, 127.300(CH-Ph), 128.5 (C-ipso PMB),
127.6 (C-3a00i), 122.1 (C-6 i), 121.7 (C-200i), 119.1 (C-500i),
118.9 (C-400i), 114.0 (C-30CC-50), 113.1 (C-300i), 110.9
(C-700i), 60.3 (C-2), 55.3 (C-5), 55.0 (O–CH3), 49.0 (N–CH2
of PMB), 43.2 (CH2–NH), 30.2 (CH2-Ph), 25.2 (C-4),
24.6 (CH3); 23.0 (C-3), 20.9 (CH2-ind); EIMS m/z (%): 553
(MC%, 3), 410 (MC%KCH2CH2indol, 31), 365 (MC%K
NHCH2CH2indol–OCH3, 5), 121 (CH3OPhCHC2 , 100);
HRMS: Calcd for C33H36N4O4: 552.2736; found: 552.2746.
H-20CH-60), 7.010(s, 1H,0 NH), 6.92 (s, 1H, H-200i), 6.80 (d,
JZ8 Hz, 1H, H-3 CH-5 ), 5.47 (d, JZ14 Hz, 1H, N–CH2
of PMB), 3.98 (br s, 1H, H-2), 3.82 (d, JZ14 Hz, 1H,
N–CH2 of PMB), 3.74 (s, 3H, O–CH3), 3.57 (s, 2H, CH2–
CO), 2.41 (br t(dd), JZ17 Hz, 1H, CH2CH2), 2.18 (br d, JZ
17 Hz, 1H, CH2CH2), 1.87 (br t(dd), JZ16 Hz, 1H,
CH2CH2), 1.53 (br d, JZ17 Hz, 1H, CH2CH2), 1.24 (s,
3H, CH3); 13C NMR (CDCl3, 75 MHz, ppm): 173.1 (CO),
172.8 (CO), 159.7 (C–OCH3 (PMB)), 137.400 (C-7a00i),
130.1 (C-3a00i), 128.2 (C-20CC-600)0 , 124.6 (C-2 i), 122.09,
120.4 (C-600iCC-500i), 119.0 (C-4 i), 114.6 (C-30CC-5 ),
111.9 (C-700i), 108.3 (C-300i), 59.8 (C-2), 56.0 (C-5),
55.6 (O–CH3), 50.0 (N–CH2 of PMB), 33.3 (CH2–CO),
31.3 (C-4), 25.5 (CH3), 23.4 (C-3); CIMS m/z (%): 450
(MHC), 432 (MHC –H2O, 9), 404 (MHC –CO2H, 4), 275
(MHC –COCH2indol–H2O, 100).
4.4. Amide formation: addition of the fourth amino acid
mimicking residue
General procedure: to a solution of acid compound 7
(0.35 mmol) in 10 mL of dry DMF at room temperature,
TBTU (0.14 mmol) was added followed by the correspond-
ing amine (1.05 mmol). The reaction mixture was stirred for
16 h at room temperature. After extraction with ethyl
acetate (3!25 mL), the combined organic layers were dried
over magnesium sulphate and evaporated under reduced
pressure. Chromatographic separation of the residue on a
silica gel column eluting with a dichloromethane/ethyl
acetate mixture gave the target compounds I–V.
(2S*,5S*) 5-Benzoylamino-1-(4-methoxybenzyl)-5-methyl-
6-oxo-piperidine-2-carboxylic acid {2-(1H-indol-3-yl)-
ethyl}-amide III. Yield: 58%; mp 215 8C (EtOH); IR
(KBr) cmK1: 3286 (w, NH), 1638 (s, CO); 1H NMR
(CDCl3, 400 MHz, ppm): 8.45 (t, JZ5 Hz, 1H, NH–CH2),
8.20 (s, 1H, NH-ind), 7.76 (d, JZ7 Hz, 2H, ortho Ph), 7.63
(d, JZ7 Hz, 1H, H-400i), 7.50 (t, JZ7 Hz, 1H, para Ph),
(2S*,5S*) 1-(4-Methoxybenzyl)-5-methyl-6-oxo-5-[(3-
phenylpropionyl) amino]-piperidine-2-carboxylic acid
[-2(1H-indol-3-yl) ethyl]-amide I. Yield: 96%; mp 120 8C
1
(EtOH); IR (KBr) cmK1: 3276 (w, NH), 1639 (s, CO); H

9562
L. Kamoune et al. / Tetrahedron 61 (2005) 9555–9562
7.43 (t, JZ7 Hz, 2H, meta Ph), 7.25 (d, JZ7 Hz, 1H,
H-7000i), 7.14 (t, J0Z0 7 Hz,001H, H-600i), 7.09–7.04 (m, 4H,
H-20CH-60CH-5 iCH-2 i), 6.79 (d, JZ8 Hz, 2H, H-30C
H-5 ), 6.49 (s, 1H, NH-C5), 5.25 (d, JZ14 Hz, 1H, N–CH2
of PMB), 3.85 (br d, JZ6 Hz, 1H, H-2), 3.73 (s, 3H,
O–CH3), 3.70 (q, JZ7 Hz, 2H, CH2–NH), 3.31 (d, JZ
14 Hz, 1H, N–CH2 of PMB), 3.04 (td, JZ7 Hz, 2H,
CH2-ind), 2.32 (td(ddd), JZ14 Hz, 3 Hz, 1H, H-4ax), 1.97
(br d, JZ14 Hz, 1H, H-3eq), 1.88 (tdd(dddd), JZ14 Hz,
3 Hz, 1H, H-3ax), 1.41 (br d, JZ14 Hz, 1H, H-4eq), 1.31 (s,
3H, CH3);13C NMR (CDCl3, 100 MHz, ppm): 171.1 (CO),
170.7 (CO), 170.6 (CO), 159.1 (C–OCH3 (PMB)), 136.3,
133.5 (C-ipso Ph CC-7a00i), 131.9, 128.5, 127.1 (CH-Ph),
129.6 (C-2000CC-60), 128.7 (C-3a00i), 127.7 (C-ipso PMB),
121.8 (C-2 i), 1210.1 (C-600i), 119.8 (C-400i), 118.6 (C-500i),
114.2 (C-30CC-5 ), 113.4 (C-300i), 111.0 (C-700i), 61.0
(C-2), 55.7 (C-5), 55.2 (O–CH3), 49.0 (N–CH2 of PMB),
40.1 (CH2–NH), 30.3 (C-4), 25.6 (CH3), 24.7 (CH2-ind),
23.1 (C-3); EIMS m/z (%): 538 (MC%, 4), 396 (MC%K
CH2CH2indole, 31), 351 (MC%KNHCOCH2CH2indole, 5);
HRMS: Calcd for C32H34N4O4: 538.2580; found: 538.2585.
CO), 3.67–3.54 (m, 2H, CH2–NH), 3.36 (d, JZ13 Hz, 1H,
N–CH2 of PMB), 2.94 (dt, JZ7 Hz, 2H, CH2-Ph), 2.35
(td(ddd), JZ14, 3 Hz, 1H, H-4ax), 1.95 (br d, JZ14 Hz,
1H, H-3eq), 1.83 (tdd(dddd), JZ14, 6, 3 Hz, 1H, H-3ax),
1.42 (br d, JZ14 Hz, 1H, H-4eq), 1.22 (s, 3H, CH3); 13C
NMR (CDCl3, 100 MHz, ppm): 171.2 (CO), 171.1 (CO),
170.7 (CO), 159.1 (C–OCH3 (PMB)), 139.2 (C-ipso Ph),
136.4 (C-7a00i), 129.7 (C-20CC-60), 128.9, 128.3, 126.1
(CH-Ph), 128.6 (C-3a00i), 126.9 (C-ipso PMB), 123.8, 122.6,
120.1, 118.7 (CH-ind), 114.0 (C-30CC-50), 111.0 (C-700i),
108.6 (C-300i), 61.0 (C-2), 55.3 (C-5), 55.2 (O–CH3), 49.0
(N–CH2 of PMB), 40.7 (CH2-Ph), 35.0 (CH2–NH), 33.3
(CH2–CO), 30.3 (C-4), 25.2 (CH3), 23.0 (C-3); EIMS m/z
(%): 552 (MC%, 20), 396 (MC%KCOCH2indole, 28), 121
(CH3OPhCHC2 , 100); HRMS: Calcd for C33H36N4O4:
552.2736; found: 552.2742.
Acknowledgements
We wish to thank the Johnson and Johnson Pharmaceutical
research Foundation, the Institute for the promotion of
Innovation through Science and Technology in Flanders
(IWT-Vlaanderen) and the FWO (Fund for Scientific
Research-Flanders (Belgium)) for financial support. We
are grateful to R. De Boer for HRMS measurements and to
S. Toppet for 2D NMR. WMDB (Postdoctoral Fellow of the
FWO-Vlaanderen) thanks the FWO and BMPV thanks the
IWT for the fellowship received.
(2S*,5S*) 5-(3-1H-Indol-3-yl-propionylamino)-1-(4-meth-
oxybenzyl)-5-methyl-6-oxo-piperidine-2-carboxylic acid
phenethyl-amide IV. Yield: 50%; mp 130 8C (EtOH); IR
(KBr) cmK: 3280 (w, NH), 1640 (s, CO); 1H NMR (CDCl3,
400 MHz, ppm, 328 K): 8.48 (t, JZ5 Hz, 1H, NH–C0O0 –C2),
8.31 (br s, 1H, NH-ind), 7.56 (d, JZ7 Hz, 1H, H-4i ), 7.36
(d, JZ7 Hz, 1H, H-700i), 7.26–7.23 (m, 5H, Ph-H), 7.16 (t,
JZ7 Hz, 1H, H-6000i), 7.008 (t, JZ7 Hz, 1H, H-500i), 7.050(0 d,
JZ8 Hz, 2H, H-2 CH-6 ), 06.95 (b0r d, JZ2 Hz, 1H, H-2 i),
6.78 (d, JZ8 Hz, 2H, H-3 CH-5 ), 6.08 (s, 1H, NH-C5),
5.28 (d, JZ14 Hz, 1H, N–CH2 of PMB), 3.84 (br d, JZ
6 Hz, 1H, H-2), 3.74 (s, 3H, O–CH3), 3.60 (q, JZ13 Hz, 2H,
CH2–NH), 3.31 (d, JZ14 Hz, 1H, N–CH2 of PMB), 3.05 (t,
JZ7 Hz, 2H, CH2-ind), 2.95 (q, JZ13 Hz, CH2-Ph), 2.48
(dt, JZ7, 2 Hz, 2H, CH2–CO), 2.25 (td(ddd), JZ14, 3 Hz,
1H, H-4ax), 1.92 (br d, JZ14 Hz, 1H, H-3eq), 1.79
(tdd(dddd), JZ14, 6, 3 Hz, 1H, H-3ax), 1.32 (br d, JZ
14 Hz, 1H, H-4eq), 1.28 (s, 3H, CH3); 13C NMR (CDCl3,
100 MHz, ppm, 328 K): 172.2 (CO), 171.1 (CO), 170.9
(CO), 159.1 (C–OCH3 (PMB)), 139.1 (C-7a00i), 135.3
(C-ipso Ph), 129.4 (C-20CC-60), 128.5 (C-3a00i), 128.8,
References and notes
1. Nachman, R. J.; Moyna, G.; Williams, H. J.; Tobe, S. S.; Scott,
A. I. Bioorg. Med. Chem. 1998, 6, 1379–1388.
2. Nachman, R. J.; Zabrocki, J.; Olczak, J.; Williams, H. J.;
Moyna, G.; Scott, A. I.; Coast, G. M. Peptides 2002, 23,
709–716.
3. Nachman, R. J.; Kaczmarek, K.; Williams, H. J.; Coast, G. M.;
Zabrocki, J. Biopolymers 2004, 75, 412–419.
4. De Borggraeve, W. M.; Verbist, B. M. P.; Rombouts, F. J. R.;
Pawar, V. G.; Smets, W. J.; Kamoune, L.; Alen, J.; Van der
Eycken, E. V.; Compernolle, F.; Hoornaert, G. J. Tetrahedron
2004, 60, 11597–11612.
¨
5. Vekemans, J.; Pollers-Wieers, C.; Hoornaert, G. J. Heterocycl.
Chem. 1983, 20, 919–923.
00
128.3, 126.1 (CH-Ph), 127.0 (C-ipso PMB), 121.9 (C-6 i),
00
121.8 (C-2000i), 119.1 (C-500i), 118.4 (C-400i), 114.4 (C-3 i),
114.0 (C-3 CC-50), 111.3 (C-700i), 61.0 (C-2), 55.1 (C-5),
55.0 (O–CH3), 49.0 (N–CH2 of PMB), 40.7 (CH2–NH), 36.5
(CH2–CO), 34.9 (CH2-Ph), 30.3 (C-4), 25.1 (CH3), 22.9
(C-3), 20.6 (CH2-ind); EIMS m/z (%): 566 (MC%, 20), 418
(MC%KNHCOCH2CH2Ph, 19); HRMS: Calcd for
C34H38N4O4: 566.2893; found: 566.2921.
6. Rogiers, J.; De Borggraeve, W. M.; Toppet, S. M.;
Compernolle, F.; Hoornaert, G. J. Tetrahedron 2003, 59,
5047–5054.
7. Loosen, P. L.; Tutonda, M. G.; Khorasani, M. F.; Compernolle,
F.; Hoornaert, G. J. Tetrahedron 1991, 47, 9259–9268.
8. De Borggraeve, W. M.; Rombouts, F. J.; Hoornaert, G. J.
Tetrahedron Lett. 2001, 42, 5693–5695. Verbist, B. M. P.;
Smets, W. J.; De Borggraeve, W. M.; Compernolle, F.;
Hoornaert, G. J. Tetrahedron 2004, 45, 4371–4374.
9. Actual concentrations: 8571, 9.05 mM; 8587, 9.29 mM; 8588,
8.83 mM; 8514, 9.71 mM. All compounds were tested as
racemates.
(2S*,5S*) 5-(2-1H-Indol-3-yl-acetylamino)-1-(4-methoxy-
benzyl)-5-methyl-6-oxo-piperidine-2-carboxylic acid phen-
ethyl-amide V. Yield: 43%; mp 201 8C (EtOH); IR (KBr)
cmK1: 3280 (w, NH), 1640 (s, CO); 1H NMR (CDCl3,
400 MHz, ppm): 8.53 (t, JZ5 Hz, 1H, NH–CH2), 8.35 (s,
1H, NH-ind), 7.65 (d, JZ7 Hz, 1H, H-400i), 7.39 (d, 00JZ
7 Hz, 1H, H-700i), 7.29–7.15 (m, 8H, Ph-HCH-6 iC
H-500iCH-200i), 7.090 (d, JZ8 Hz, 2H, H-20CH-60), 6.80
(d, JZ8 Hz, 2H, H-3 CH-50), 6.04 (s, 1H, NH-C5), 5.33 (d,
JZ13 Hz, 1H, N–CH2 of PMB), 3.85 (br d, JZ6 Hz, 1H,
H-2), 3.75 (s, 3H, O–CH3), 3.73 (2!d, JZ13 Hz, 2H, CH2–
10. We assume the diuretic effect is due to stimulation of the same
receptors the natural peptides bind to.
11. Broad signals probably due to aggregation effects.