Application of tandem Ugi multi-component reaction/ring closing metathesis to the synthesis of a conformationally restricted cyclic pentapeptide

Article abstract of DOI:10.1039/b414374d

A conformationally restricted cyclic pentapeptide, containing an unsaturated 9-membered lactam as a semi-rigid scaffold, was prepared in a very convergent manner, through tandem Ugi reaction/ring closing metathesis.

Full text of DOI:10.1039/b414374d

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A R T I C L E
Application of tandem Ugi multi-component reaction/ring closing
metathesis to the synthesis of a conformationally restricted cyclic
pentapeptide
Silvia Anthoine Dietrich,a Luca Banfi,*a Andrea Basso,a Gianluca Damonte,b Giuseppe Guantia
and Renata Rivaa
a Department of Chemistry and Industrial Chemistry, via Dodecaneso 31, 16146, Genova, Italy.
E-mail: banfi@chimica.unige.it; Fax: +390103536118; Tel: +390103536119
b Department of Experimental Medicine, Biochemistry Section, c/o Center of Excellence for
Biomedical Research, University of Genova, v. le Benedetto XV, 16132, Genova, Italy
Received 17th September 2004, Accepted 19th October 2004
First published as an Advance Article on the web 23rd November 2004
A conformationally restricted cyclic pentapeptide, containing an unsaturated 9-membered lactam as a semi-rigid
scaffold, was prepared in a very convergent manner, through tandem Ugi reaction/ring closing metathesis.
component reactions followed by secondary transformations22–26
Introduction
in diversity-oriented synthesis,27 we decided to design and
synthesize a new family of “external” reverse-turn scaffolds
characterized by an unsaturated 9-membered lactam (general
formula 1, Scheme 1). According to our plan, various diverse
members of this family could be prepared convergently in two
steps by coupling a multi component reaction (the Ugi 4CR)
with a subsequent ring closing metathesis (RCM). In this way,
not only could the scaffolds be synthesized in just 2 steps, but
several different substituents could be placed at will, as R1, R2,
and R3. Although coupling of Ugi MCR with RCM has also
been recently described by other groups, 17,28–31 the synthesis of
9-membered rings by this strategy was unprecedented. Also a
related tandem Passerini/RCM was reported.30 In a preliminary
communication32 we have described the successful synthesis
of a series of compounds of general formula 1 through this
approach. Now we report full details on the synthesis of a
particular member of this family of scaffolds (namely 2) by two
alternative synthetic routes, and its transformation into the first
cyclic pentapeptides 3. The conformations of both 2 and 3 will
be also thoroughly discussed, on the basis of NMR data.
The interaction of peptides with receptors1 or with other
proteins2 has a strong influence on several physiological pro-
cesses. Therefore, small molecules have been used as antagonists
of protein function in the pharmaceutical industry since its very
beginning. The effectiveness of protein–receptor or protein–
protein interaction is strongly dependent on the secondary
and tertiary structure of the peptides. Thus, small oligopep-
tides characterized by the same recognition sequence, but
lacking the conformational restrictions typical of the natural
macromolecules, may be completely inactive. Among the most
important secondary motifs, the reverse-turns (e.g. the b-turns,
the hairpins and so on) play a particularly important role. For
this reason various research groups have tried to design and
synthesize reverse-turn mimetics.
This has been successfully accomplished by using cyclic
penta- or hexapeptides,3,4 obtained by assembling proteinogenic
amino acids. However, turn mimics composed of only amino
acids are not ideal, because compounds of this kind may have
unfavourable bioavailability properties.2
Another strategy involves the attachment of small peptide
chains to an unnatural rigid or semi-rigid template (scaffold).
In some cases this template represents the reverse-turn itself
(and has been defined as “internal reverse-turn mimetic”).5 In
other cases, the rigid scaffold serves to force an attached cyclic
peptide chain to adopt the desired reverse-turn conformation
(and has been defined as an “external reverse-turn mimetic”).5
This latter strategy seems particularly promising. The fact that
the scaffold is “unnatural” should grant it a higher metabolic
stability, compared to the cyclic penta- or hexapeptides. On the
other hand, the fact that the recognition sequence is not placed
on the template itself (as for “internal reverse-turn scaffolds”)2,6,7
allows for a higher degree of freedom in testing several different
oligopeptides or peptidomimetics as recognition motifs.
In the past, various conformationally restricted reverse-turn
scaffolds have been designed and synthesized. Most of them
were fused bicyclic8–10 lactams, but also bridged bicyclic,11,12
spirocyclic,13 or monocyclic (with ring sizes from 3 to 7)14–18
lactams have been explored. However, only a few reports dealt
with mesocyclic compounds.19–21
Scheme 1
Results and discussion
A drawback connected to most of the reverse turn scaffolds
reported to date is that the synthetic approaches to them are
poorly flexible. Even small variations of the substituents deco-
rating the basic scaffold, which could be useful in order to finely
tune the biological activity, require de novo syntheses. In the
course of a project on the exploitation of isocyanide-based multi-
Synthesis
For the preparation of the required scaffold 2, we first utilized
the 2-step strategy already reported in our preliminary com-
munication. It involved Ugi condensation of Boc-glycine 11
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 9 7 – 1 0 6
9 7
©

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with isocyanoacetate 7 and the preformed imine derived from 5-
hexen-2-one and benzylamine, followed by RCM of the resulting
diene 12. The required isocyanide 7 was prepared, as a racemic
mixture, in 4 high-yielding steps, starting from commercially
available diethyl formamidomalonate, as described in Scheme 2.
The Ugi condensation (Scheme 3) proceeded in high yield, but
with no stereoselectivity at all, giving an inseparable mixture of
the two diastereoisomers in a 1 : 1 ratio.
explained by the insufficient equilibrium concentration of the syn
rotamer at the amide bond in the acyclic diene (only this rotamer
is able to cyclize). It was suggested that, for the same reason,
also secondary 9-membered lactams could not be obtained
by RCM.28 On the other hand, both 8-membered28,33,34 and
9-membered lactams28 can be obtained starting from tertiary
amides; in this case the relative abundance of the acyclic rotamers
is expected to be closer to 1 : 1. However, while both secondary
and tertiary unsaturated 8-membered lactams exist as the syn
conformers, the previous report on the synthesis of tertiary
unsaturated 9-membered lactams28 does not give information
on the actual conformational equilibrium between syn and anti
rotamers in the cyclic compounds.
On the basis of preliminary computer aided analysis,32 we
expected the anti rotamer at the amide to be more stable than
the syn one in secondary 9-membered unsaturated lactams
1.35 Therefore, the anti rotamer of the acyclic diene should,
in this case, undergo cyclization even faster than the syn one.
This prediction was later confirmed by NMR conformational
analysis on the metathesis products (vide infra).
The yield of RCM was relatively good when carried out
with Grubbs’ 1st generation catalyst in refluxing CH2Cl2, even
when at a concentration not dramatically low (4 mM). It is
worth noting that formation of a 10-membered lactam by RCM
was reported to afford satisfactory yields only at much lower
concentrations (0.8 mM).19 It was difficult to drive the reaction
to completion, mainly because the catalyst tended to become
deactivated after 24–48 hours (the colour of the solution turned
from pink-orange to black). This may be caused, as previously
suggested,29,36 by interaction of the catalyst with one of the
polar amidic groups. The overall 53% yield was raised to 65% if
based upon recovered starting material. We tried to optimize the
reaction by changing the solvent in order to increase the reaction
temperature. However, in refluxing benzene, dichloroethane, or
THF, the catalyst was rapidly deactivated. At lower temperatures
these solvents were less satisfactory than CH2Cl2 anyway. We
also tested Grubbs’ 2nd generation catalyst. The reaction was
indeed faster, and it was possible now to drive it to near-
completion. However, the relative percentage of intermolecular
products was increased. An analogous outcome was recently
reported by Creighton and Reitz.34
Scheme 2 Reagents and conditions: a) NaH, allyl-Br, r.t. b) 1. NaOH,
EtOH; 2. Dioxane, reflux. c) POCl3, Et3N, −30 ◦C. d) BnNH2, benzene,
Dean–Stark.
The reaction was completely stereoselective with regard to the
double bond, giving only Z compounds. The two diastereoiso-
mers 2a,b were easily separated from each other, but were
contaminated by several acyclic dimeric by-products derived
from intermolecular processes, easily identified as such in the
13C NMR-DEPT spectra by the presence of terminal double
bonds. It is worth noting that, even if only the less encumbered
double bond (the one derived from the ketone), is involved in
dimerization, and only E double bonds are formed, 6 different
diastereoisomeric acyclic dimers may be obtained! Thus, in order
to obtain 2a and 2b in pure form, 2–3 chromatographies with
different eluents were necessary.
For this reason we decided to explore an alternative strategy,
using this time, as the isocyanide component, the achiral
malonate 8. In this approach the second stereogenic centre
was planned to be generated only after the RCM reaction,
thus reducing the number of diastereoisomeric products and
by-products, and therefore simplifying the purification process.
The Ugi condensation with 8 was slower, as expected, and
required a higher temperature. However, the yield was still high
enough. On the other hand, the yield and conversion of RCM
were remarkably better in this case. Interestingly, the catalyst
seemed to be much more stable and the solution did not change
colour even after 3–4 days. The percentage of intermolecular
products was also lower, and this allowed us, for preparative
purposes, to increase the concentration to 6.5 mM. Purification
of the product was much easier as well, since we had now only
one (racemic) product, and only 2 by-products (reasonably well
separated from 14) were detected by TLC. In this case, if only the
Scheme 3 Reagents and conditions: a) EtOH, r.t. b) 4 mM in CH2Cl2,
PhCH Ru(PCy3)2Cl2, reflux. c) EtOH, 65 ◦C. d) 6.5 mM in CH2Cl2,
=
=
PhCH Ru(PCy3)2Cl2, reflux. e) 1. NaOH, EtOH; 2. Dioxane, reflux.
Yields in parentheses are based upon recovered starting material.
More critical was the ensuing ring closing metathesis. There
were no examples in the literature of RCM that gives 9-
membered secondary lactams. It is well known that the anal-
ogous cyclization of secondary amides to afford 8-membered
lactams by RCM is unfeasible.28,33 This behaviour has been
9 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 9 7 – 1 0 6

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less encumbered double bond is involved in dimerization, and E
double bonds are formed, only 2 diastereoisomeric dimers are
expected. Compound 14 was converted into a 6 : 4 mixture of
2a,b by saponification followed by decarboxylation.
at the level of 2b. Because of the difference in cyclization yield,
about 1% of 2a contaminating 2b may justify the presence of 8%
3a in crude 3b.
The advantages of this alternative route are: a) The purifi-
cation of RCM product 14 is much easier than that of RCM
products 2a,b. b) A higher concentration may be used in the
RCM step. c) The decarboxylation is moderately stereoselective
favouring the cis isomer 2a, while from RCM of 12 a slightly
higher percentage of the less wanted trans isomer 2b is obtained.
e) the overall yield of the pure cis compound 2a from 5 is 24%
instead of 20%; as discussed below, the cis scaffold is the most
useful one.
With a good methodology for the rapid obtainment of both
2a and 2b in hand, we went on to transform these scaffolds into
cyclic peptides. Since 2a and 2b were obtained in racemic forms,
we chose, as the first example, to attach an achiral dipetide
formed by two additional glycine units. Although the cyclic
peptide 3 is not very attractive from the point of view of its
biological properties, it has allowed us to check the ease of
macrocycle formation for the two different diastereoisomers and
to do a first preliminary conformational analysis in the absence
of additional biases derived from side-chains of more complex
amino acids.
Both 2a and 2b were elaborated to the acyclic triglycine deriva-
tives 15a,b. Removal of the terminal protecting groups followed
by cyclization in DMF–CH2Cl2 at 6 mM with HATU/collidine
afforded the two diastereoisomeric cyclopeptides 3a and 3b
(Scheme 4). In the case of the cis compound, the yield of 3a
was quite good (55%) and the compound could be obtained
in high purity by silica gel chromatography, as evidenced by
NMR and HPLC/MS analysis. On the other hand, formation
of the trans adduct 3b was much more troublesome. A crude
product giving nearly a single spot in tlc was isolated in only
13% yield. Moreover, HPLC-MS analysis showed that, although
the expected monomer 3b was the main component, the 2
diastereoisomers of the cyclodimer were also present (38%),
as well as a small amount of 3a (8%). The actual yield of 3b
was therefore only 7–8%. This result is not unexpected, since
conformational analysis of 2b (vide infra) has indicated that
the virtual dihedral angle between the nitrogen attached at C-3
and the carboxyl attached at C-9 as well as the distance
between these two atoms is not favourable for cyclopeptide
cyclization. Therefore, formation of 3b is probably disfavoured
both enthalpically and entropically. The presence of 3a is
probably due to traces of cis isomer not completely separated
Conformational analysis
The double bond configuration in metathesis adducts 2 and
14 as well as in their derivatives was demonstrated to be Z by
=
the CH CH vicinal coupling constants (which were around
9–10 Hz). In compounds 2a and 2b only one set of signals
was detected by NMR at r.t. This means that only one of the
two possible rotamers around the ring amide bond is present.
This rotamer is in both cases the anti one, as demonstrated by
the presence of a strong NOE between the NH and the ortho
hydrogens of the benzyl group in 2a and of a very strong NOE
between the NH and the CH3 bonded at C-3 in 2b. These NOEs
are obviously possible only for the anti rotamer and also allow
us to determine without doubt the relative configuration of the
two diastereoisomers.
From the coupling constants and NOEDIFF experiments it
is also possible to get a fairly accurate idea of the preferred
conformation of the 9-membered ring. In order to get some hints
on the possible conformations of these tetrahydroazoninones we
carried out an MM2 minimization on the parent compound with
the software Chem3D (v. 4.5) from CSC, Cambridge (USA),
looking for all local minima. These compounds were found to
be not completely rigid. Four conformations with a comparable
energy were found and three of them are depicted in Fig. 1. In the
absence of substituents on the ring, conformations 16a and 16b
are enantiomeric and therefore isoenergetic. The same applies
for 17a,b (only one of the two conformations is shown). The
difference between 16 and 17 is the position of the double bond.
In 17 it is on the same side of NH; in 16 it is on the opposite
side.
In the following discussion, for the sake of clarity, we will
consider the (9S) enantiomers of 2a and 2b, as depicted in
Scheme 3. An examination of the NMR data indicates that,
in both cases, a conformation analogous to 16a, placing the
CO2Et in pseudo-equatorial (b) position, is actually preferred
(see also Scheme 5). For example, in 2a, there is a high J1–9 of
9.3 Hz, which excludes conformation 16b, with the CO2Et in
pseudo-axial position. The J8–9 (3.9 and 7.2 Hz) and J7–8 (6.7
and 9.0 Hz) are perfectly compatible with conformations 16,
but not with 17. In 17 the dihedral angles related to J8–9 should
be 41◦ and 76◦, whereas those related to J7–8 should be 64◦ and
31◦ and so they are clearly not in agreement with the detected
values. Moreover, NOEs between the ring NH and one of the
H-8 and one of the H-5, and an NOE between H-9 and H-
7 were detected. They are possible in 16, but not in 17. Also,
other J values and NOEs are in agreement with the proposed
conformation (see the Experimental section).
For the trans isomer 2b, similar arguments demonstrate that
also in this case the preferred conformation is 16a, with the
CO2Et group in the pseudo-equatorial position.
The equilibrium between 16a and 16b is expected to be slow
enough to allow detection of both conformations at r.t., as
demonstrated in the case of malonate 14 (vide infra). The fact
1
that only one set of signals is evident in the H and 13C NMR
means that the preference for the conformations having the
CO2Et in the pseudo-equatorial position should be quite high
in both diastereoisomers.
It would be interesting to know which of the two di-
astereoisomers 2a and 2b is more stable. At first sight, the trans
compound 2b is expected to be more stable, since the bulkier
nitrogen substituent is in the pseudo-equatorial position. On the
other hand, the fact that the nitrogen is planar (while CH3 is
tetrahedral) and that a hydrogen bond may be formed between
the carbonyl bonded to it and the ring NH could favour a
pseudo-axial position of the nitrogen. The 1 : 1 ratio of the two
conformers in malonate 14 (vide infra) and the only moderate
Scheme 4 Reagents and conditions: a) 1. NaOH, EtOH, r.t.; 2.
H-GlyGlyOMe, BOP, Et3N, CH2Cl2. b) 1. LiOH, THF–H2O; 2. TFA;
3. HATU, sym-collidine, DMF–CH2Cl2 (6 mM).
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 9 7 – 1 0 6
9 9

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out minimization, noticing that addition of the substituents
does not change considerably the various bond angles and bond
lengths of the ring (Scheme 6). We then compared the minimized
structure with various types of b-turns. b-Turns are usually
defined by two u and two w angles. However, our templates
have been designed in order to function as “external” reverse-
turn scaffolds. So the coincidence of all of these 4 angles is not
mandatory. On the contrary, it is more important to compare the
virtual dihedral angles between NHAc, C-3, C-9 and CO2Me,
as well as the distances between NHAc and CO2Me (Scheme 6).
These values turned out to be quite similar to those determined
for a type IIꢀ b-turn (obviously if we consider the enantiomer of
18 they will be similar to a type II b-turn). Kessler has shown that
a IIꢀ b-turn may be ideal as an “external” template in preparing
conformationally restricted RGD peptides to be employed as
integrin ligands. In one of the most effective cyclic pentapeptides,
this IIꢀ b-turn was assured by a D-Phe-L-Val unit bonded to the
RGD recognition sequence.37
Scheme 6 Conformational comparison of a simplified analogue of
scaffold 2a and type IIꢀ b-turn. The conformation of 18 was minimized
with Chem3D and corresponds to conformation 16a (Fig. 1). Virtual
dihedral angles and distances a–d for the type IIꢀ b-turn were determined
by applying standard bond length and angles and the dihedral angles
typical of this b-turn.
Using the same approach we calculated, for the trans (3R,9S)
isomer of 18, a virtual dihedral angle and a distance of about
◦
˚
+115 and 5.98 A. While the angle is not far from the one typical
ꢀ
˚
of a type III b-turn, the distance is 1.4–1.5 A higher. From these
data it is clear that cis scaffold 2a is the most promising among
the two isomers as an external reverse-turn mimetic. On the
other hand, the low yield in the cyclization of trans 15b may be
explained by the excessive distance between the nitrogen at C-3
and the carbonyl at C-9 (which are the two attachment points
for the tripeptide chain).
Fig. 1 Conformations of the parent tetrahydroazoninones as mini-
While 2a and 2b showed just one set of signals in the
1H and 13C NMR spectra, the malonate 14 gave two sets of
signals, in a 1 : 1 ratio. These are most probably due to a
slow conformational equilibrium between 16a and 16b. An
alternative hypothesis would be a conformational equilibrium
between the two rotamers at the tertiary amide, but in our
opinion this is unlikely because: a) we do not see the reason
why this equilibrium should be present in 14 and not in 2a,b; b)
the equilibrium at the amide bond is not expected to influence in
such a strong way the d values of the ring protons and carbons;
and finally, c) the NOEDIFF experiments showed, for example,
that in one conformer the ring NH is close to the CH3 at C-3,
while in the other one it is close to the aromatic protons. These
NOEs are quite similar to those detected for 2a and 2b. All
other NMR data (NOEs and J values) are in agreement with
an equilibrium between the two conformations 16a and 16b.
The fact that the ratio is 1 : 1 shows that the two groups at C-3
(CH3 and the nitrogen substituent) occupy the pseudo-equatorial
and the pseudo-axial positions equally, suggesting also a similar
energy for 2a and 2b.
mized with Chem3D (MM2).
Scheme 5 Simplified alternative conformations of 2a and 2b.
stereoselectivity in the decarboxylation reaction suggest that the
two stereoisomers should be quite close in energy.
Having established by NMR spectroscopy the approximate
preferred conformation of the ring in 2a and 2b it was possible
to check if they are well suited or not to act as external reverse-
turn mimetics. Using Chem3D (MM2), we applied to 18 (a
simplified analogue of 2a) the ring conformation 16a and carried
Finally, the cis cyclic peptide 3a was carefully studied by NMR
spectroscopy with the aid of NOEDIFF, COSY, and double
resonance experiments, as well as measuring the difference in
d between d6-DMSO and CDCl3. A first important fact is that
only one set of signals is present at r.t., either in d6-DMSO or
1 0 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 9 7 – 1 0 6

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in CDCl3. As already stated above, it is very unlikely that the
two ring conformations related to 16a and 16b may interconvert
fast enough at r.t. to give single signals. Also the two syn and
anti rotamers of the tertiary amide would surely give different
signals. The presence of a single set of signals means therefore
that the equilibrium strongly favours a single conformer at the
ring and at the tertiary amide bond.
Once again the J values and the NOEs related to the ring
hydrogens gave results quite similar to those collected for
the acyclic scaffolds 2a and 14. It seems therefore that the
conformation of the 9-membered lactam is not strongly modified
by the attachment to the tripeptide.
The signals of the NH groups of the three glycines were
assigned thanks to NOEDIFF experiments: there is a NOE
between the NH of glycine b and the CH2N of glycine a, and
a (small) NOE between the NH of glycine c and the CH2N of
glycine b (Scheme 7). The presence of a NOE between one of the
CH2N groups of glycine c and the aromatic protons is consistent
with an anti conformation around the tertiary amide bond.
Conclusions
The cyclic peptidomimetic 3a, including a conformationally
biased scaffold, has been straightforwardly prepared in only 5
steps thanks to a tandem Ugi reaction–ring closing metathesis.
NMR studies have allowed us to suggest a preferred confor-
mation for the scaffolds 2a,b and for the cyclic compound 3a.
The formation of the trans cyclic peptide 3b in only low yield
confirms that the corresponding scaffold is not a good external
reverse-turn mimetic. On the other hand, the cis scaffold 2a
mimics well a type IIꢀ b-turn and appears to be well suited
for the synthesis of conformationally biased cyclopeptides or
cyclopeptidomimetics. The promising properties of 2a have been
demonstrated by the good yield achieved in macrocyclization,
by the fact that the macrocyclic peptide 3a seems to be
conformationally defined, and by the probable presence of
strong intramolecular hydrogen bonds corresponding to b- and
c-turns.
Although all the compounds described in this work are
racemic, and GGG is not a biologically relevant recognition
sequence, these results open the way to the preparation of other
cyclic peptides including enantiomerically pure scaffolds 1 and
more useful tripeptide sequences (such as the RGD one)attached
to it. Studies directed toward this goal are in progress in our
laboratory.
Experimental
NMR spectra were taken at 200 or 300 MHz (1H), and 50 or
75 MHz (13C), using as internal standard: TMS for 1H NMR in
CDCl3, the central peak of DMSO (at 2.506 ppm) for 1H NMR
in d6-DMSO, the central peak of CDCl3 (at 77.02 ppm) for 13
C
Scheme 7
NMR in CDCl3, the central peak of DMSO (at 39.429 ppm)
for 13C NMR in d6-DMSO. Chemical shifts are reported in
ppm (d scale), coupling constants are reported in hertz. Peak
On passing from d6-DMSO to CDCl3 (0.01 M) the behaviour
of the four NH protons is strikingly different: while the ring NH
and the NH of glycine b displayed a lower d in CDCl3 (−1.15
and −0.23 ppm respectively), for the NH of glycines c and a we
observed a surprisingly higher d in CDCl3 than that in DMSO
(+0.91 compared to +1.00 ppm).
1
assignment in H NMR spectra was also made with the aid
of double resonance and COSY experiments. In some cases
coupling constants were not directly determined from normal
spectra, but with the aid of double resonance or D2O exchange
experiments. In these cases they are indicated with a * symbol. In
AB systems, the proton A is considered downfield and B upfield.
Peak assignment in 13C spectra was made with the aid of DEPT
experiments.
In order to gain further insight, we also measured the tem-
perature dependance of these NH chemical shifts (in DMSO).38
The thermal coefficients (negative parts per billion per K) turned
out to be 3.9 for ring NH, 4.9 for glycine b, 1.7 for glycine c,
and −1.0 for glycine a. Moreover, the ring NH undergoes a very
slow exchange with D2O (in DMSO). It takes more than 1 hour
for the signal to disappear completely at r.t., while the signals of
the other three NH disappear in less than 10 minutes.
The negative thermal coefficient for glycine a, and the higher
d in CDCl3 than in DMSO suggest its participation in a strong
intramolecular hydrogen bond. Also for the NH of glycine c,
the particularly high d in CDCl3 and the low thermal coefficient
may indicate the involvement in an intramolecular hydrogen
bond. Finally, all data (including NOEs) suggest an insignificant
participation of NH of glycine b in intramolecular hydrogen
bonds.
GC-MS were carried out on an HP-5971A instrument, using
an HP-1 column (12 m long, 0.2 mm wide), electron impact at
70 eV, and a mass temperature of about 170 ◦C. Only m/z > 33
were detected. All analyses were performed with a constant He
flow of 0.9 mL min−1, and (unless otherwise stated) with initial
◦
temperature of 100 ◦C, initial time 2 min, rate 20 C min−1,
final temperature 260 ◦C, final time 4 min, injection temperature
250 ◦C, detector temperature 280 ◦C. Rt are in min.
HPLC-MS were carried out with an Agilent 1100 LC/MSD
Trap SL instrument (electrospray ion trap analysis) with a C18
reverse phase Polarity column (Waters Corporation, MA, USA).
In all cases, before introducing the eluent in the MS, a detection
at 220 nm was performed using a diode array detector integrated
in the system. The MS electrospray ion source parameters were
set to maximize, from time to time, the interesting m/z ratios.
IR spectra were measured with a Perkin–Elmer 881 instru-
ment as CHCl3 solutions. Melting points were measured on
a Bu¨chi 535 apparatus and are uncorrected. TLC analyses
were carried out on silica gel plates and developed with I2
vapour. Rf values were measured after an elution of 7–9 cm.
Chromatographies were carried out on 220–400 mesh silica gel
using the “flash” methodology. Petroleum ether (40–60 ◦C) is ab-
breviated as PE. In extractive work-up, aqueous solutions were
always reextracted thrice with the appropriate organic solvent.
Organic extracts were washed with brine, dried over Na2SO4
and filtered before evaporation of the solvent under reduced
pressure. Dry solvents were purchased from Fluka. All reactions
As for the ring NH, both the temperature coefficient, as well
as the CDCl3 − DMSO d difference, point towards its non-
involvement in hydrogen bonds. The very slow D2O exchange is
somehow in contradiction with this assumption. However, this
may be simply due to the fact that this NH is rather buried inside
the molecule. Actually, a hydrogen bond between this NH and
the carbonyl of glycine c or b would indeed prevent the observed
NOEs with H-5 and H-8.
A tentative favoured conformation that seems in agreement
with the collected NMR data is shown in Scheme 7 and involves
a strong b-turn between glycines c (acting as amino acid i) and
a (acting as aminoacid i + 3) (in this b-turn the aminoacids i + 1
and i + 2 are those included in the scaffold) and a c-turn between
glycines c (i + 2) and a (i).
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 9 7 – 1 0 6
1 0 1

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employing dry solvents were carried out under a nitrogen (or
argon when specified) atmosphere. The purity of all compounds
was established by TLC, 1H and 13C NMR, and by GC-MS or
HPLC or HPLC-MS. Diethyl formamidomalonate, 5-hexen-2-
one, Boc-glycine, sodium hydride and Grubbs’ catalyst were all
purchased from Fluka or Aldrich and used as such.
62.73; H, 7.24; N, 9.14%; GC-MS Rt 1.55; m/z: 125 (M+
−
28, 16.8), 124 (3.9), 108 (28.4), 107 (20.2), 98 (28.4), 97 (28.0),
85 (8.3), 84 (20.0), 81 (27.2), 80 (74.8), 79 (27.7), 70 (22.6), 69
(19.0), 68 (10.1), 67 (8.5), 57 (7.0), 56 (12.9), 55 (21.1), 54 (88.5),
53 (100.0), 52 (25.2), 51 (17.1), 50 (7.3), 45 (6.3), 43 (13.6), 42
(30.5), 41 (38.7), 39 (71.5). 1H NMR (CDCl3, 200 MHz): d 5.81
=
[1 H, ddt, CH CH2, Jd 9.8, 17.2, Jt 7.2]; 5.32–5.19 [2 H, m,
=
CH2 CH]; 4.28 [2 H, q, CH2CH3, J 7.2]; 2.76–2.50 [2 H, m,
Diethyl (allyl)(formamido)malonate 5
=
CH2CH CH2]; 1.32 [3 H, t, CH3CH2, J 7.2]. IR: mmax 2151,
Sodium hydride (60% in mineral oil) (1.652 g, 41.31 mmol)
was suspended in dry dimethylformamide (DMF) (40 mL)
1754, 1433, 1371, 1331, 1270, 1242, 1190, 1020, 925 cm−1.
◦
and cooled to 0 C. A solution of diethyl formamidomalonate
Diethyl (allyl)(isocyano)malonate 8
(7.994 g, 39.34) in dry DMF (20 mL) was slowly added over
5 min. After 10 min, the cooling bath was removed and the
mixture stirred for 30 min at r.t. Allyl bromide (3.57 mL, 4.997 g,
41.31 mmol) was then added via a syringe. After stirring for 3 h,
and cooling in an ice bath, the reaction was quenched with
saturated aqueous NH4Cl (100 mL). The mixture was extracted
with Et2O (70 mL + 50 mL) and with AcOEt (2 × 50 mL). The
united organic extracts were washed with H2O and evaporated
to give an oil. It was taken up with CH2Cl2 and n-octane and
evaporated again. After removing the solvent overnight at 8 ×
10−2 mbar, a solid (9.92 g) was obtained. Trituration with n-
pentane gave pure 5 as a white solid (8.904 g, 93%). Rf 0.57
(PE–AcOEt 1 : 1); mp 64.5–65.8 ◦C. Found: C, 54.45; H, 7.0; N,
5.7%. C11H17NO5 requires: C, 54.31; H, 7.04; N, 5.76%; GC-MS
Rt 5.48; m/z 243 (M+, 1.8), 198 (28.2), 174 (25.7), 170 (47.1), 151
(3.4), 146 (5.9), (44.1), 124 (80.1), 123 (7.1), 118 (6.6), 114 (7.3),
112 (6.2), 97 (8.2), 96 (43.4), 74 (5.6), 69 (13.8), 68 (100.0), 67
(11.0), 56 (8.2), 47 (16.2), 43 (9.4), 42 (14.9), 41 (65.7), 39 (19.0).
1H NMR (CDCl3, 200 MHz): d 8.17 [1 H, s, CHO]; 6.93 [1 H, s,
Prepared from 5 (6.386 g, 26.25 mmol) following the same
procedure employed for 7. Yield after chromatography: 5.192 g
(88%) of a colourless oil that had some tendency to darken on
standing, Rf 0.56 (PE–AcOEt 8 : 2). Found: C, 58.8; H, 6.9; N,
6.0%. C11H15NO4 requires: C, 58.66; H, 6.71; N, 6.22%; GC-MS
Rt 4.04; m/z 226 (M+ + 1, 0.1), 199 (M+ − 26, 1.1), 198 (1.0),
197 (2.7), 169 (64.5), 153 (15.8), 152 (21.9), 151 (100.0), 134
(9.1), 133 (73.9), 129 (8.1), 125 (27.5), 124 (61.6), 123 (10.7), 111
(35.9), 110 (5.0), 108 (16.4), 107 (17.1), 106 (28.9), 105 (16.7), 101
(8.0), 99 (5.7), 98 (9.8), 97 (19.2), 96 (32.2), 81 (24.3), 89 (68.5),
79 (45.5), 78 (48.7), 69 (45.0), 68 (75.7), 67 (22.7), 57 (67.0), 54
(43.6), 53 (71.5), 52 (60.3), 41 (60.4), 39 (44.3). 1H NMR (CDCl3,
=
200 MHz): d 5.79 [1 H, ddt, CH CH2, Jd 9.8, 17.2, Jt 7.2]; 5.35–
=
5.20 [2 H, m, CH2 CH]; 4.32 [4 H, q, CH2CH3, J 7.1]; 2.90 [2
=
H, d, CH2CH CH2, J 7.0]; 1.32 [3 H, t, CH3CH2, J 7.1]. IR:
mmax 2142, 1752, 1368, 1298, 1192, 1147, 1095, 1036, 924 cm−1.
N-(Hex-5-en-2-ylidene)(phenyl)methanamine 10
=
NH]; 5.58 [1 H, ddt, CH CH2, Jd 9.4, 17.2, Jt 7.4]; 5.27–5.08
A solution of 5-hexenone (10.16 mL, 87.73 mmol) and benzy-
lamine (8.547 g, 79.76 mmol) in benzene was refluxed in a Dean–
Stark apparatus for 4 h. Evaporation of benzene at 16 mbar,
followed by distillation at 0.55 mbar, afforded 10 (pure by GC-
MS) as a colourless liquid (bp 85–90 ◦C), which tended to darken
if left in the light (13.20 g, 88%). NMR spectroscopy indicated
a trans : cis ratio of 3.9 : 1. GC-MS (injection temperature:
200 ◦C, initial temperature: 45 ◦C, initial time: 2 min, rate:
20 ◦C min−1): Rt 7.97; m/z 187 (M+, 5.1), 186 (15.0), 172 (4.5),
=
[2 H, m, CH2 CH]; 4.27 [4 H, q, CH2CH3, J 7.1]; 3.10 [2 H, d,
CH2CH CH2, J 7.4]; 1.27 [6 H, t, CH3, J 7.1].
=
Ethyl (allyl)(formamido)acetate 6
A solution of diethyl (allyl)(formamido)malonate 5 (8.33 g,
34.24 mmol) in 96% EtOH (300 mL), was treated, at r.t., with 6 M
aqueous NaOH (6.85 mL, 41.09 mmol) and stirred for 140 min.
Then a mixture of 32% HCl (2.69 mL, 27.39 mmol) and 96%
EtOH (10 mL) was added. After evaporation of nearly all the
EtOH, the residue was taken up with 0.5 M citric acid (20 mL),
saturated NaCl (50 mL) and Et2O (100 mL). The phases were
separated and the aqueous one re-extracted twice with Et2O and
twice with AcOEt. The organic phases gave, on evaporation, a
white solid (6.876 g). It was taken up in 1,4-dioxane and refluxed
for 6 h. Evaporation gave an oil that was chromatographed on
90 g of silica gel (PE–AcOEt 1 : 1 to 4 : 6) to give pure 6 as a
slightly yellow oil (4.629 g, 79%). Rf 0.62 (PE–AcOEt 1 : 1 + 5%
AcOH). Found: C, 56.1; H, 7.75; N, 7.9%. C8H13NO3 requires:
C, 56.13; H, 7.65; N, 8.18%; GC-MS Rt 3.54; m/z: 171 (M+,
1.5), 130 (59.1), 126 (45.2), 125 (16.3), 102 (16.4), 98 (100.0), 97
(12.9), 81 (9.6), 74 (86.4), 71 (6.4), 70 (44.4), 68 (8.7), 54 (6.0),
53 (6.7), 47 (5.6), 46 (21.4), 43 (23.3), 42 (12.0), 41 (19.5), 39
(15.7). 1H NMR (CDCl3, 200 MHz): d 8.21 [1 H, s, CHO]; 6.20
1
104 (19.6), 91 (100.0), 65 (13.6), 42 (5.9), 41 (4.9), 39 (5.5). H
NMR (200 MHz): d 7.33 (cis), 7.31 (trans) [5 H, s, arom.]; 5.98–
=
=
5.70 [1 H, m CH CH2]; 5.14–4.93 [2 H, m, CH CH2]; 4.52 (cis),
4.49 (trans) [2 H, s, CH2Ph]; 2.50–2.27 [4 H, m, CH2CH2CH ];
=
2.09 (cis), 1.91 (trans) [3 H, s, CH3].
Ugi adducts 12a,b
A solution of isocyanide 7 (1.784 g, 11.66 mmol) in absolute
EtOH (12 mL), was treated at room temperature with imine
10 (2.609 g, 13.93 mmol) and with Boc-glycine 11 (2.442 g,
13.94 mmol). After stirring under N2 for 48 h, the solvent
was evaporated, and the crude product chromatographed (PE–
AcOEt 1 : 1 to 4 : 6) to give a pure 1 : 1 mixture of 12a,b
as a foam (5.714 g, 95%). Found: C, 64.9; H, 8.1; N, 7.95%.
1
C28H41N3O6 requires: C, 65.22; H, 8.01; N, 8.15%. H NMR
=
[1 H, broad s, NH]; 5.69 [1 H, ddt, CH CH2, Jd 9.0, 17.6, Jt
7.1]; 5.25–5.08 [2 H, m, CH2 CH]; 4.76 [1 H, dt, CHNH, Jd
(CDCl3, 200 MHz): d 7.53–7.24 [5 H, m, arom.]; 6.35 [0.5 H,
d, NH, J 7.3]; 6.32 [0.5 H, d, NH, J 7.3]; 5.84–5.68 [2 H, m,
=
8.0, Jt 6.0]; 4.32–4.12 [2 H, m, CH2CH3]; 2.73–2.46 [2 H, m,
CH2CH CH2]; 1.30 [3 H, t, CH3, J 7.2].
=
CH CH2]; 5.43 [1 H, broad s, NHBoc]; 5.20–4.90 [4 H, m,
=
=
CH CH2]; 4.73–4.54 [3 H, m, CHN and CH2Ph]; 4.30–4.12 [2
H, m, CH2CH3]; 4.03–3.91 [1.5 H, CH2NHBoc]; 3.84 [0.5 H,
dd, CHHNHBoc, J 4.0, 17.2]; 2.70–2.48 [2 H, m, allylic CH2];
Ethyl (allyl)(isocyano)acetate 7
=
A solution of 6◦(2.983 g, 17.4 mmol) in dry CH2Cl2 (50 mL) was
cooled to −30 C and treated with Et3N (8.2 mL, 59.4 mmol)
and POCl3 (1.75 mL, 19.2 mmol). After 25 min the reaction
was quenched with saturated aqueous NaHCO3 (80 mL). The
mixture was allowed to warm to r.t., extracted with Et2O, and
evaporated to dryness. Chromatography (PE–Et2O 8 : 2) gave
pure 7 as a colourless oil (2.510 g, 94%). Rf 0.54 (PE–Et2O 6
: 4). Found: C, 62.45; H, 7.3; N, 9.0%. C8H11NO2 requires: C,
2.30–1.70 [4 H, m, allylic CH2 and CH2CH2CH ]; 1.45 and 1.44
[3 H, 2 s, CH3]; 1.40 [9 H, s, (CH3)3C]; 1.284 [1.5 H, t, CH3CH2,
J 7.2]; 1.280 [1.5 H, t, CH3CH2, J 7.2]. 13C NMR (50 MHz,
CDCl3)(the number of diastereoisomers is given in brackets):
=
d 173.23 (1d), 173.01 (1d), 171.84 (2d), 169.94 (2d) [C O];
155.65 (2d) [urethane C O]; 137.51 (2d) [quat. arom.]; 137.38
=
=
(2d), 132.51 (2d) [CH CH2]; 129.19 (2d), 127.61 (2d), 125.96
(2d) [arom. CH]; 119.04 (2d), 115.21 (2d) [CH CH2]; 79.54
=
1 0 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 9 7 – 1 0 6

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(2d) [C(CH3)3]; 65.66 (1d), 65.59 (1d) [quat. C–N]; 61.48 (2d)
[CH2CH3]; 51.74 (1d), 51.70 (1d) [CHN]; 47.58 (1d), 47.35 (1d)
[CH2Ph]; 43.36 (2d) [CH2N]; 36.50 (1d), 36.43 (1d), 35.32 (1d),
conf. + C(CH3)3 of 1 conf.]; 1.37 [4.5 H, s, C(CH3)3 of 1 conf.];
1.36–1.23 [6 H, m, CH3CH2]. From COSY we could establish
that the signal at d 5.14 is coupled with those at d 3.39 and 2.59.
Irradiating the NH at d 6.74 there was an NOE of 3.2% on the
methyl at d 1.67, and an NOE of 0.5% on H-8 at d 2.50. On
irradiating the NH at d 6.89 there was a NOE of 7% on the
ortho aromatic H (doublet at d 7.28 with J 7.0) and a NOE of
1% on H-7 at d 5.14 ppm. Therefore the signals at d 6.89, 5.14,
3.39, 2.59, 1.41 are all due to the conformer with the CH3 on the
opposite side of the ring NH; the signals at d 6.74, 5.55–5.42,
3.35, 2.50, 1.67 are due to the conformer with the CH3 group
on the same side as the ring NH. 13C NMR (CDCl3, 50 MHz):
171.68, 171.25, 170.26, 169.51, 168.60, 168.00, 167.25, 166.96
=
34.93 (1d) [allylic CH2]; 28.32 (2d) [C(CH3)3 + CH2CH2CH ];
21.45 (1d), 20.99 (1d) [CH3]; 14.20 (2d) [CH3CH2].
Ugi adduct 13
A solution of isocyanide 8 (3.869 g, 17.18 mmol) in absolute
EtOH (17 mL), was treated at room temperature with imine
10 (2.0875 g, 11.15 mmol) and with Boc-glycine 11 (2.000 g,
◦
11.41 mmol). After stirring under N2 at 65 C for 11 h, and at
r.t. for 48 h, the solvent was evaporated, and the crude product
chromatographed (PE–AcOEt 6 : 4 to 1 : 1) to give pure 13
as a foam (5.130 g, 77%). Found: C, 63.05; H, 7.8; N, 7.05%.
=
=
[C O]; 155.64 [urethane C O, 2 conf.]; 137.53 [quat. arom.,
=
2 conf.]; 137.31, 135.71 [CH CH]; 129.48, 129.10, 127.74,
1
C31H45N3O8 requires: C, 63.35; H, 7.72; N, 7.15%. H NMR
=
127.37, 125.71, 125.39 [arom. CH]; 124.04, 121.75 [CH CH];
79.50, 79.27 [C(CH3)3]; 65.48, 64.80, 64.19, 64.08 [quat. CN];
63.00, 62.76, 62.17 (2 conf.) [CH2CH3]; 46.99, 46.07 [CH2Ph];
43.51, 42.93 [CH2NHBoc]; 38.62, 37.78 [CH2CH ]; 31.92, 29.19
[CH2CH ]; 28.30 [C(CH3)3]; 23.84, 19.88 [CH2CH2CH ];
22.40, 20.50 [CH3]; 14.03, 13.98 [CH3CH2].
(CDCl3, 200 MHz): 7.52–7.22 [5 H, m, arom.]; 7.05 [1 H, s,
=
NH]; 5.92–5.62 [2 H, m, CH CH2]; 5.41 [1 H, broad s, NHBoc];
5.24–4.91 [4 H, m, CH CH2]; 4.60 and 4.56 [2 H, AB system,
=
=
CH2Ph, J 19.4]; 4.30–4.15 [4 H, m, CH2CH3]; 4.02 and 3.80
[2 H, AB part of ABX system, CH2NHBoc, JAB 17.2, JAX 4.4,
JBX 4.0]; 3.10 [2 H, d, C–CH2CH , J 7.4]; 2.37–1.70 [4 H, m,
=
=
=
=
CH2CH2CH ]; 1.45 [3 H, s, CH3]; 1.40 [9 H, s, C(CH3)3]; 1.25
(Z,3R*,9R*) and (Z,3R*,9S*)-3-(((N-Benzyl-t-butoxycarbonyl)-
amino)acetamido)-9-(ethoxycarbonyl)-3-methyl-4,5,8,9-tetra-
hydro-1H-azonin-2(3H)-ones 2a and 2b
and 1.23 [6 H, 2 t, CH3CH2, J 7.2]. 13C NMR (CDCl3, 50 MHz):
=
=
d 172.11, 169.78, 167.82, 167.71 [C O]; 155.61 [urethane C O];
137.53 [quat. arom.]; 137.40, 132.05 [CH CH2]; 129.19, 127.50,
125.92 [arom. CH]; 119.53, 115.28 [CH CH2]; 79.56 [C(CH3)3];
66.30, 65.39 [quat. CN]; 62.48, 62.38 [CH2CH3]; 47.64 [CH2Ph];
43.31 [CH2NHBoc]; 36.97, 34.99 [allylic CH2]; 28.34 [C(CH3)3];
=
=
Method A: RCM of 12. A solution of dienes 12a,b (1.549 g,
3.00 mmol) in dry CH2Cl2 (750 mL) was placed under argon, and
treated with benzylidene bis(tricyclohexylphosphine)ruthenium
dichloride (489 mg, 0.591 mmol). In order to ensure removal of
all oxygen present, a series of vacuum/argon cycles was carried
out. The orange solution was refluxed for 62 h, by which time
it had become black. Et3N (1 mL) was added, and most of
the solvent was evaporated, until about 100 mL was left. This
solution was introduced in a column of silica gel packed with
CH2Cl2. Elution with CH2Cl2–AcOEt–EtOH–Et3N 74 : 20 : 5 :
1 to 69 : 25 : 5 : 1 gave a crude product containing both substrate
and products. Two other chromatographies (PE–AcOEt 3 :
7 + 2% 96% EtOH to PE–AcOEt 15 : 85 + 2% 96% EtOH)
separated the substrate, the cis product, and the trans product.
The cis compound was still contaminated with an intermolecular
by-product and was further purified by chromatography using
CH2Cl2–AcOEt 6 : 4 + 2% 95% EtOH. Overall, substrate 12a,b
(270 mg, 17.4%, grey foam), cis adduct 2a (351 mg, 24.0%,
slightly grey foam), and trans adduct 2b (432 mg, 29.5%, grey
foam) were obtained. The overall yield based upon recovered
starting material was 65%.
=
28.20 [CH2CH2CH ]; 21.36, 14.02 [CH3].
(Z)rac-3-(((N-Benzyl-t-butoxycarbonyl)amino)acetamido)-9,9-
bis(ethoxycarbonyl)-3-methyl-4,5,8,9-tetrahydro-1H-azonin-
2(3H)-one 14
A solution of diene 13 (2.250 g, 3.83 mmol) in dry CH2Cl2
(600 mL) was placed under argon, and treated with benzyli-
dene bis(tricyclohexylphosphine)ruthenium dichloride (393 mg,
0.478 mmol). In order to ensure removal of all oxygen present,
a series of vacuum/argon cycles was carried out. The orange
solution was refluxed for 48 h. Et3N (1 mL) was added, and
most of the solvent was evaporated, until about 100 mL were
left. This solution was introduced in a column of silica gel packed
with CH2Cl2. Elution with CH2Cl2–AcOEt–EtOH–Et3N 74 : 20
: 5 : 1 to 69 : 25 : 5 : 1 gave a black crude product containing
both substrate and products (2.362 g). It was chromatographed
with AcOEt–PE–EtOH 34.5 : 64.5 : 0.5 to 64.5 : 34.5 : 0.5
to give pure 14 as a grey foam (1.278 g, 60%). Also 382 mg of
starting material (17%) were recovered. Yield based on recovered
starting material was 72%. Apart from 14 and the substrate 13,
the crude product showed the presence of 2 impurities (faster
eluting than 14 in PE–AcOEt). They were both intermolecular
dimers, characterized by the presence of terminal double bonds
(clearly detected with 13C/DEPT NMR spectroscopy). Rf 0.18
(AcOEt–PE 6 : 4), 0.74 (AcOEt–PE 9 : 1). Found: C, 62.2; H,
7.4; N, 7.45%. C29H41N3O8 requires: C, 62.24; H, 7.38; N, 7.51%.
1H NMR (CDCl3, 300 MHz) (the spectrum shows 2 distinct
conformers in a 1 : 1 ratio): d 7.52–7.22 [5 H, m, arom.]; 6.89,
6.74 [1 H, 2 s, NH of 2 conf.]; 5.74–5.55 [1 H, m, H-6 of 2 conf.];
5.52 [1 H, broad s, NHBoc]; 5.55–5.42 [0.5 H, m, H-7 of 1 conf.];
5.14 [0.5 H, dt, H-7 of 1 conf., Jd 7.2, Jt 10.3]; 4.62 and 4.55
[1 H, slightly broad AB system, CH2Ph of 1 conf., JAB 17.1];
4.50 [1 H, s, CH2Ph of 1 conf.]; 4.46–4.22 [4 H, m, CH2CH3];
4.15–4.01 [1 H, m, CHHNHBoc of 2 conf.]; 3.85 [0.5 H, broad
d, CHHNHBoc of 1 conf., J 15.9]; 3.50 [0.5 H, slightly broad
dd, CHHNHBoc of 1 conf., J 1.9, 17.0]; 3.39 [0.5 H, t, pseudo-
eq. H-8 of 1 conf., J 13.8]; 3.35 [0.5 H, t, pseudo-eq. H-8 of 1
conf., J 13.8]; 2.59 [0.5 H, dd, pseudo-ax. H-8 of 1 conf., J 6.9,
13.8]; 2.50 [0.5 H, dd, pseudo-ax. H-8 of 1 conf., J 6.5, 13.8];
2.45–2.34 [0.5 H, m, H-5 of 1 conf.]; 2.10–1.80 [3.5 H, m, H-5,
H-4]; 1.67 [1.5 H, s, CH3 of 1 conf.]; 1.41 [6 H, s, CH3 of 1
Method B: decarboxylation of 14. A solution of malonate 14
(505 mg, 0.902 mmol) in abs. EtOH (11.0 mL), was cooled to
0 ◦C, and treated with 2.96 N aq. NaOH (385 lL, 1.14 mmol).
The solution was stirred for 2 h. Solid (NH4)H2PO4 (100 mg)
was added. The mixture was allowed to warm to r.t., diluted
with 5% aq. (NH4)H2PO4 (20 mL), with 1 N HCl (1 mL), and
with AcOEt. The aqueous phase was saturated with NaCl. After
separation of the phases, the aqueous phase was extracted 3
more times with AcOEt. The solution was evaporated, taken up
in 1,4-dioxane (5 mL) and refluxed for 3 h. After evaporation, the
crude product was chromatographed with PE–AcOEt–EtOH
34.5: 64.5 : 0.5 to give pure cis diastereoisomer 2a (202.4 mg,
46.0%), and pure trans diastereoisomer 2b (152.9 mg, 34.8%).
Overall yield: 81%. The diastereoisomeric ratio was determined
by 1H NMR spectroscopy on the crude product and turned out
to be 60 : 40 (cis : trans).
2a (cis): Rf 0.26 (PE–AcOEt 3 : 7). Found: C, 63.9; H, 7.7;
N, 8.4%. C26H37N3O6 requires: C, 64.05; H, 7.65; N, 8.62%. 1H
NMR (300 MHz, CDCl3): d 7.42 [2 H, t, meta-H, J 7.5]; 7.32
[1 H, t, para-H, J 7.5]; 7.25 [2 H, d, ortho-H, J 7.7]; 6.30 [1 H,
d, NH, J 9.3]; 5.54 [1 H, broad s, NHBoc]; 5.54–5.40 [1 H, m,
H-6]; 5.33 [1 H, slightly broad q, H-7, J ≈ 9.0]; 4.74–4.52 [2
H, m, CH2Ph]; 4.58 [1 H, ddd, CHN, J ≈ 3.9, 7.2, 9.3]; 4.22
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 9 7 – 1 0 6
1 0 3

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=
[2 H, q, CH2CH3, J 7.2]; 4.10 and 4.02 [2 H, AB part of ABX
system, CH2NHBoc, JAB 16.9, JAX 4.7, JBX 3.9]; 2.79 [1 H, broad
ddd, H-8 a, J7–8 ≈ 9.0*]; 2.17 [1 H, dt, H-8 b, Jd 13.5, Jt 6.7];
2.10–1.88 [3 H, m, H-5 (2) and H-4 (1)]; 1.88–1.74 [1 H, m, H-4
(1)]; 1.44 [3 H, s, CH3C]; 1.41 [9 H, s, (CH3)3C]; 1.30 [3 H, t,
CH3CH2, J 7.1]. NOEDIFF: on irradiating the NH at d 6.30:
5.0% overall NOE on benzyl ortho-H; 2.4% NOE on H-8 b; 4.6%
NOE on one of the H-5; no NOE on H-8 b or on CH3C. On
irradiating the CH3 at d 1.44: 1.2% NOE on benzyl ortho-H;
0.9% NOE on H-6; 1.2% on H-4 downfield; no NOE on NH.
On irradiating H-7: 1.2% NOE on H-9. 13C NMR (75 MHz,
6.87 [1 H, t, NH, J 5.7]; 5.43 [1 H, slightly broad q, CH CH,
J 9.0]; 5.25 [1 H, dt, CH CH, Jd 3.5, Jt 11.1]; 4.98 [1 H, d,
=
CHHPh, J 18.3]; 4.26 [1 H, d, CHHPh, J 18.3]; 4.16–3.78 [7 H,
m, CHN and CH2N]; 3.64 [3 H, s, OCH3]; 2.60–2.40 [2 H, m,
CH2]; 2.35–1.95 [4 H, m, CH2]; 1.74–1.57 [1 H, m, CH2]; 1.38 [9
H, s, C(CH3)3]; 1.15 [3 H, s, CH3]. 13C NMR (DMSO, 50 MHz):
=
d 172.86, 170.88, 169.93, 169.06, 168.80 [C O]; 156.17 [urethane
=
=
C
O]; 137.94 [quat. arom.]; 133.39 [CH CH]; 128.56, 127.29,
126.60 [arom. CH]; 124.56 [CH CH]; 77.74 [C(CH3)3]; 64.40
[quat. CN]; 52.37 [CHN]; 51.68 [CH3O]; 47.82 [CH2Ph]; 43.25,
41.98, 40.37 [CH2N]; 38.28, 29.67 [CH2CH ]; 28.09 [(CH3)3C];
23.01 [CH2CH2CH ]; 22.80 [CH3]. IR: mmax 3360 (broad), 2953,
=
=
=
=
CDCl3): d 171.68, 171.56, 170.39 [C O]; 155.65 [urethane
=
=
C
O]; 136.92 [quat. arom.]; 135.28 [CH CH]; 129.36, 127.92,
2864, 2816, 1740, 1650, 1543, 1487, 1448, 1368, 1326, 1279, 1145,
125.71 [arom. CH]; 122.51 [CH CH]; 79.57 [C(CH3)3]; 65.68
[CNBn]; 61.47 [CH2CH3]; 51.12 [CHN]; 47.45, 43.65 [CH2N];
1079, 1037, 983, 949 cm−1.
=
=
=
38.83 [CH2CH ]; 29.86 [CH2CH ]; 28.26 [C(CH3)3]; 21.91
(Z,3R*,9R*)-3-(((N-Benzyl-t-butoxycarbonyl)amino)acetamido)-
9-(((((methoxycarbonylmethyl)amino)carbonylmethyl)amino)car-
bonyl)-3-methyl-4,5,8,9-tetrahydro-1H-azonin-2(3H)-one 15b
=
[CH2CH2CH ]; 21.56 [CH3]; 14.11 [CH3CH2]. IR: mmax 3427,
2971, 1728, 1680, 1489, 1448, 1402, 1369, 1163, 1054 cm−1.
2b (trans): Rf 0.18 (PE–AcOEt 3 : 7). Found: C, 63.85; H, 7.6;
N, 8.35%. C26H37N3O6 requires: C, 64.05; H, 7.65; N, 8.62%. 1H
NMR (300 MHz, CDCl3): d 7.54 [2 H, d, ortho-H, J 7.6]; 7.38 [2
H, t, meta-H, J 7.3]; 7.26 [1 H, t, para-H, J 7.4]; 6.18 [1 H, d, NH,
Prepared in 80% yield by the same procedure described above
for 15a. Rf 0.45 (AcOEt–CH2Cl2–MeOH 69 : 30 : 1). Found: C,
58.9; H, 7.2; N, 11.6%. C29H41N5O8 requires: C, 59.27; H, 7.03; N,
11.92%. 1H NMR (CDCl3, 200 MHz): d 8.41 [1 H, t, NH, J 5.9];
8.27 [1 H, broad s, NH]; 7.62 [2 H, d, ortho-H, J 7.4]; 7.44–7.19
[3 H, m, meta- and para-H]; 6.75 [1 H, d, ring NH, J 10.0]; 6.57
=
J 9.6]; 5.65–5.30 [3 H, m, CH2CH2CH CH and NHBoc]; 4.71
[1 H, dt, CHN, Jd 3.0, Jt 9.8]; 4.56 [2 H, s, CH2Ph]; 4.32–4.06 [3
H, m, CH2CH3 and CHHNHBoc]; 3.59 [1 H, dd, CHHNHBoc,
J 3.0, 17.2] 2.62–2.42 [1 H, m H-8 a, small J with H-9 (from
COSY)];2.40–2.10[2 H, m, H-8 b+ H-5];2.10–1.80[3H, m, H-5
(1) and H-4 (2)]; 1.67 [3 H, s, CH3C]; 1.37 [9 H, s, (CH3)3C]; 1.31
[3 H, t, CH3CH2, J 7.1]. NOEDIFF: on irradiating the NH at d
6.18: 3.4% NOE on C–CH3; 5.0% NOE on H-8 b. On irradiating
the CH3 at d 1.65: 1.1% NOE on benzyl ortho-H; 17.5% NOE
on NH; 6.0% NOE on one of the H-5. 13C NMR (50 MHz,
=
[1 H, broad t, NHBoc]; 5.43 [1 H, slightly broad q, CH CH,
=
J 9.1]; 5.21–5.02 [1 H, broad m, centre 5.11, CH CH]; 4.62 [2
H, s, CH2Ph]; 4.41 [1 H, t, CHN, J 9.9]; 3.95–3.77 [4 H, m,
CH2N]; 3.64 [3 H, s, OCH3]; 3.70–3.60 [1 H, m, CHHNHBoc];
3.29 [1 H, dd, CHHNHBoc, J 5.4, 16.7]; 2.50–2.16 [3 H, m,
CH2]; 2.00–1.85 [2 H, m, CH2]; 1.74–1.57 [1 H, m, CH2]; 1.59 [3
H, s, CH3]; 1.33 [9 H, s, C(CH3)3]. 13C NMR (DMSO, 50 MHz):
=
d 172.31, 170.89, 170.15, 169.06, 168.80 [C O]; 155.35 [urethane
=
CDCl3): d 172.26, 171.62, 169.73 [C O]; 155.65 [urethane
=
=
C
O]; 139.50 [quat. arom.]; 133.53 [CH CH]; 128.35, 126.55,
125.87 [arom. CH]; 124.83 [CH CH]; 77.72 [C(CH3)3]; 63.74
[quat. CN]; 52.20 [CHN]; 51.62 [CH3O]; 44.80 [CH2Ph]; 42.25,
=
=
C
O]; 137.57 [quat. arom.]; 134.40 [CH CH]; 129.05, 127.36,
=
=
125.79 [arom. CH]; 124.06 [CH CH]; 79.33 [C(CH3)3]; 64.22
[CNBn]; 61.65 [CH2CH3]; 51.66 [CHN]; 45.87, 42.99 [CH2N];
38.14 [CH2CH ]; 33.00 [CH2CH ]; 28.26 [C(CH3)3]; 23.02
=
41.63, 40.45 [CH2N]; 37.77, 32.24 [CH2CH ]; 28.06 [(CH3)3C];
=
=
=
21.94 [CH2CH2CH ]; 19.97 [CH3].
=
[CH2CH2CH ]; 20.90 [CH3]; 14.13 [CH3CH2].
Cyclic peptide 3a
(Z,3R*,9R*)-3-(((N-Benzyl-t-butoxycarbonyl)amino)acetamido)-
9-(((((methoxycarbonylmethyl)amino)carbonylmethyl)amino)car-
bonyl)-3-methyl-4,5,8,9-tetrahydro-1H-azonin-2(3H)-one 15a
A solution of compound 15a (110.1 mg, 0.187 mmol) in
tetrahydrofuran (5 mL), was treated at r.t. with 0.5 M aq.
LiOH (1.13 mL, 0.565 mmol) and stirred for 1 h. The solution
was poured into 5% aq. (NH4)H2PO4 (15 mL) + 1 N HCl
(0.5 mL). After saturation with NaCl, the mixture was extracted
3 times with AcOEt and evaporated to dryness. It was taken
up in CH2Cl2 (1 mL) and treated with CF3CO2H (250 lL).
After stirring for 90 min at r.t., the solvents were evaporated.
In order to remove completely the CF3CO2H, the residue was
taken up twice with n-heptane and evaporated again. Then it
was finally taken up in dry DMF (13 mL) and dry CH2Cl2
(17 mL). Sym-collidine (104 lL, 0.786 mmol) and HATU [(O-7-
azabenzotriazol-1-yl)-N,N,Nꢀ,Nꢀ-tetramethyluronium hexaflu-
orophosphate] (150.6 mg, 0.396 mmol) were added in sequence
and the mixture stirred for 2 days at r.t. The solvents were
evaporated. Then the residue was taken up in CHCl3, and
washed with 5% aq. (NH4)H2PO4 saturated with NaCl, and with
10% K2CO3 (15 mL) + 0.1 M NaOH (5 mL). Evaporation and
chromatography (CH2Cl2–EtOH 9 : 1) gave pure 3a as a white
solid (47.2 mg, 55%). Rf 0.42 (CH2Cl2–MeOH 9 : 1). Found: C,
60.4; H, 6.4; N, 15.25%. C23H29N5O8 requires: C, 60.65; H, 6.42;
N, 15.37%.
N-Boc-glycylglycine methyl ester (179.4 mg, 0.728 mmol) was
dissolved in CH2Cl2 (1.2 mL), and treated, at r.t., with CF3CO2H
(400 lL). After 1 h 15 min, the solvent was evaporated to
dryness. The residue was taken up in CH2Cl2 and evapo-
rated again three times to give crude glycylglycine methyl
ester TFA salt. Meanwhile, a solution of ester 2a (213.2 mg,
0.437 mmol) in EtOH (5.5 mL), was treated, at r.t., with 3
N aq. NaOH (190 lL, 0.57 mmol). After stirring for 30 min,
the solution was poured into 5% aq. (NH4)H2PO4 (30 mL) +
1 N HCl (1.3 mL). After saturation with NaCl, the mixture
was extracted 4 times with AcOEt. Evaporation furnished
the crude acid. It was taken up in CH2Cl2 (3 mL) and
added to the flask containing the crude glycylglycine methyl
ester. Et3N (273 lL, 1.96 mmol) and BOP (benzotriazol-1-
yloxy-tris(dimethylamino)phosphonium hexafluorophosphate)
(298.2 mg, 0.674 mmol) were added in this order. The solution
was stirred at r.t. overnight and then poured into 5% aq.
(NH4)H2PO4 saturated with NaCl (30 mL), and extracted with
AcOEt (3 times). The organic layer was washed with saturated
aq. NaHCO3 and evaporated to dryness. Chromatography with
CH2Cl2–AcOEt–EtOH 46.5 : 46.5 : 7 to 45 : 45 : 10 followed
with another chromatography with AcOEt–MeOH 98 : 2 gave
pure 15a as a white solid (203 mg, 79%). Rf 0.52 (AcOEt–
CH2Cl2–MeOH 69 : 30 : 1). Found: C, 59.1; H, 7.1; N, 11.8%.
1H NMR (DMSO, 300 MHz): d 8.70 [1 H, t, NH-b, J 5.7];
7.64 [1 H, d, ring NH, J 13.2]; 7.46–7.35 [3 H, m, meta-H and
NH-c]; 7.34–7.26 [3 H, m, ortho- and para-H]; 6.92 [1 H, d,
NH-a, J 7.3]; 5.43 [1 H, slightly broad q, H-7, J 9.0]; 5.27 [1 H,
dt, H-6, Jd 5.2, Jt 10.8]; 5.00 [1 H, d, CHHPh, J 18.3]; 4.38 [1
H, dd CHHN of glycine c, J 5.1, 15.3*] 4.35 [1 H, d, CHHPh, J
18.3]; 4.15–4.03 [2 H, m, H-9 and CHHN of glycine a]; 3.81 [1
H, dd, CHHN of glycine b, J 6.0, 15.6]; 3.60* [1 H, dd, CHHN
1
C29H41N5O8 requires: C, 59.27; H, 7.03; N, 11.92%. H NMR
(DMSO, 300 MHz): d 8.58 [1 H, t, NH, J 5.7]; 7.75 [1 H, d,
NH, J 10.2]; 7.50–7.25 [5 H, m, arom.]; 7.09 [1 H, broad t, NH];
1 0 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 9 7 – 1 0 6

View Article Online
of glycine c, JAB 15.6*]; 3.57* [1 H, dd, CHHN of glycine b, JAB
15.6*]; 3.35* [1 H, dd, CHHN of glycine a, JAB 14.1*]; 2.47 [1
H, ddd, H-8 a, J ≈ 2.0, 7.8, 12.6]; 2.55–2.10 [3 H, m, H-5 b,
H-8 b, H-4 (1)]; 2.10–1.95 [1 H, m, H-5 a]; 1.33 [1 H, dd, H-4
(1), J 9.9, 15.6]; 1.18 [3 H, s, CH3]. NOEDIFF: on irradiating
the signal at d 3.65 (H of glycines c and b): 3.3% NOE on ortho-
H (doublet at d 7.27); on irradiating the signal at d 3.81 (H of
glycine b): 1% NOE of NH of glycine c; on irradiating the ring
NH at d 7.64: 4.44% NOE on H-5 b (at d 2.35) and 3.33% NOE
on H-8 b (at d 2.16); on irradiating the signal at d 4.10 (CHN
and CH of glycine a): 3% NOE on H-7, 1.4% NOE on NH of
glycine b; on irradiating NH of glycine b (at d 8.70): 1.9% NOE
on CHHN of glycine a at d ≈ 4.06 ppm and 1.5% NOE on
CHHN of glycine a at d 3.35 ppm. On irradiating the signal of
the methyl at d 1.18 ppm: 3.7% NOE on ortho-H at d 7.27 and
1% on meta-H at d 7.36. 1H NMR (anhydrous CDCl3, 300 MHz,
0.01 M)(attribution of ring NH is certain (COSY). Attribution
of glycine a-c NH is only guessed): d 8.47 [1 H, broad t, NH b,
J ≈ 5.0]; 8.31 [1 H, broad s, NH c]; 7.92 [1 H, d, NH a, J 8.1]; 7.42
[2 H, t, meta-H, J 7.2]; 7.36–7.24 [3 H, m, ortho- and para-H];
6.49 [1 H, d, ring NH, J 9.9]; 5.55 [1 H, slightly broad q, H-7, J
9.1]; 5.34 [1 H, dt, H-6, Jd 4.5, Jt 10.8]; 5.29 [1 H, d, CHHPh, J
18.0]; 4.61 [1 H, d, CHHPh, J 18.0]; 4.66–4.36 [4 H, m, H-9 + 3
CHH glycines]; 3.52 [1 H, dd, CHH glycine b, J 4.5, 15.3]; 3.42
[1 H, slightly broad d, CHH glycine a or c, J 14.0]; 3.41 [1 H,
slightly broad d, CHH glycine a or c, J 14.1]; 2.59 [1 H, slightly
broad q, H-8 b, J 11.4]; 2.42–2.22 [3 H, m, (from left to right):
H-5, H-4 b, H-8 a]; 2.06–1.90 [1 H, m, H-5]; 1.58 [1 H, dd, H-4
a, J 10.5, 15.9]; 1.47 [3 H, s, CH3]. Dd (CDCl3 − DMSO): ring
NH: −1.15; NH-a: +0.91; NH-b: −0.23; NH-c: +1.00.
Biosystems MSD Sciex, Toronto, Canada) equipped with a
MALDI ion source. 2,5-Dihydroxybenzoic acid at a final
concentration of 5 mg mL−1in 70 : 30 0.1% TFA in H2O–0.05%
TFA in CH3CN was used as matrix. All the measures were
carried out mixing 500 fmol of an in-house obtained hexapeptide
(ALELFR, MW = 747.4272) with 500 fmol of sample. Internal
instrument calibration was performed using the main mono-
charged matrix fragment at m/z = 137.0239 (from DHB) and
mono-charged ion at m/z = 748.4352 of the above mentioned
hexapeptide. The main peak obtained was that of the mono-
sodium adduct of the monomer at m/z = 478.2034. Calculated
for C23H29N5O5Na: 478.2066 (accuracy: 6 ppm)
Cyclic peptide 3b
Prepared from 15b following the same procedure above de-
scribed for 3a. After careful chromatographic purification, a
product apparently giving a single spot in tlc (Rf 0.38 (CH2Cl2–
MeOH 9 : 1) was obtained in 13% yield. Both 1H and 13C
NMR showed the presence of 3b as the major product, but
it was contaminated by other three similar substances. HPLC-
MS (see 3a above for the conditions) showed a main sharp peak
corresponding to the expected monomer 3b (Rt 32.2 min), a
peak corresponding to the cis isomer 3a (Rt 31.4 min), and
two broader peaks (Rt 37.5 and 39.5 min), corresponding to
the 2 diastereoisomeric cyclodimers. Attempts to isolate pure
3b by silica gel chromatography with various eluents failed.
The relative ratio of 3b, 3a and cyclodimers in this mixture,
determined by HPLC/UV, is 55 : 8 : 37. Therefore the yield of
3b is 7%.
HPLC-MS [for the conditions see 3a]: the peak at Rt 32.2 gave
m/z (positive mode): 456.22 (M + H+) (base peak). Also other
smaller peaks (<10%) were detected: 478 (M + Na+), 494 (M +
K+), 911 (dimeric artifact), 933 and 949 (Na+ and K+ of dimeric
artifact). MS/MS of the peak at 456: 428.0 (42%), 411.0 (100),
399.9 (69), 382.8 (18), 370.9 (17), 353.7 (15), 320.8 (26), 296.7
(10), 292.8 (11), 284.8 (26), 263.7 (26), 251.7 (10), 246.6 (10),
228.8 (12), 211.8 (10), 206.9 (19), 178.8 (18), etc. MS/MS of the
peak at 911 of the cyclodimer with Rt 37.5: 740.6 (100), 712.6
(10), 696.6 (40), 563.3 (20), 456.0 (38), 428.0 (11), 411.0 (5), 399.9
(13), 284.8 (14). MS/MS of the peak at 911 of the cyclodimer
with Rt 39.5: 740.6 (100), 712.6 (10), 696.6 (44), 563.3 (24), 456.0
(52), 428.0 (13), 411.0 (4), 399.9 (13), 284.8 (12). HRMS: (for
conditions see 3a) the main peak obtained was that of the mono-
sodium adduct of the monomer at m/z = 478.2118. Calculated
for C23H29N5O5Na: 478.2066 (accuracy: 10 ppm).
13C NMR (DMSO, 75 MHz): d 172.01, 170.84, 170.21, 169.57,
=
=
168.96 [amidic C O]; 137.91 [quat. arom.]; 135.51 [CH CH];
128.60, 127.31, 126.76 [arom. CH]; 124.35 [CH CH]; 64.95
=
[quat. CN]; 51.80 [CHN]; 48.37 [CH2Ph]; 43.44, 43.32, 41.77
[CH2N]; 38.15, 28.89 [allylic CH2]; 22.92 [CH2]; 22.42 [CH3].
13C NMR (CDCl3, 75 MHz): d 172.53, 172.44, 172.28, 171.90,
=
=
171.23 [amidic C O]; 137.27 [quat. arom.]; 133.26 [CH CH];
129.25, 127.96, 126.07 [arom. CH]; 124.47 [CH CH]; 65.74
=
[quat. CN]; 51.91 [CHN]; 49.28 [CH2Ph]; 44.50, 42.73, 42.48
[CH2N]; 39.02, 30.03 [allylic CH2]; 24.08 [CH2]; 23.04 [CH3].
HPLC-MS was performed with the following eluent: 5 min
isocratic 100% A (0.1% TFA in H2O); then linear gradient from
0% to 70% B (0.1% TFA in CH3CN) over 20 min. Temperature:
15 ◦C for 15 min; then gradient to 5 ◦C in 1 min; then 5 ◦C
for the rest of analysis; flow: 0.35 mL min−1 for 16 min; then
0.1 mL min−1. Detection by TIC and DAD (220, 254 nm) showed
only 1 peak, with Rt = 31.4 min: m/z (positive mode): 456.24
(M + H+) (base peak) (calculated mass: 456.22). MS/MS of
this peak: 428.0 (62%), 411.0 (44), 399.9 (100), 382.8 (9), 370.9
(8), 353.7 (13), 320.8 (54), 296.7 (5), 292.8 (12), 284.8 (12), 263.7
(14), 228.8 (13), 211.8 (16), 206.9 (11), 178.8 (5), etc. In addition,
other smaller peaks (<10%) were detected: m/z = 478 (M +
Na+), 494 (M + K+), 911 (dimeric artifact), 933 and 949 (Na+
and K+ of dimeric artifact). The fact that the 911 peak was an
instrument artifact is demonstrated by the following evidence: a)
This dimeric artifact was present also in the trans cyclic peptide
3b, where the real dimer was detected at different Rt (see below).
b) The fragmentation energy used to perform MS/MS analysis
on the 911 ion co-eluting with the monomers was lower (0.6 V)
and therefore consistent with a non-covalent interaction, than
that needed to fragment the 911 ion of the real trans dimer
(9 V). c) The obtained MS/MS mass spectrum of the dimeric
artifact revealed a poor fragmentation: 456.0 (100%), 428.0 (31),
411.0 (4), 399.9 (8); on the contrary, the MS/MS spectrum of
the real trans dimers gave a rich fragmentation (see below). d)
The dimeric artifact gaved a poorly resolved isotopic cluster,
probably due to the presence of multicharged (e.g. tetramers)
instrumental overlapping signals.
We tried to separate a mixture of 3b and 3a (8 : 2) from
the cyclodimers by reverse-phase preparative HPLC (conditions
were similar to those employed for analytical HPLC). The
separation was successful but, surprisingly, on evaporation to
dryness (at 30–40 ◦C), extensive decomposition of 3b was
observed giving hydrolysed adducts (as demonstrated by the
presence in the MS of peaks with m/z = 474). After a further
silica gel chromatography, we isolated a small amount of an 83
: 17 mixture of 3a and 3b. Thus it seems that 3b is much more
sensitive to hydrolysis than 3a.
From careful analysis of the NMR spectra of the above
mentioned mixtures we could extract the signals of 3b: 1H NMR
(DMSO, 300 MHz): d 8.44 [1 H, t, NH, J 6.1]; 7.74 [1 H, d, NH,
J 6.3]; 7.48–7.22 [6 H, m, arom. and NH]; 7.11 [1 H, d, NH,
=
J 6.9]; 5.66–5.36 [2 H, m, CH CH]; 4.71 and 4.59 [2 H, AB
system, CH2Ph, J 17.7]; 4.45–3.60 [6 H, m, CHN and CH2N];
3.43–3.30 [1 H, m, CHHN]; 2.50–1.60 [5 H, m, allylic CH2 and
other CHH]; 1.40–1.20 [1 H, m, CHH]; 1.01 [3 H, s, CH3].
13C NMR (DMSO, 75 MHz): d 171.68, 171.35, 170.94, 170.17,
=
=
169.21 [amidic C O]; 138.11 [quat. arom.]; 134.57 [CH CH];
128.68, 127.28, 126.81 [arom. CH]; 125.87 [CH CH]; 64.39
=
HRMS was carried out on an ibrid q-TOF geometry tandem
mass spectrometer (Q-STAR XL MS/MS system – Applied
[quat. CN]; 53.02 [CHN]; 46.04 [CH2Ph]; 44.72, 43.90, 42.15
[CH2N]; 36.62, 23.11 [allylic CH2]; 19.63 [CH3]; 19.23 [CH2].
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 9 7 – 1 0 6
1 0 5

View Article Online
16 K. Brickmann, P. Somfai and J. Kihlberg, Tetrahedron Lett., 1997,
38, 3651.
17 A. D. Piscopio, J. F. Miller and K. Koch, Tetrahedron, 1999, 55, 8189.
18 A. Nouvet, M. Binard, F. Lamaty, J. Martinez and R. Lazaro,
Tetrahedron, 1999, 55, 4685.
19 B. E. Fink, P. R. Kym and J. A. Katzenellenbogen, J. Am. Chem.
Soc., 1998, 120, 4334.
20 A. J. Souers and J. A. Ellman, Tetrahedron, 2001, 57, 7431.
21 J. Bondebjerg, Z. Xiang, R. M. Bauzo, C. Haskell-Luevano and M.
Meldal, J. Am. Chem. Soc., 2002, 124, 11046.
22 L. Banfi, G. Guanti and R. Riva, Chem. Commun., 2000, 985.
23 L. Banfi, G. Guanti, R. Riva, A. Basso and E. Calcagno, Tetrahedron
Lett., 2002, 43, 4067.
24 A. Basso, L. Banfi, R. Riva, P. Piaggio and G. Guanti, Tetrahedron
Lett., 2003, 44, 2367.
25 L. Banfi, A. Basso, G. Guanti and R. Riva, Mol. Diversity, 2003, 6,
227.
26 L. Banfi, A. Basso, G. Guanti and R. Riva, Tetrahedron Lett., 2004,
Acknowledgements
We gratefully thank Federico De Pellegrini and Carlo Scapolla
for their very valuable collaboration, and MIUR (PRIN 00 and
02) for financial support.
References
1 T. R. Gadek and J. B. Nicholas, Biochem. Pharm., 2003, 65, 1.
2 K. Burgess, Acc. Chem. Res., 2001, 34, 826.
3 R. Haubner, D. Finsinger and H. Kessler, Angew. Chem., Int. Ed.
Engl., 1997, 36, 1375.
4 M. A. Dechantsreiter, E. Planker, B. Matha¨, E. Lohof, G.
Ho¨lzemann, A. Jonczyk, S. L. Goodman and H. Kessler, J. Med.
Chem., 1999, 42, 3033.
5 A. Golebiowski, J. Jozwik, S. R. Klopfenstein, A. O. Colson, A. L.
Grieb, A. F. Russell, V. L. Rastogi, C. F. Diven, D. E. Portlock and
J. J. Chen, J. Comb. Chem., 2002, 4, 584.
6 W. Qiu, X. Gu, V. A. Soloshonok, M. D. Carducci and V. J. Hruby,
Tetrahedron Lett., 2001, 42, 145.
7 M. Eguchi, M. S. Lee, M. Stasiak and M. Kahn, Tetrahedron Lett.,
2001, 42, 1237.
8 S. Hanessian, G. McNaughton-Smith, H.-G. Lombart and W. D.
Lubell, Tetrahedron, 1997, 53, 12789.
9 L. Halab, F. Gosselin and W. D. Lubell, Biopolymers, 2000, 55, 101.
10 L. Belvisi, A. Caporale, M. Colombo, L. Manzoni, D. Potenza, C.
Scolastico, M. Castorina, M. Cati, G. Giannini and C. Pisano, Helv.
Chim. Acta, 2002, 85, 4353.
45, 6637.
27 M. D. Burke and S. L. Schreiber, Angew. Chem., Int. Ed., 2004, 43,
46.
28 G. Vo-Thanh, V. Boucard, H. Sauriat-Dorizon and F. Guibe´, Synlett,
2001, 37.
29 C. Hebach and U. Kazmaier, Chem. Commun., 2003, 596.
30 B. Beck, G. Larbig, B. Mejat, M. Magnin Lachaux, A. Picard, E.
Herdtweck and A. Do¨mling, Org. Lett., 2003, 5, 1047.
31 R. Krelaus and B. Westermann, Tetrahedron Lett., 2004, 45, 5987.
32 L. Banfi, A. Basso, G. Guanti and R. Riva, Tetrahedron Lett., 2003,
44, 7655.
33 C. J. Creighton and A. B. Reitz, Org. Lett., 2001, 3, 893.
34 C. J. Creighton, G. C. Leo, Y. Du and A. B. Reitz, Bioorg. Med.
Chem., 2004, 12, 4375.
11 A. Trabocchi, E. G. Occhiato, D. Potenza and A. Guarna, J. Org.
Chem., 2002, 67, 7483.
12 T. K. Chakraborty, A. Ghosh, A. Ravi Sankar and A. C. Kunwar,
Tetrahedron Lett., 2002, 43, 5551.
35 S. R. Wilson and R. A. Sawicki, J. Org. Chem., 1979, 44, 330.
36 A. Fu¨rstner and K. Langemann, Synthesis, 1997, 792.
37 R. Haubner, R. Gratias, B. Diefenbach, S. L. Goodman, A. Jonczyk
and H. Kessler, J. Am. Chem. Soc., 1996, 118, 7461.
38 N. J. Baxter and M. P. Williamson, J. Biomol. NMR, 1997, 9, 359.
13 M. F. Bran˜a, M. Garranzo, B. de Pascual-Teresa, J. Pe´rez-Castells
and M. Rosario Torres, Tetrahedron, 2002, 58, 4825.
14 S. N. Filigheddu and M. Taddei, Tetrahedron Lett., 1998, 39, 3857.
15 T. K. Chakraborty, A. Ghosh, R. Nagaraj, A. Ravi Sankar and A. C.
Kunwar, Tetrahedron, 2001, 57, 9169.
1 0 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 9 7 – 1 0 6