Synthesis of sidechain adapted β-turn mimics for modifying the C-terminus of substance P

Article abstract of DOI:10.1016/j.tetlet.2005.01.056

Sidechain adapted β-turn mimics of type 1 characterised by a fixed cis-conformation of the peptide chain in the aminopiperidinonecarboxylate scaffold have been synthesised from pyrazinones in order to perform a β-turn scan of the messenger region of substance P. The synthesis of a substance P peptide analogue is also described.

Full text of DOI:10.1016/j.tetlet.2005.01.056

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 1707–1710
Synthesis of sidechain adapted b-turn mimics for
modifying the C-terminus of substance P
b
b
Vijaykumar G. Pawar,a Wim M. De Borggraeve,a, Veronique Maes, Dirk A. Tourwe,
*
´
Frans Compernollea and Georges J. Hoornaerta,*
aLaboratory for Organic Synthesis, K.U. Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium
bLaboratory of Organic Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, B-1050, Brussels, Belgium
Received 23 November 2004; accepted 12 January 2005
Available online 29 January 2005
Abstract—Sidechain adapted b-turn mimics of type 1 characterised by a fixed cis-conformation of the peptide chain in the amino-
piperidinonecarboxylate scaffold have been synthesised from pyrazinones in order to perform a b-turn scan of the messenger region
of substance P. The synthesis of a substance P peptide analogue is also described.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
APCscaffolds, which enables one to do a b-turn/cis-pep-
tide bond scan of the C-terminal sequence of the neuro-
peptide substance P (SP). Our approach is illustrated by
the synthesis of a first APCmodified substance P
analogue.
The synthesis of constrained peptidomimetics is an
important field in synthetic organic chemistry. Incorpo-
ration of rigidified scaffolds in a peptide sequence and
further assessment of its biological activity can provide
information about the bioactive conformation of the
system. We recently published a general synthesis for
systems of type 1 (Fig. 1) and classified them as Ôtype-
VI-likeÕ b-turn mimetics.2,1 In these aminopiperidinone-
carboxylate (APC) scaffolds, the peptide bond is fixed in
a cis-conformation and different sidechain functional-
ities can be incorporated in these systems. So far how-
ever, we only synthesised model compounds, which are
not suitable for incorporation into the peptide chain.
In this paper, we report the synthesis of functionalised
Substance P (SP) is the undecapeptide neurotransmitter
Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-Phe8-Gly9-Leu10-
Met11-NH2.12 It has been implicated in several diseases
including arthritis, asthma, inflammatory bowel disease
1
and depression.8,7 According to H NMR experiments,
a b-turn is present in the C-terminal hexapeptide se-
quence Gln6-Phe7-Phe8-Gly9-Leu10-Met11-NH2. This
C-terminal hexapeptide sequence (messenger region)
shows similar activity as the native sequence, however
it lacks its selectivity. A Ôtype VI-like turnÕ containing
a cis-peptide bond was observed at the Phe7-Phe8 posi-
tion of a bioactive [pGlu6, NMe-Phe8, Aib9] (6–11) ana-
logue of substance P.6,9 In this context, replacement of
the Phe7-Phe8 residues with a turn inducing substructure
that contains a type VI-like, constrained cis-peptide
bond, seems an interesting research goal. Also other
turn types centered at Phe7-Phe8, Phe8-Gly9 and
Gly9-Leu10 residues have been proposed by various
authors.10,13,3 Interestingly, application of our APC
scaffold methodology would enable one to perform a
cis-peptide bond/b-turn scan of the whole C-terminal re-
gion of the peptide. An important feature of our meth-
odology in this regard is that the sidechain of the
corresponding amino acid residue (through which bind-
ing to the receptor might occur) can be retained in the
Figure 1. Aminopiperidinonecarboxylate.
Keywords: cis-Peptide bond; b-Turn; Peptidomimetics; Substance P;
Pyrazinones.
*
Corresponding authors. Tel.: +32 16 327404/09; fax: +32 16
327990; e-mail addresses: wim.deborggraeve@chem.kuleuven.ac.be;
georges.hoornaert@chem.kuleuven.ac.be
0040-4039/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.01.056

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V. G. Pawar et al. / Tetrahedron Letters 46 (2005) 1707–1710
Figure 2. b-Turn scanning of the messenger region of substance P.
scaffolds (principle shown in Fig. 2). Boc and Fmoc pro-
tection strategies have been elaborated to render these
scaffolds amenable for peptide synthesis.
this stage, the N-terminus of the Boc and Fmoc deriva-
tives 7a–d is already suitably protected, but the methyl
ester and the PMB function still have to be removed
in order to make the analogues suitable for peptide
work. Model compound 7e was initially used to study
these subsequent deprotection reactions.
2. Synthesis of bicyclic precursors
The bicyclic precursors 5a–c for the APCsystems, with
correct sidechain functionality in place, were synthesised
using our established method. This implies functionali-
sation of dichloropyrazinones 2a–c, followed by cyclo-
addition of 3a–c with ethene at high pressure. The C2-
bridge introduced in the Diels–Alder reaction eventually
serves as a conformational restriction in the APCsys-
tems. Finally, the reactive imidoyl chloride adducts
4a–c were hydrolysed to give the bislactam systems 5a–c
(Scheme 1). As the R1 substituent, a PMB group was
chosen instead of the benzyl group that was used
previously because the latter could not be removed.
4. Removal of the PMB group and cleavage of the methyl
ester, a test case
To deblock the carboxyl function of model compound
7e, a simple saponification was tried using aqueous so-
dium hydroxide or lithium hydroxide (Scheme 3). The
desired compound 8e was formed only in minor amount.
The main product isolated was the cyclised bislactam 5b.
This means that the cleavage of the amide was the faster
reaction (and not the hydrolysis of the hindered ester)
resulting in regeneration of cyclic compound 5b. Treat-
ment of methyl ester 7e with lithium iodide in EtOAc
however led to a very clean reaction with the formation
of only the desired product 8e in a good yield.4 The soft
nucleophile iodide selectively attacks the methyl group.
It also allows cleavage of the ester in the presence of
base labile groups like Fmoc.
3. Methanolysis of bislactam systems and formation of
Boc/Fmoc protected scaffolds
The strained bislactam systems 5a–c, when treated with
an HCl–saturated methanol solution, open selectively at
the secondary lactam position to yield hydrochloride
salts 6a–c (Scheme 2).11,2 The corresponding amines
were trapped by subsequent reaction with a tertiary
amine and Boc2O/Fmoc-Cl or an acid chloride to yield
compounds 7a–d. NMR and mass spectral analysis of
these compounds confirm the structures proposed. At
Different conditions were tried on the model compound
7e to remove the PMB group (Scheme 4). Treating 7e
with trifluoroacetic acid at reflux temperature nor a
reductive procedure using H2/Pd–C(10%) gave satis-
factory yields of the desired compounds. However
Scheme 1. Synthesis of bicyclic precursors.

V. G. Pawar et al. / Tetrahedron Letters 46 (2005) 1707–1710
1709
oxidative cleavage by ceric ammonium nitrate (CAN)
was very successful.
5. Removal of the PMB group and cleavage of the methyl
ester in the APC systems
Based on the work performed on the model compound
7e, demethylation of the methyl ester and removal of
the PMB group in turn was effected for the Boc and
Fmoc protected APCsystems ( Scheme 5). In general,
the oxidative PMB removal gives satisfactory results
for all the Fmoc protected analogues. We soon found
that CAN also is an effective reagent to remove a Boc
protective group,5 so the selective oxidative removal of
PMB on Boc protected compound 9c was not successful.
Scheme 2. Methanolysis of bicyclic adducts. Reagents and conditions:
(a) (1) MeOH/HCl, 50 °C, 16 h; (2) MeOH/HCl, rt, 2 h; (b) (1) Fmoc-
Cl, DIEA, DMAP, CHCl3, 2 h; (2) Boc2O, Et3N, CHCl3, 1.5 h; (3)
Ph(CH2)2COCl, Et3N, CHCl3, 1 h.
In contrast to our test case 7e, we were able to remove
the PMB group from the APCsystems with R 6 = isobu-
tyl using trifluoroacetic acid (TFA) at reflux tempera-
ture. This TFA procedure does not work in any of the
other cases.
As shown in Scheme 5 and the accompanying table we
tried two different sequences: first removal of the PMB
group followed by demethylation and vice versa. The
yield of the final products in both sequences are compa-
rable, path 1 was more practical for 10b and path 2 for
10d (Table 1).
Scheme 3. Deprotection of the carboxyl function, a test case.
As can be seen from the table, we managed to synthes-
ise all the Fmoc-protected derivatives 10a,b and 10d
that were initially proposed for the modification of
SP. The synthesis of the Boc analogue 10c was not
successful, due to the unselective nature of the CAN
deprotection. The partially deprotected intermediate 9c
obtained by demethylation of methyl ester 7d however
is also expected to be useful in Boc-based peptide
synthesis.
6. Incorporation of APC systems into the C-terminal
hexapeptide region of SP, a first example
A substance P peptide analogue Gln6-Phe7-Phe8-(Gly-
Leu)APC-Met11-NH2 was synthesised using solid-phase
peptide techniques (Scheme 6). Rink amide resin was
Scheme 4. Removal of PMB group. Reagents and conditions: (a)
TFA, reflux, 16 h; (b) H2/Pd–C(10%), ethanol, 16 h; (c) CAN,
acetonitrile, À15 °C–rt.
Scheme 5. Synthesis of fully deprotected APCanalogues. Reagents and conditions: (a) CAN/water, acetonitrile, 0 °Cto rt, 2 h; (b) TFA, reflux,
overnight; (c) LiI, ethyl acetate, reflux, overnight.

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V. G. Pawar et al. / Tetrahedron Letters 46 (2005) 1707–1710
Table 1. Deprotection of the scaffolds
R3
R6
R7
Path 1
Path 2
Product
Product
Yieldmethod (%)
Product
Yield (%)
Product
Yield (%)
Yieldmethod (%)
Bn
Bn
Bn
H
Bn
H
Fmoc
Fmoc
Boc
8a
8b
8c
8d
67a
66a
—
55a
70b
10a
10b
10c
10d
52
68
—
64
9a
9b
9c
9d
42
71
90
69
10a
10b
10c
10d
—
70a
00a
60a
73b
H
Isobutyl
Fmoc
a See method a in Scheme 5.
b See method b in Scheme 5.
Fmoc based scaffolds seem to be the more feasible ones.
A first peptide analogue has been prepared demonstrat-
ing that the APC-scaffold can be introduced into the
peptide sequence with an appropriate coupling reagent
and using a longer coupling time. The synthesis of the
other substance P analogues and biological testing is un-
der current investigation.
Acknowledgements
The authors thank the F.W.O. (Fund for Scientific Re-
search–Flanders (Belgium)) and the Johnson and John-
son Pharmaceutical Research Foundation for financial
support. W.M.D.B. (Postdoctoral Fellow of the
F.W.O.) and V.M. (Research Assistant of the F.W.O.)
thank the F.W.O. for the fellowship received.
Scheme 6. Synthesis of SP (6–11) APCanalogue.
used in order to obtain the C-terminal carboxamide.
Successive couplings with the common N-Fmoc pro-
tected amino acids (used in standard threefold excess)
were accomplished by reaction with HOBT/DICin
DMF and were completed within 2 h. However, attach-
ment of the more hindered Gly-Leu APCscaffold 10d
(twofold excess) required activation with TBTU in
DMF and took 16 h for completion (ninhydrin test).
The N-terminal Gln residue (with a trityl protected side-
chain) was capped with acetic anhydride. Finally, the
peptide was cleaved from the resin and detritylated
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7. Conclusion
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