A convenient strategy of dimerization by microwave heating and using 2,5-diketopiperazine as scaffold

Article abstract of DOI:10.1016/S0040-4039(02)02836-8

A novel and convenient microwave-assisted dimerization of an active peptide compound using the DKPs as scaffold is described. The key reaction giving rise to the diketopiperazine scaffold is the intermolecular coupling. No epimerization was detected in the reactions used. Conventional and microwave heating of the reactions are compared. Synthesis by microwave irradiation gave the desired compounds in higher yields and in shorter reaction times than those obtained by conventional heating.

Full text of DOI:10.1016/S0040-4039(02)02836-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 1145–1148
A convenient strategy of dimerization by microwave heating and
using 2,5-diketopiperazine as scaffold
Vincenzo Santagada,a,* Ferdinando Fiorino,a Elisa Perissutti,a Beatrice Severino,a Sara Terracciano,a
Giuseppe Cirinob and Giuseppe Caliendoa
aDipartimento di Chimica Farmaceutica e Tossicologica, Universita` di Napoli ‘Federico II’, Via D. Montesano, 49, 80131 Naples,
Italy
bDipartimento di Farmacologia Sperimentale, Universita` di Napoli ‘Federico II’, Via D. Montesano, 49, 80131 Naples, Italy
Received 8 October 2002; accepted 16 December 2002
Abstract—A novel and convenient microwave-assisted dimerization of an active peptide compound using the DKPs as scaffold is
described. The key reaction giving rise to the diketopiperazine scaffold is the intermolecular coupling. No epimerization was
detected in the reactions used. Conventional and microwave heating of the reactions are compared. Synthesis by microwave
irradiation gave the desired compounds in higher yields and in shorter reaction times than those obtained by conventional heating.
© 2003 Elsevier Science Ltd. All rights reserved.
In both lead identification and lead optimization pro-
cesses there is an acute need for new organic small
molecules. Traditional methods of organic synthesis are
orders of magnitude too slow to satisfy the demand for
these compounds. The fields of combinatorial and auto-
mated medicinal chemistry (i.e. the simultaneous or
parallel synthesis of libraries)1 have been developed to
meet the increasing requirement of new compounds for
drug discovery; within these fields, speed is of the
essence. The efficiency of microwave flash-heating
chemistry in dramatically reducing reaction times
(reduced from days and hours to minutes and seconds)
has recently been proven in several different fields of
organic chemistry.2–7 The preponderance of heterocyclic
frameworks in historical samplings of known drug
structures provides strong rational for the inclusion of
these scaffolds in lead discovery programs. In fact, one
current strategy for the design of new drugs consists of
the display, on a suitable rigid scaffold, of functional
groups that are known to be involved in interactions
with the corresponding receptor. Among several hetero-
cycles, 2,5-diketopiperazines (DKPs) have attracted
considerable attentions in the recent years.8 They can
be considered as the smallest cyclic peptides, derived
from the folding head-to-tail of a linear unprotected
dipeptide.9 DKPs scaffolds are a common motif in
several natural products with therapeutic properties.
Included are inhibitors of the mammalian cell cycle,10
of the plasminogen activator-1,11 and of the topoiso-
merase I,12 as well as competitive antagonists to Sub-
stance P at the neurokinin-1 receptor.13 Furthermore,
DKPs have been shown to be useful scaffolds for
rational design of several drugs14 since are typically
stable to proteolysis, one important consideration when
designing potential drug scaffolds. DKPs are often
formed by a side reaction, arising from intramolecular
aminolysis at the dipeptidyl level, in both solution and
solid-phase synthesis.15 DKPs formation has been
established to be either acid15b,e,16 or base catalysed15a,c–g
and it is strongly dependent on the nature and the
sequence of the amino acids.15b Several methods for the
intentional synthesis of 2,5-diketopiperazines on solid
support have been proposed and they are based mainly
on the cleavage-induced cyclization of linear dipep-
tides.8,17 Dimerization of an active compound often
results in enhanced binding and improved pharmaco-
logical properties. This potency is attributed to a higher
concentration of pharmacophores in proximity to
recognition sites. Usually, this bivalent ligand approach
supposes to use a symmetrical bifunctional linker X to
anchor substrates P, giving rise to the general structure
P-X-P.18,19 Knowing the importance of the DKPs in
medicinal chemistry our attention has turned to
improve the reaction of dimerization19 of an active
peptide compound using the DKPs as scaffold and
Keywords: synthesis of diketopiperazine; dimerization; microwave
irradiation in solution.
* Corresponding author. Tel.: +0039-81-678648; fax: +0039-81-
678649; e-mail: santagad@unina.it
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02836-8

1146
V. Santagada et al. / Tetrahedron Letters 44 (2003) 1145–1148
bearing in mind new synthetic methodologies. The
application of microwave energy to organic compounds
for conducting synthetic reactions at highly accelerated
rates is an emerging technique.2–7 These observations
led us to check into the possibility for improvement in
methods used for the synthesis on solution of the
substituted 2,5-DKP. The challenge was to build differ-
ent dimeric structures, involving a Gly-Gly diketopiper-
azine scaffold, by activating C-terminal glycine
monomers (Scheme 1). Our strategy started with intro-
ducing a C-terminal glycine moiety on a pharma-
cophore, followed by an activation step to provide the
diketopiperazine bearing the two substrates. Two path-
ways have to be considered depending on the nature of
the active substances (Scheme 1a and b). In fact, as
substrate we considered the synthetic hexapeptide
(Scheme 1a) corresponding to the proteolitically
revealed amino terminal sequence Ser-Leu-Ile-Gly-Arg-
Leu (PAR-2 AP) of rat PAR-2, that is able to efficiently
activate the PAR-2 receptor,20 and some dipeptide
derivatives obtained by us21 corresponding the follow-
ing general structure Na-benzoyl-Arg(NO2)-Leu-NH2
(Scheme 1b) and containing the minimal structural
requirements for agonist activity of PAR-2 receptor.
The pathophysiological role of PAR-2 is not clearly
established, and only recently this receptor has been
recognized to play a role in inflammatory,22,23 gas-
trointestinal,23 and vascular24 diseases. The targeted
heptapeptide was synthesized by standard solution
methods, as a C-terminal glycine, by 3+4 fragment
condensation.25 a-Amino functions were protected by
Fmoc group except for the N-terminal amino acid that
was protected by Boc group. The side chain protecting
group for arginine was Pbf (Scheme 1a). DCC/HOBt
was employed for the coupling method.26 The different
masking groups, Boc, Pbf and Fmoc were removed,
cases, the key reaction giving rise to the diketopiper-
azine scaffold is the intermolecular coupling.30 In fact
the intermolecular coupling was influenced by the par-
ticular activator employed (DCC/DMAP and HBTU/
DMAP). DCC/DMAP method produced no cyclization
when the reaction was performed both with conven-
tional heating or microwave heating. In contrast, the
intermolecular coupling under microwave heating was
obtained with high yields using HBTU/DMAP (Scheme
1a, 90%, 5 min, mV, 400 W, 40°C; Scheme 1b, range
between 85 and 92%, 8 min, mV, 400 W, 40°C) while
the yield was lower when the reaction was performed in
the same conditions by conventional heating (Scheme
1a, 33%, 24 h, 40°C and Scheme 1b, range between 35
and 42%, 24 h, 40°C). No epimerization was detected in
the reactions above described. The conditions utilized
with the microwave energy represents the optimum
profile of power/time and temperature. The tempera-
ture of the stirred reaction mixture was monitored
directly by a microwave-transparent fluoroptic probe
inserted into the solution. The overall reaction times of
the reactions performed by microwave irradiation were
dramatically reduced. Final compounds were tested for
its ability to relax isolated rat aorta as described previ-
ously.21 The ability to relax pre contracted rat aorta
with phenylephrine was tested using the final com-
pound in a dose varying between 10−7 and 10−4 M. The
tested compound did not cause any relaxation up to a
dose of 10−4 M.
In conclusion, we have shown that the application of
microwave irradiation improves the intermolecular cou-
pling yields and significantly reduces reaction times in
the solution synthesis of dimerization using DKPs as
scaffold and HBTU/DMAP as coupling reagents.
27
respectively, by TFA/CH2Cl2 and 20% piperidine in
DMF.28 The tripeptide derivatives Na-benzoyl-
Arg(NO2)-Leu-Gly-OH and analogues were prepared
by standard liquid phase peptide synthesis.29 All final
compounds were purified by preparative RP-HPLC25
and analysed on a LCQ Ion-Trap mass spectrometer,
yielding mass values in agreement with the expected
chemical composition within 10 ppm accuracy. In both
Acknowledgements
This work was supported by a grant from Regione
Campania ai sensi della L. R. 31 dicembre 1994, N° 41,
art. 3, 1° comma.
Scheme 1. Dimerization of PAR-2AP and dipeptide derivatives (Na-benzoyl-Arg(NO2)-Leu-NH2 and analogues) using DKPs as
scaffold.

V. Santagada et al. / Tetrahedron Letters 44 (2003) 1145–1148
1147
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V. Santagada et al. / Tetrahedron Letters 44 (2003) 1145–1148
26. DCC/HOBt method: To a solution of the carboxylic
component (1 mmol) in DMF (5 mm) at 0°C were added
the amino acid component (1 mmol), 1-hydroxybenzotri-
azole (HOBt) (1 mmol), and DCC (1.1 mmol) in the
above order. The reaction mixture was stirred for 2 h at
0°C and for 24 h at room temperature. Then N,N%-dicy-
clohexylurea (DCU) was filtered off, and the solution was
diluted with EtOAc (100 ml). The solution was washed
consecutively with brine, 0.5N KHSO4, brine, 5%
NaHCO3, and brine. The organic phase was dried
(MgSO4), was crystallized from appropriate solvents or
purified by column chromatography.
27. Removal of Boc- and Pbf-protecting groups: The pro-
tected compound (1 mmol) was treated with TFA (2.5
ml) for 0.5 h at room temperature. Et2O was added to the
solution until the product precipitated. The crude
product was purified by preparative RP-HPLC as
reported in Ref. 25.
28. Removal of Fmoc-protecting group: Standard cleavage
procedure was used to remove Fmoc protecting group.
The peptide (1 mmol) was dissolved in 20% piperidine/
DMF (15 ml) and the reaction was allowed to proceed at
room temperature for 45 min. The solvent was evapo-
rated in vacuo, the residue was triturated with diethyl
ether (25 ml), the solid was collected, washed with diethyl
ether, and dried in vacuo.
HOBt method.26 Successively the Boc protecting group of
the N-terminal residue was removed by TFA/CH2Cl2
27
and coupled with the appropriate Na-benzoyl-Arg(NO2)-
OH derivatives by DCC/HOBt method to obtain the
protected tripeptides that were purified by crystallization
(diethylether/hexane, 8:2). The resulted compounds were
characterized and converted to their carboxylic form by
saponification with 1N NaOH,25 successively purified by
RP-HPLC and characterized by LCQ Ion-Trap mass
spectrometer.
30. General procedure for intermolecular cyclization: Synthesis
was performed using a microwave oven ETHOS 1600,
Milestone especially designed for organic synthesis. The
experiments were carried out in standard Pyrex glassware
chamber. A solution of the free carboxylic heptapeptide
or Na-benzoyl-Arg(NO2)-Leu-Gly-OH derivatives (3.16
mmol), DMAP (4-(dimethylamino)pyridine, 3.47 mmol)
and HBTU (3.47 mmol) in 6 mL anhydrous DMF was
treated by microwave irradiation for 5 or 8 min, respec-
tively (UV, 400 W, 40°C) or by conventional heating for
24 h at 40°C, respectively. The reaction mixture was
separated from the solvent, the crude residue was taken
up in ethyl acetate and washed successively three times
with citric acid (5%), sodium bicarbonate (5%), and a
saturated solution of sodium chloride. The final crude
products, after removal of the Boc and Pbf protecting
groups (Scheme 1a), were purified and characterized by
preparative RP-HPLC as reported in Ref. 25.
29. General procedure for acyl-tripeptide derivatives: The
starting dipeptide Boc-Leu-Gly-OCH3 was produced
from Boc-Leu-OH and HCl·H-Gly-OCH3 by the DCC/