Siamese depsipeptides: Constrained bicyclic architectures

Article abstract of DOI:10.1002/anie.200904135

The first members of a new class of depsipeptides with tartaric acid as the core unit, called Siamese depsipeptides, are described. These compounds were synthesized from a branched precursor in only one cyclization step. The structural manipulation of a n

Full text of DOI:10.1002/anie.200904135

Communications
DOI: 10.1002/anie.200904135
Depsipeptides
Siamese Depsipeptides: Constrained Bicyclic Architectures**
Javier Ruiz-Rodrꢀguez, Jan Spengler,* and Fernando Albericio*
Dedicated to Professor Klaus Burger
Bioactive cyclic peptides and depsipeptides which are isolated
from natural sources provide a range of key lead structures
for the design of new drugs, as reflected by the notable
increase in peptide- and depsipeptide-based drugs during
recent years.[1] However, peptides as drugs show some
drawbacks that are inherently associated with their structure.
Thus, linear peptides can be degraded rapidly by proteases,
and receptor affinity is low owing to the large number of
conformations which they can adopt. Cyclization can over-
come these problems to provide both conformational restric-
tion and enhanced proteolytic stability.[2] Natural cyclic
peptides adopt mainly a “head to tail” or “head to side
chain” architecture. Bicyclic peptides are mostly formed by
disulfide bridges, such in the conotoxins, or by a combination
of a “head to side chain” and disulfide bridges, such in the
case of thiocoralines and related natural compounds, which
show a C2 symmetry.[3] The creation of new cyclic peptide
architectures is of special interest with respect to possible
improvement of pharmacological properties. Consequently,
several other types of cyclic peptides, such as “homodetic side
chain to side chain”, “backbone to backbone”, as well as a
combinations of these have been described.[4]
has been designed. Cyclic depsipeptide dimers connected by a
CC bond were constructed. Two kind of possible connections
can be envisaged, those that share a side chain bond or those
that share a backbone bond. Owing to the resulting proximity
of both structurally identical cycles, they can be called
Siamese depsipeptides. For Siamese depsipeptides, the hy-
droxy acid of the natural compound was replaced with tartaric
acid and the second cycle was constructed on its additional
OH and CO2H function. Both modes of connection prevent
steric interaction between the two cycles, and H-bond
interactions are expected to be minimal because ester bonds
are not H-bond donors and are weak H-bond acceptors.[5] The
two Siamese depsipeptides proposed are shown in Scheme 1.
The cyclic depsipeptide sansalvamide A (SA), which is
produced by a marine fungus and shows cancer cell cytotox-
icity, was selected as a model. SA is composed of four amino
acids (2 ꢀ Leu, Phe, Val) and one hydroxy acid (leucic acid).[6]
More than 80 peptidic analogues, wherein the hydroxy acid
and the amino acid constituents were substituted for d-amino
acids and N-methyl amino acids with preserved or altered side
chains, are already reported. Some of them exhibit greater
activity and better selectivity than the natural SA.[7] However,
although SA is a relatively small and structurally simple
depsipeptide, an active sequence has not been disclosed to
date.
As part of one of our ongoing research programs devoted
to the discovery of active depsipeptides—modeled on natural
depsipeptides—a new concept of depsipeptide architecture
The synthesis of Siamese depsipeptides is challenging
because two cyclization steps are required and peptide
cyclizations are often low-yielding steps. Five-membered all-
[*] Dr. J. Ruiz-Rodrꢀguez, Dr. J. Spengler, Prof. F. Albericio
Institute for Research in Biomedicine, Barcelona Science Park (PCB)
Baldiri Reixac 10, 08028-Barcelona (Spain)
Fax: (+34)93-403-7126
E-mail: jan.spengler@irbbarcelona.org
fernando.albericio@irbbarcelona.org
Homepage: http://www.pcb.ub.es/fama/
Dr. J. Spengler, Prof. F. Albericio
CIBER-BBN, Networking Centre on Bioengineering, Biomaterials
and Nanomedicine, PCB
Baldiri Reixac 10, 08028-Barcelona (Spain)
Prof. F. Albericio
Department of Organic Chemistry, University of Barcelona
Martꢀ i Franquꢁs 1–11, 08028-Barcelona (Spain)
[**] We kindly acknowledge Dr. Miguel Moreno (PCB) and Andres M.
Francesc from PharmaMar, S.A.U., 28770-Colmenar Viejo, Madrid
(Spain) for the performance of the cell assays. We thank the
Barcelona Science Park for the facilities (Confocal Laboratory, and
Nuclear Magnetic Resonance Laboratory). This study was sup-
ported by CICYT (CTQ2006-03794/BQU, CTQ2008-00177), the
Generalitat de Catalunya (2005SGR 00662), the Barcelona Science
Park, and the Institute for Research in Biomedicine.
Supporting information (full experimental details, together with
copies of 1H NMR spectra of the depsipeptides 1, 2, 5, 6, and 7) for
this article is available on the WWW under http://dx.doi.org/10.
1002/anie.200904135.
Scheme 1. Replacement of the leucic acid in sansalvamide A (SA) with
tartaric acid provides Siamese depsipeptides 1 and 2.
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(S)-peptides (as is the case for SA, where all five residues
have S configuration) are more difficult to obtain than those
bearing at least one residue with R configuration.[8] Therefore,
we tested several cyclization strategies with d-tartaric acid
(S,S configuration) as well as with l-tartaric acid (R,R confi-
guration). Stepwise cyclizations resulted in low yields of
monocycles and were therefore discarded. Finally, the sym-
metrically, doubly branched acyclic depsipeptide 3, with l-
tartaric acid as core unit, was found to be a key for the
preparation of the Siamese depsipeptides in just one cycliza-
tion step. However, the reaction produced insufficient
product when d-tartaric acid was used as the core
(Scheme 2). The precursor 3 was prepared on 2-chlorotrityl
resin.[9] The amino acids Val and Leu were coupled on the
resin using standard protocols for Fmoc solid-phase peptide
synthesis. The tartaric acid was introduced as monoamide 4 by
activation as the HOBt ester. These reaction conditions
tolerate unprotected secondary hydroxy groups.[10] The mono-
esterification of the hydroxy groups of tartaric acid in the
solid phase was performed by coupling a 11-fold excess of
Fmoc-Leu-OH for six hours under the conditions developed
by Neises and Steglich.[11] Probably because of the steric
hindrance of the Fmoc group, the bisesterification proceeded
very slowly at this stage: extensive repetitions of esterification
(two times more for 15 h) only resulted in moderate diester
formation (50–60%). However, when the Fmoc group of the
Leu ester was removed and Boc-Phe-OH was coupled, the
bisesterification step (16 h) proceeded with a higher yield
(80%). After coupling the second Boc-Phe-OH unit (activa-
tion with PyAOP/DIEA, 30 min), precursor 3 was cleaved
from the resin and the protecting groups were completely
removed (TFA/CH2Cl2 (9:1), 1 h). The resulting product was
subjected to the double macrocyclization with HATU/DIEA
in solution (2 h) without prior purification. Two products, with
the expected mass for the Siamese depsipeptides, were
isolated separately by semipreparative HPLC methods in
3.4% (1) and 5.1% (2) overall yield and in 96% and 91%
purity by HPLC analysis, respectively.[12]
The proton signals of the tartaric acid residue and of the
1
amide group in the H NMR spectra of the two cyclization
products allowed assignation of the structures. One com-
pound showed two proton signals with the same integration
and coupling constant for tartaric acid (d = 5.66 ppm, J =
1.3 Hz, 1H; 5.70 ppm, J = 1.3 Hz, 1H), and at least seven
amide proton signals. We assigned this signal pattern to 1
À
because a rotation of the two cycles along the C C bond was
possible. The other compound showed a single proton signal
for tartaric acid (d = 5.82 ppm, 2H) and four amide protons.
We assigned this signal pattern to the compound with
structure 2 because of its C2-symmetry (see the NMR spectra
in the Supporting Information).
With respect to SA, the configuration of the hydroxy acid
residue in the Siamese depsipeptides was changed from S to
R. Therefore, the SA analogue 5 with (R)-leucic acid was
synthesized by an optimized procedure.[7a] The cycles within
the Siamese depsipeptide 2 contained an additional carbon
atom. The analogue 6 bearing a CH2-homologated (R)-leucic
acid, and 7, bearing a CH2-homologated (S)-leucic acid, were
prepared to evaluate the influence of ring size on activity. The
required b-hydroxy acids were introduced in the peptide
chain as dipeptide building blocks prepared from the
corresponding leucic acid by an Arndt–Eistert protocol
developed in our laboratory (Scheme 3).[13]
The inhibitory activity of the full set of depsipeptides SA,
1, 2, and 5–7 against three human cancer cell lines (MDA-
MB-231, A-549, HT-29) was tested by routine screening.
Activity was observed for 1, 2, and SA in low micromolar
range and was similar for all three cell lines, however, the
monocyclic analogues 5–7 were found to be inactive.
Remarkably, the Siamese depsipeptides showed significantly
higher activity than SA. In addition, we performed in-house
cell-growth inhibition assays with A-549 cells, and the trend of
activities was reproduced. The curves shown in Figure 1 are
averaged from three independent assays and correspond to 1
as the compound with greatest activity (IC50 = (1.1 Æ 0.8) mm),
followed by 2 (IC50 = (4.0 Æ 2.3) mm), and SA (IC50 = (8.3 Æ
1.5) mm). No significant activity at a concentration of 100 mm
was observed for the other monocycles 5–7.
Scheme 2. Synthesis of the Siamese depsipeptides 1 and 2 through
the symmetrically branched precursor 3 as the key intermediate.
Boc=tert-butoxycarbonyl, DIEA=N,N-diisopropylethylamine, DIPC-
DI=diisopropylcarbodiimide, DMAP=4-dimethylaminopyridine,
DMF=N,N-dimethylformamide, Fmoc=9-fluorenylmethyloxycarbonyl,
HATU=O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexa-
fluorophosphate, HOBt=1-hydroxybenzotriazole, PyAOP=(7-azaben-
zotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate,
TFA=trifluoroacetic acid.
These results show that neither the ring expansion (an
additional CH2 group in the ring at the position of leucic acid)
nor the inversion of the configuration at the chiral center of
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Communications
conformational constriction caused by the tartaric acid core,
or whether phenomena like statistical effects (the active part
is presented twice in one molecule) or multivalent binding are
involved.[15]
In summary, we have described the preparation of
Siamese depsipeptides, which are the first members of a
new class of depsipeptide architecture with tartaric acid as the
core unit. Remarkably, these compounds were synthesized in
reasonable yields from a branched precursor in just one
cyclization process. We have shown that the structural
manipulation of the natural bioactive depsipeptide SA gives
rise to analogues with greater activity, thereby providing
additional information on structure–activity relationships
with a small sets of compounds. This concept is currently
being applied to other bioactive peptides in our laboratory
and we are testing the effects of other core units.
Received: July 26, 2009
Revised: September 3, 2009
Published online: October 6, 2009
Scheme 3. Analogues of depsipeptide SA prepared for comparison of
biological activities.
Keywords: biological activity · bioorganic chemistry ·
depsipeptides · structure–activity relationships · tartaric acid
.
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~
&
*
Figure 1. Effect of Siamese depsipeptides 1 ( ), 2 ( ), and SA ( ) on
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