Solid-phase synthesis of oxathiocoraline by a key intermolecular disulfide dimer

Article abstract of DOI:10.1021/ja0686312

Oxathiocoraline, a member of the quinoxaline antibiotic family, has been synthesized on solid-phase. The depsipeptide exhibits high synthetic complexity owing to the presence of consecutive NMe amino acids, two ester moieties, a disulfide bridge, and two

Full text of DOI:10.1021/ja0686312

Published on Web 04/10/2007
Solid-Phase Synthesis of Oxathiocoraline by a Key Intermolecular Disulfide
Dimer
Judit Tulla-Puche,*,† Nu´ria Bayo´-Puxan,† Juan A. Moreno,† Andre´s M. Francesch,‡ Carmen Cuevas,‡
Mercedes AÄ lvarez,†,§ and Fernando Albericio*,†,|
Institute for Research in Biomedicine, Barcelona Science Park, 08028-Barcelona, Spain, PharmaMar, S.A.U.,
28770-Colmenar Viejo, Madrid, Spain, and Laboratory of Organic Chemistry, Faculty of Pharmacy, and
Department of Organic Chemistry, UniVersity of Barcelona, 08028-Barcelona, Spain
Received December 7, 2006; E-mail: jtulla@pcb.ub.es; albericio@pcb.ub.es
Triostin,1 Echinomycin,2 Thiocoraline,3 and BE-221794 (Figure
Scheme 1. Formation of the Key Intermolecular Disulfide Dimer
1) belong to a family of potent antitumoral bicyclic peptide
antibiotics, all of marine origin but proceeding from distinct
actinomycetes. These octadepsipeptides share a common general
structure with two key heterocyclic units which act as bisinter-
calators to DNA.5 The central disulfide or thioacetal bridge is
flanked by two ester or thioester moieties which give shape to the
symmetrical bicycle. The unusually high number of nonproteino-
genic amino acids on such a small structure (NMe and D-aa) adds
further difficulty to the synthesis of these depsipeptides. In the
search for new compounds with anticancer activity, the preparation
of new quinoxaline antibiotics was undertaken. Here we report the
first and unique solid-phase synthesis of a member of this family,
the oxathiocoraline 1,6 which exhibits high synthetic complexity
[NMe amino acids, ester bonds, Cys(Me)].7
Scheme 2. Synthesis of Oxathiocoralinea
Figure 1.
From a synthetic point of view, the ester moiety imposes several
additional drawbacks to the regular amide bond,8 the most important
being the risk of diketopiperazine (DKP) formation both at the resin
level and within the chain. Furthermore, cysteines are prone to
racemization when forming part of an ester. Moreover, NMe-Cys-
(Me) is prone to â-elimination to give didehydroalanine under basic
conditions and to be easily oxidized. Finally, the presence of both
a NMe-Cys(Me) residue and consecutive NMe amino acids calls
for a delicate balance in the coupling conditions; while NMe-Cys-
(Me) would require no basic conditions throughout the elongation
of the peptide, the coupling of NMe amino acids usually needs
harsh (highly basic) conditions.9 Initially, several attempts which
covered a broad number of conventional strategies (stepwise
synthesis, 4 + 4 fragment-coupling approach, choice of different
starting points) were examined. Because of the strong propensity
of the two consecutive NMe-Cys to form DKP and owing to the
a (i) Fmoc-Gly-OH, DIPCDI, DMAP, CH2Cl2-DMF (9:1); (ii) piperi-
dine-DMF (1:4); (iii) Fmoc-D-Ser(Trt)-OH, HATU, HOAt, DIEA, DMF;
(iv) pNZ-Cl, DIEA, DMF; (v) TFA-TIS-CH2Cl2 (2:2.5:95.5); (vi) Alloc-
NMe-Cys(Me)-OH, DIPCDI, DMAP, CH2Cl2-DMF; (vii) Pd(PPh3)4,
PhSiH3, CH2Cl2; (viii) Boc-NMe-Cys(Acm)-OH, HATU, HOAt, DIEA,
DMF; (ix) I2 in DMF; (x) TFA-H2O-CH2Cl2 (3:1:7); (xi) PyBOP, HOAt,
DIEA, DMF; (xii) Na2S2O4, ACN-EtOH-H2O; (xiii) 3-hydroxy-
quinaldic acid, EDC‚HCl, HOSu, CH2Cl2.
lability of the ester bond, the linear octadepsipeptide was never
obtained. We reasoned that the presence of the disulfide in an earlier
stage of the synthesis may restrict the flexibility of the chain, thereby
minimizing DKP formation (Scheme 1). With this in mind, efforts
were directed at obtaining a linear protected tetradepsipeptide of
good purity, to achieve the intermolecular disulfide dimer prior to
removal of the key Cys-protecting group.
For the construction of the tetradepsipeptide (Scheme 2), Gly
was chosen as the starting point since in this case the occurrence
of DKP formation is minimized along the chain. As a solid support,
Wang resin, was used.10
† Institute for Research in Biomedicine.
‡ PharmaMar, S.A.U.
§ Faculty of Pharmacy, University of Barcelona.
| Department of Organic Chemistry, University of Barcelona.
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10.1021/ja0686312 CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
the solid-phase formation of the disulfide dimer peptide, which
prevents the formation of internal DKPs. Other drawbacks have
been overcome by the use of a myriad of protecting groups such
as Fmoc, pNZ, Alloc, and Boc for N-protection, Trt for the OH,
and Acm for the SH, in combination with a number of coupling
reagents: DIPCDI/DMAP for the ester formation, HATU /HOAT/
DIEA for the sequential incorporation of the amino acids, PyBOP/
HOAt/DIEA for the bis lactamization, and EDC‚HCl/HOSu for the
final incorporation of the cromophore. We presume that this strategy
will be of general application for the preparation of other important
bicyclic depsipeptides.
Figure 2. HPLC of (a) tetradepsipeptide; (b) oxathiocoraline.
Acknowledgment. We thank Dr. Oscar Millet from the CIC
bioGUNE and Dr. Margarida Gair´ı from the Barcelona Scientific
Park (BSP) for NMR technical support. This study was partially
supported by CICYT (CTQ2006-03794/BQU, PETRI), the Gen-
eralitat de Catalunya (2005SGR 00662), and the BSP. J.T.P. is a
Beatriu de Pino´s fellow (Generalitat de Catalunya).
As we started the synthesis of the tetradepsipeptide, we became
aware that apart from the choice of the starting point, the choice
of protecting groups was also crucial. A range of different protecting
groups were required because of the fragility of the ester bond
during chain elongation:11 (a) Fmoc for the residues preceding the
ester functionality;11a (b) p-nitrobenzyloxycarbonyl (pNZ) in the
place of the heterocylic moiety;11b (c) trityl (Trt) for the protection
of the side chain of Ser;11c (d) allyloxycarbonyl (Alloc) for the
residue forming part of the ester;11d and (e) Boc for the N-terminal
Cys.11e Other choices of protecting groups gave the tetradepsipeptide
in poor yields. The optimal conditions for the synthesis of the
tetradepsipeptide were thus as follows: Fmoc-Gly-OH was loaded
onto Wang resin via symmetrical anhydride/DMAP; after removal
of the Fmoc group, Fmoc-D-Ser(Trt)-OH was introduced using
HATU/HOAt/DIEA in DMF. Next, the Fmoc group was removed
before construction of the ester bond, and the free amine was
reprotected with pNZ-Cl/DIEA. The pNZ group in this position
was compatible with the ester bond, and thus could be removed at
the tetradepsipeptide level on solid-phase or at a later stage in
solution.
Next, the trityl group was cleaved by treatment with TFA-TIS-
CH2Cl2 and elongation was continued through the side chain of
Ser. To make the ester bond, Alloc-NMe-Cys(Me)-OH was
introduced with DIPCDI/DMAP overnight.12 Next, the Alloc group
was removed under neutral conditions by using Pd(PPh3)4/PhSiH3
in CH2Cl2. The introduction of Boc-NMe-Cys(Acm)-OH was
accomplished with two treatments of HATU/ HOAt/DIEA.13 In
these conditions the tetradepsipeptide was obtained in 89% purity14
(Figure 2a).
At this point the key intermolecular disulfide dimer was formed
using I2 in DMF.15 Finally, to avoid sulfoxide formation, cleavage
of the dimer from the resin was performed in the presence of H2O
with two treatments of TFA-H2O-CH2Cl2 (3:1:6), with concomi-
tant cleavage of the Boc group. After evaporation of the solvent,
the dimer was lyophilized. Next, the crude dimer was dissolved in
CH2Cl2-DMF (19:1) and preactivated with HOAt. After adjusting
the pH to slightly basic conditions with DIEA, solid PyBOP was
added to obtain, after 6 h, the pNZ-protected bicycle, with no signs
of internal DKP formation.16 The pNZ group was removed with
Na2S2O4 in ACN-EtOH-H2O (4:1:1), and the 3-hydroxyquinaldic
acid17 was introduced with the rather mild coupling method, EDC‚
HCl and HOSu in CH2Cl2, to avoid over incorporation of carboxylic
acid. The crude oxathiocoraline was obtained in 7% overall yield,
after purification by semipreparative HPLC (Figure 2b).
In vitro activity of oxathiocoraline was evaluated in three tumor
cell lines (MDA-MB-231, A549, HT29) and showed growth
inhibitory effect, similar to other depsipeptides presently in clinical
trials (see SI).
Supporting Information Available: Experimental procedures,
characterization material, and abbreviations. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
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Gravalos, D.; De la Calle, F.; Ferna´ndez-Puentes, J. L. J. Antibiot. 1997,
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(6) Oxathiocoraline is more polar and stable than its parent thiocoraline and
therefore it could show improved pharmacokinetic properties.
(7) Solution syntheses for triostin and thiocoraline have been reported: (a)
Chakravarty, P. K.; Olsen, R. K. Tetrahedron Lett. 1978, 19, 1613-1616.
(b) Shin, M.; Inouye, K.; Otsuka, H. Bull. Chem. Soc. Japan 1984, 57,
2203-2210. (c) Boger, D. L.; Ichikawa, S. J. Am. Chem. Soc. 2000, 122,
2956-2957. Boger, D. L.; Ichikawa, S.; Tse, W. C.; Hedrick, M. P.; Jin,
Q. J. Am. Chem. Soc. 2001, 123, 561-568.
(8) Syntheses of the less complex azaderivatives have been reported. (a)
Azathiocoraline by solid-phase methodology: Bayo´-Puxan, N.; Ferna´ndez,
A.; Tulla-Puche, J.; Riego, E.; Cuevas, C.; AÄ lvarez, M.; Albericio, F.
Chem.sEur. J. 2006, 12, 9001-9009. (b) Azatriostin and des-N-
(tetramethyl)azatriostin by solution strategies: Boger, D. L.; Lee, J. K. J.
Org. Chem. 2000, 65, 5996-6000. Dietrich, B.; Diederichsen, U. Eur. J.
Org. Chem. 2005, 147-153.
(9) A solution and a solid-phase synthesis of triostin analogues lacking the
NMe amino acids have been reported: (a) Lorenz, K. B.; Diederichsen,
U. J. Org. Chem. 2004, 69, 3917-3927. (b) Malkinson, J. P.; Anim, M.
K.; Zloh, M.; Searcey, M. J. Org. Chem. 2005, 70, 7654-7661.
(10) Wang resin could be used in this case since the incorporation of the third
residue was through the side chain of Ser, which avoided the formation
of diketopiperazines at the resin level.
(11) The use of alternative protection was accompanied by dramatic side
reactions. For instance (a) cleavage of the ester bond was observed with
the use of piperidine. (b) The pNZ in this position was compatible with
the ester bond. In other positions of the chain (third and fourth residue),
the removal conditions (6 M SnCl2, 1.6 mM HCl) effected cleavage of
the ester functionality. (c) TBDMS could also be used at this point but
the amino acid was not commercial. (d) Both the use of Fmoc and pNZ
groups resulted in cleavage of the ester bond. (e) The use of other acid-
labile groups (such as Trt) were also investigated but abandoned because
of the difficulty in obtaining the protected NMe amino acid.
(12) A certain degree of racemization (2%) was observed; shorter times or
reagents that minimize racemization (e.g., MSNT) gave lower conversions
or lower purity products (MSNT requires large excess of base).
(13) Less equivalents of base were used to avoid side reactions.
(14) Alternatively, the heterocycle moiety can be introduced on solid-phase
with DIPCDI/HOAt in DMF (see SI).
(15) This intermolecular disulfide dimer formation was showed to be also
possible on another kind of resins, showing the robustness of this strategy.
(16) To exclude a 13-membered ring cyclization in this strategy, reduction of
the disulfide bridge has been carried out showing the mass corresponding
to the reduced peptide and not to the half structure (see SI).
(17) Riego, E.; Bayo´, N.; Cuevas, C.; Albericio, F.; AÄ lvarez, M. Tetrahedron
2005, 61, 1407-1411.
In conclusion, here we describe an optimal synthetic strategy
for the solid-phase preparation of oxathiocoraline. The key step is
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