Tentoxin as a scaffold for drug discovery. Total solid-phase synthesis of tentoxin and a library of analogues

Article abstract of DOI:10.1021/ol0345273

(Matrix presented) A solid-phase method for the synthesis of tentoxin has been developed. Two key steps - dehydration and N-alkylation - are carried out while the peptide is anchored to the resin. The method, which has been validated by the preparation of

Full text of DOI:10.1021/ol0345273

ORGANIC
LETTERS
2003
Vol. 5, No. 12
2115-2118
Tentoxin as a Scaffold for Drug
Discovery. Total Solid-Phase Synthesis
of Tentoxin and a Library of Analogues
Jose C. Jime´nez,§,‡ Bibiana Chavarr´ıa,‡ AÅ ngel Lo´pez-Macia`,‡,| Miriam Royo,‡,
,§,‡
Ernest Giralt,§,‡ and Fernando Albericio*
Barcelona Biomedical Research Institute and Combinatorial Chemistry Unity,
Barcelona Science Park, UniVersity of Barcelona, Josep Samitier 1,
08028-Barcelona, Spain, Department of Organic Chemistry,
UniVersity of Barcelona, Mart´ı i Franque´s 1, 08028-Barcelona, Spain,
and PharmaMar, AVda Reyes Cato´licos 1, 28770-Colmenar Viejo, Madrid, Spain
albericio@pcb.ub.es
Received March 26, 2003
ABSTRACT
A solid-phase method for the synthesis of tentoxin has been developed. Two key stepssdehydration and N-alkylationsare carried out while
the peptide is anchored to the resin. The method, which has been validated by the preparation of a library of tentoxin analogues, should be
applicable to the generation of further libraries that have the tentoxin scaffold structure, as well as other structures containing N-alkylated
didehydroamino acids.
The recent developments in genomics, proteomics, and
molecular biology are providing a large number of new
targets associated with the most important diseases.1 Al-
though computational molecular modeling methods have
evolved enormously in the past few years, rational drug
design methods based on these are far from being routinely
implemented.2 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 interac-
tions with the corresponding receptor.3 Scaffolds or templates
can be prepared through de novo design. Furthermore, an
alternative and attractive strategy involves taking advantage
of the rich source of structural diversity and biological
activity shown by natural products as a result of evolution
and natural selection.4,5 In this sense, a number of natural
products can be considered as priVileged structuressas
defined by Evans and co-workers6sbecause their function
may be related to binding to more than one protein, different
from that involved in their biosynthesis.7 Natural products
with contrasting biological activities could therefore be
considered as unique scaffolds for the discovery of new drugs
aimed at targets that differ from their primary activity. The
(4) Henkel, T.; Brunne, R. M.; Mu¨ller, H.; Reichel, F. Angew. Chem.,
Int. Ed. 1999, 38, 643-647.
(5) Wrigley, S. K.; Hayes, M. A.; Thomas, R.; Chrystal, E. J. T.;
Nicholson, N., Eds. BiodiVersity. New Leads for the Pharmaceutical
and Agrochemical Industry; Royal Society of Chemistry: Cambridge, UK,
2000.
§ Barcelona Biomedical Research Institute
‡ Department of Organic Chemistry
| PharmaMar
Combinatorial Chemistry Unity
(6) Evans, B. E.; Rittle, K. E.; Bock, M. G.; DiPardo, R. M.; Freidinger,
R. M.; Whitter, W. L.; Lundell, G. F.; Veber, D. F.; Anderson, P. S.; Chang,
R. S. L.; Lotti, V. J.; Cerino, D. J.; Chen, T. B.; Kling, P. J.; Kunkel, K.
A.; Springer, J. P.; Hirshfield, J. J. Med. Chem. 1988, 31, 2235-2246.
(7) Breinbauer, R.; Vetter, I. R.; Waldmann, H. Angew. Chem., Int. Ed.
2002, 41, 2878-2890.
(1) Hruby, V. J. J. Pept. Res. 2002, 60, 305-306.
(2) Shimada, J.; Ekins, S.; Elkin, C.; Shakhnovich, E. I.; Wery, J.-P.
Targets 2002, 1, 196-205.
(3) Tan, D. S.; Foley, M. A.; Stockwell, B. R.; Shair, M. D.; Schreiber,
S. L. J. Am. Chem. Soc. 1999, 121, 9073-9087.
10.1021/ol0345273 CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/09/2003

drug discovery process can be facilitated further by modify-
ing these scaffolds by exploiting combinatorial techniques.8
Tentoxin (1), which is a phytotoxic metabolite isolated
from the pathogenic fungus Alternaria tenuis Ness,9,10 can
be assessed as a scaffold because it fits all of the requirements
described above. For example, tentoxin induces chlorosis in
many dicotyledone plants, except cereals, tomatoes, and
members of the species cruciferae and graminae,11 thus
making it a potential selective herbicide.12 Furthermore, it
has been demonstrated that tentoxin has at least two sites
that are capable of forming strong interactions with
substrates.13-16 The first site, which shows great affinity, is
the cause of the chlorosis effect whereas the second, which
has a lower affinity constant, is related to an effect involving
growth stimulation.
Tentoxin exhibits structural features not commonly found
in natural peptides: (i) the presence of a didehydroamino
acid (DDAA), (ii) the strained 12-membered-ring system,
and (iii) the fact that two of the four residues are methylated,
including the nonproteinogenic didehydroamino acid. These
factors all contribute to the challenge for this system as a
synthetic target.
To adopt the tentoxin structure as a scaffold in a combi-
natorial program it is necessary to have a rapid and efficient
method for its synthesis, preferably with the potential for
automization. Although the preparation of compound libraries
may be conducted in solution or in solid phase, the latter
method is often preferred for reasons highlighted by the
syntheses of biomolecules such as peptides25 and oligonucle-
otides.26 The first objective of the present work was there-
fore to develop a solid-phase method for the synthesis of
tentoxin. Syntheses described in the literature are mainly
carried out in solution,10 with the exception of the example
reported by Rich and Mathiaparanam, in which the linear
tetrapeptide containing a precursor of the DDAA was
prepared in the solid phase before the dehydration and
methylation stages.27
On the other hand, with the exception of diketopiperazines,
cyclic tetrapeptides are the smallest cyclic peptides that can
be synthesized.17 These compounds have a molecular weight
below 500, which makes them suitable for modification and
to fulfill the Lipinski18 and Tice19 rules. Finally, NMR studies
have shown that the tentoxin cycle is rather rigid.20,21
The key steps of the strategy presented here are (i) the
formation of the DDAA, (ii) the N-methylation of the DDAA,
(iii) the choice of the linear precursor, and (iv) cyclization.
(i) The synthesis of didehydropeptides (DDP) such as
tentoxin by the introduction of the protected NR-DDAA
cannot be carried out. This is because the weak nucleophi-
licity of the corresponding enamine function prevents an
efficient elongation in the C to N direction when the NR-
protecting group is removed.28,29 The synthesis of DDP can
only be accomplished by forming the double bond once the
peptide sequence has been assembled or by incorporation
of a shorter peptide, preferably a dipeptide, containing the
DDAA.30 However, in this case the second strategy suffers
from several drawbacks. For example, it would involve
carrying out the dehydration step in solution, which is not
appropiate in a combinatorial scheme if different analogues
are required. Furthermore, selective protection of the carboxyl
group of the â-hydroxyphenylalanine [phenylserine, Phe(â-
OH)] in the presence of amino and hydroxyl groups must
be carried out.
Tentoxin also contains motifs of other therapeutically
interesting peptides, including the presence of N-methyl
residues and the didehydroamino group present in cyclospo-
rine,22 thiocoraline,23 or kahalalide F.24
(8) Nicolaou, K. C.; Hanko, R.; Hartwig, W., Eds. Handbook of
Combinatorial Chemistry; Wiley-VCH: Weinheim, Germany, 2002; Vols.
1 and 2.
(9) Fulton, N. D.; Bollenbacher, K.; Templeton, G. E. Phytopathology
1965, 55, 49-51.
(10) Cavelier, F.; Verducci, J.; Andre, F.; Haraux, F.; Sigalat, C.; Traris,
M.; Vey, A. Pestic. Sci. 1998, 52, 81-89 and references therein.
(11) Durbin, R. D.; Uchytil, T. F. Phytopathology 1977, 67, 602-603.
(12) Lax, A. R.; Shepherd, H. S.; Edwards, J. V. Weed Technol. 1988,
2, 540-544.
(13) Steele, J. A.; Uchytil, T. F.; Durbin, R. D.; Bhatnagar, P.; Rich, D.
H. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 2245-2248.
(14) Hu, N.; Mills, D. A.; Huchzermeyer, B.; Richter, M. L. J. Biol.
Chem. 1993, 268, 8536-8540.
In the present work the DDP was prepared in the solid
phase by a method developed in our laboratory.31 The route
(15) Dahse, I.; Pezennec, S.; Girault, G.; Berger, G.; Andre, F.;
Liebermann, B. J. Plant Physiol. 1994, 143, 615-620.
(16) Sigalat, C.; Pitard, B.; Haraux, F. FEBS Lett. 1995, 368, 253-
256.
(17) In the literature, only the synthesis of a cyclotripeptide containing
concatenated R-amino acids through the R-function [c(D-Phe-L-Pro-L-Pro),
which contains two Pro and a D residue that favors the cyclization] has
been described. Rothe, M.; Faehnle, M.; Maestle, W. In Peptides: Synthesis,
Structure, Function, Proceedings of the American Peptide Symposium, 7th;
Rich, D. H., Gross, E. Eds.; Pierce Chem. Co.: Rockford, IL, 1981; pp
89-92.
(18) Lipinski, C. A.; Fiese, E. F.; Korst, R. J. Quant. Struct.-Act. Relat.
1991, 10, 109-117.
(19) Tice, C. M. Pest Manag. Sci. 2001, 57, 3-16.
(20) Rich, D. H.; Bhatnagar, P. K. J. Am. Chem. Soc. 1978, 100, 2212-
2218.
(21) Pinet, E.; Neumann, J.-M.; Dahse, I.; Girault, G.; Andre, F.
Biopolymers 1995, 36, 135-152.
(22) Borel, J. F.; Kis, Z. L.; Beveridge, T. Search Anti-Inflammatory
Drugs 1995, 27-63.
(23) Romero, F.; Espliego, F.; Perez Baz, J.; Garcia de Quesada, T.;
Gravalos, D.; De la Calle, F.; Fernandez-Puentes, J. L. J. Antibiot. 1997,
50, 734-737.
(24) Hamann, M. T.; Scheuer, P. J. J. Am. Chem. Soc. 1993, 115, 5825-
5826.
(25) Lloyd-Williams, P.; Albericio, F.; Giralt, E. In Chemical Approaches
to the Synthesis of Peptides and Proteins; CRC Press: Boca Raton, FL,
1997.
(26) Bellon, L.; Wincott, F. Oligonucleotide Synthesis. In Solid-Phase
Synthesis. A Practical Guide; Kates, S. A., Albericio, F., Eds.; Marcel
Dekker: New York, 2000; pp 475-528.
(27) Rich, D. H.; Mathiaparanam, P. Tetrahedron Lett. 1974, 15, 4037-
4040.
(28) Moriya, T.; Yoneda, N.; Miyoshi, M.; Matsumoto, K. J. Org. Chem.
1982, 47, 94-98.
(29) Shin, C. G.; Yonezawa, Y.; Yamada, T. Chem. Pharm. Bull. 1984,
32, 3934-3944.
(30) Bayo´, N.; Jime´nez, J. C.; Rivas, L.; Nicola´s, E.; Albericio, F. Chem.,
Eur. J. 2003, 9, 1096-1103.
2116
Org. Lett., Vol. 5, No. 12, 2003

Table 1. Linear Peptides Enclosed in the Tentoxin Sequence
risk of
difficulty of
cyclizationa
DKP
sequence
epimerizationa
formationb (%)
I
II
H-L-MeAla-L-Leu-L/D-Phe(âOH)-Gly-O-Resin
H-Gly-L-MeAla-L-Leu-L/D-Phe(âOH)-O-Resin
H-L-Leu-L/D- Phe(âOH)-Gly-L-MeAla-O-Resin
H-L/D-Phe(âOH)-Gly-L-MeAla-L-Leu-O-Resin
none
none
high
-
moderate
high
34
15
III
low
48
IV
very high
not evaluated
a Assumed. b Calculated by amino acid analysis (AAA) of the peptide resin before and after the incorporation of the third residue.
involves the preparation of the peptide with the precursor
of the didehydro residue, in this case [L/D-Phe(â-OH)],
activation of the hydroxy group with 3-(3′-dimethylamino-
propyl)-1-ethylcarbodiimide (EDC, WSC) in the presence
of CuCl,32 and concomitant elimination. Although the linear
sequence was obtained with the diastereomeric mixture of
Phe(â-OH), this method gave exclusively the Z isomer, which
is the thermodynamically more stable product.
Scheme 1. Solid-Phase Strategy Developed for the
Preparation of Tentoxina
(ii) The N-methylation of the didehydrophenylalanine
residue was also carried out on the solid phase with MeI as
the alkylating agent and K2CO3 as the base in the presence
of 18-crown-6 as a phase transfer reagent in DMF. These
conditions led to regioselective methylation of the didehydro
residue due to the greater acidity of the corresponding amide
bond. The absence of 18-crown-6 or the use of 1,8-
diazobicyclo[5.4.0]undec-7-ene (DBU) as base instead of K2-
CO3 did not give the N-methylated derivative in any case.
As expected, the Fmoc group of the N-terminal residue was
unstable under these conditions and therefore these residues
have to be introduced while protected with the Boc group.
(iii) In this case, the choice is limited for several reasons.
As mentioned above, the DDAA or its precursor cannot be
situated at the N-terminal position (Sequence IV, Table 1).
On the other hand, tentoxin contains an additional N-
methylated residue (Ala) and a glycine, which the Ala residue
can epimerizase if it is at the C-terminal position during the
cyclization25 and both the Ala and glycine are prone to induce
diketopiperazine (DKP) formation if they are at the first two
positions with respect to the C-terminal.33 Synthesis of the
different possible peptides was carried out and DKP forma-
tion was evaluated. Table 1 shows the results and highlights
problematic sequences along with the corresponding DKP
formation data. Sequence I contains an N-MeAla as N-
terminal and this would be expected to make the cyclization
step more difficult. The Gly at the C-terminal should favor
both cyclization and DKP formation. Sequence II contains
a Gly at the N-terminal that should favor cyclization.
Furthemore, the didehydro residue could be advantageous
because epimerization is not applicable in this case, although
the carboxylic groups of these residues have poor reactivity.34
Sequence III should suffer from a high risk of racemization
because N-MeAla is at the C-terminal, but will have a high
probability of cyclization because the Leu at the N-terminal
(31) Royo, M.; Jime´nez, J.; Lo´pez-Macia`, AÅ .; Giralt, E.; Albericio, F.
Eur. J. Org. Chem. 2001, 45-48.
a Reagents and conditions: (a) Fmoc-Gly-OH, DIPCDI/DMAP,
DCM; (b) SPPS; (b′) piperidine/DMF (2:8); (b′′) Fmoc/Boc-aa-
OH, DIPCDI/HOBt, DMF; (c) EDC‚HCl, CuCl, DCM/DMF (9:
1); (d) MeI, K2CO3, 18-crown-6, DMF; (e′) ) (e′′) TFA/H2O (19:
1); (f) DIPCDI/HOBt/DIEA, DCM/DMF (99:1).
(32) Fukase, K.; Kitazawa, M.; Sano, A.; Shimbo, K.; Horimoto, S.;
Fujita, H.; Kubo, A.; Wakamiya, T.; Shiba, T. Bull. Chem. Soc. Jpn. 1992,
65, 2227-2240.
(33) Alsina, J.; Giralt, E.; Albericio, F. Tetrahedron Lett. 1996, 37, 4195-
4198 and references therein.
(34) Lo´pez-Macia`, AÅ .; Jime´nez, J. C.; Royo, M.; Giralt, E.; Albericio,
F. J. Am. Chem. Soc. 2001, 123, 11398-11401.
Org. Lett., Vol. 5, No. 12, 2003
2117

Tentoxin was obtained with a nonoptimized overall yield
of 25% calculated from the starting Wang resin [60% for
steps a-e′′ (Scheme 1) and 41% for cyclization and
purification steps].
Table 2. Tentoxin Analogues Synthesized by Using the
Approach Outlined in Scheme 137
The compounds shown in Table 2 were synthesized by
following a similar strategy. The preparation of compounds
3 and 4 was carried out with 1,2-L-diaminopropionic acid
(instead of Leu) bearing Fmoc and Alloc groups for the
protection of the R and â amino groups, respectively. The
Alloc group was removed after the alkylation step by a
process involving an allyl transfer to PhSiH3 in the presence
of Pd(PPh3)4, followed by acylation of the â-amino function
by the DIPCDI/HOBt method.36,37
In conclusion, the synthetic method reported here can be
applied to generate libraries of compounds that have tentoxin
as the scaffold structure, as well as other structures containing
N-alkylated didehydroamino acids. All reactions except for
the final cyclization take place on the solid phase.
Acknowledgment. M.R. is a Ramon y Cajal fellow
(MCyT, Spain). This work was partially supported by CICYT
(BQU2000-0235, BQU2002-02047, and BIO2002-02301),
Generalitat de Catalunya [Grup Consolidat, and Centre de
Refere`ncia en Biotecnologia].
is not a hindered residue. Finally, it was demonstrated that
the risk of DKP formation was high because the two
C-terminal residues were prone to promote this side reaction.
Although sequence II initially appeared to be a promising
candidate, it was ruled out because the dehydration step
carried out on the solid phase did not give the target
compound due to the formation of several byproducts and
early cleavage from the resin.35
Supporting Information Available: Experimental pro-
cedures for the synthesis of tentoxin. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL0345273
(36) Although tentoxin and its analogues were obtained after cyclization
with excellent purity, similar to that shown by tentoxin (HPLC profile X,
Scheme 1), further purification was carried out in all cases by reversed-
phase HPLC to give compounds with a purity greater than 95%.
(37) For analogues 2 and 3, Alloc removal was performed with Pd(PPh3)4
(0.1 equiv) and PhSiH3 (10 equiv) in anhydrous DCM (3 × 15 min) under
an Ar atmosphere.
Tentoxin was ultimately synthesized by using sequence I
(Scheme 1).
(35) Jime´nez, J. C.; Bayo´, N.; Chavarr´ıa, B.; Lo´pez-Macia`, AÅ .; Royo,
M.; Nicola´s, E.; Giralt, E.; Albericio, F. Lett. Pept. Sci. In press.
2118
Org. Lett., Vol. 5, No. 12, 2003