Process for the preparation of cyclo(Asp-DPhe-NMeVal-Arg-Gly)

Details Process for the preparation of cyclo(Asp-DPhe-NMeVal-Arg-Gly) Process for the preparation of cyclo(Asp-DPhe-NMeVal-Arg-Gly)

2001-09-12

2004-11-23

Patten, Patricia A (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

4 to 5 amino acid residues in defined sequence

C514S009100, C514S183000, C530S333000, C530S335000, C530S338000, C530S339000

Reexamination Certificate

active

06822074

ABSTRACT:

This application is a 371 of PCT/EP/00/01751 filed Jan. 3, 2000 which claims benefit of priority to Application number 19910727.0 filed in Germany on Mar. 11, 1999.
The invention relates to a novel process for the preparation of the cyclic pentapeptide cyclo(Arg-Gly-Asp-DPhe-NMeVal).
Cyclic pentapeptides, amongst them also cyclo(Arg-Gly-Asp-DPhe-NMeVal) and its physiologically acceptable salts, are disclosed in EP 0 770 622. The present invention is to be regarded as a selection invention in relation to EP 0 770 622.
Generally, cyclic peptides are obtained by cyclization of a linear precursor molecule under the customary conditions of peptide synthesis. In order that selective linkage of two amino acids or two segments, consisting of a number of amino acids, or alternatively a cyclization of a linear peptide, can be guaranteed, the corresponding functionalities of the amino acids which are not intended to participate in the reaction are to be blocked by appropriate protective groups. Various types of protective groups for amino, carboxyl, hydroxyl, thiol or carboxamide functions, and also for guanidine functions or for the imidazole nitrogen, were therefore developed which, in their combination, make possible a wide possibility of variation with respect to the optimization of the reactions mentioned beforehand. The synthesis of the linear precursor molecules, the linear peptides, can moreover be carried out by means of two methods, on the one hand by means of a solid-phase peptide synthesis, on the other hand in solution. In this case, stepwise couplings of the amino acids or fragment condensations of segments of amino acids are possible. The respective coupling steps can in turn be carried out using different condensation reagents, such as carbodiimides, carbodiimidazole, those of the uronium type such as TBTU, or according to mixed anhydride methods or active ester methods.
The invention was based on the object of developing a novel, improved process for the preparation of cyclo(Arg-Gly-Asp-DPhe-NMeVal), in comparison with the previously known processes.
It was surprisingly found that in the synthesis of the cyclopeptide cyclo(Arg-Gly-Asp-DPhe-NMeVal) by cyclization of a linear precursor molecule, the combination of the protective groups-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for the guanidino group in the side chain of the arginine and benzyl (Bzl) for the carboxyl group in the side chain of the aspartic acid leads to an optimization with respect to the yield.
The invention therefore relates to a process for the preparation of the cyclic pentapeptide
cyclo(Arg-Gly-Asp-DPhe-NMeVal)
by cyclization of a linear pentapeptide selected from the group consisting of
H-Arg(Pbf)-Gly-Asp(OBzl)-DPhe-NMeVal-OH,
H-Gly-Asp(OBzl)-DPhe-NMeVal-Arg(Pbf)-OH,
H-Asp(OBzl)-DPhe-NMeVal-Arg(Pbf)-Gly-OH,
H-DPhe-NMeVal-Arg(Pbf)-Gly-Asp(OBzl)-OH or
H-NMeVal-Arg(Pbf)-Gly-Asp(OBzl)-DPhe-OH,
subsequent protective group removal and, if appropriate, further conversion into its physiologically acceptable salts.
The invention furthermore relates to a process for the preparation of the cyclic pentapeptide
cyclo(Arg-Gly-Asp-DPhe-NMeVal)
as described, characterized in that the linear pentapeptide H-Asp(OBzl)-DPhe-NMeVal-Arg(Pbf)-Gly-OH is cyclized.
The reaction conditions of this cyclization of the linear peptides selected from the group consisting of
H-Arg(Pbf)-Gly-Asp(OBzl)-DPhe-NMeVal-OH,
H-Gly-Asp(OBzl)-DPhe-NMeVal-Arg(Pbf)-OH,
H-Asp(OBzl)-DPhe-NMeVal-Arg(Pbf)-Gly-OH,
H-DPhe-NMeVal-Arg(Pbf)-Gly-Asp(OBzl)-OH or
H-NMeVal-Arg(Pbf)-Gly-Asp(OBzl)-DPhe-OH
with respect to the choice of the dehydrating agent, the inert solvent and the reaction temperature and the further conversion into its physiologically acceptable salts have already been disclosed in EP 0 770 622.
The benzyl protective group on the side chain of the aspartic acid can be removed under customary conditions (for this cf.: T. W. Greene, P. G. M. Wuts, Protective Groups in Organic Chemistry, 2
nd
Ed., Wiley, New York 1991 or P. J. Kocienski, Protecting Groups, 1
st
Ed., George Thieme Verlag, Stuttgart—New York, 1994, H. Kunz, H. Waldmann in Comprehensive Organic Synthesis, Vol. 6 (Ed. B. M. Trost, I. Fleming, E. Winterfeldt), Pergamon, Oxford. 1991, pp. 631-701), e.g. by treating with hydrogen in the presence of a catalyst (e.g. of a noble metal catalyst such as palladium, expediently on a support such as carbon) Suitable solvents are, for example, alcohols such as methanol or ethanol or amides such as DMF or alternatively mixtures with further inert solvents, such as, for example, mixtures with water. As a rule, the hydrogenolysis is carried out at temperatures between approximately 0 and 100° C. and pressures between approximately 1 and 200 bar, preferably at 20-30° C. and 1-10 bar.
The Pbf protective group, which was introduced into peptide chemistry by L. A. Carpino et al., Tet. Lett. 1993, 34, 7829-7832, is removed, for example, by treating with 95% trifluoroacetic acid (TFA). The Pbf protective group in this case shows a greater lability with respect to TFA than the structurally similar protective groups 4-methoxy-2,3,6-trimethylphenylsulfonyl (Mtr) and 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), which are also possible as side-chain protective groups with respect to the synthesis of cyclo(Arg-Gly-Asp-DPhe-NMeVal). TEA is preferably used in an excess without addition of a further solvent. TFA can also be employed as a mixture with an inert solvent, such as, for example, the combination TFA/dichloromethane in the ratio 6:4. TFA can furthermore also be employed with an addition of 1-10%, preferably 2%, of water. The reaction temperature for the cleavage is expediently between approximately 0 and approximately 50° C., and the reaction is preferably carried out between 15 and 30° C. (room temperature).
The abbreviations of amino acids listed above and below stand for the radicals of the following amino acids:
Asp
Aspartic acid
Arg
Arginine
Gly
Glycine
Phe
Phenylalanine
Val
Valine
Furthermore, above and below the following have the meanings:
Boc
tert-Butoxycarbonyl
Bzl
Benzyl
CHA
Cyclohexylamine
D
Characterization of a D-amino acid
DCCI
Dicyclohexylcarbodiimide
DMAP
Dimethylaminopyridine
DMF
Dimethylformamide
EDCI
N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide
hydrochloride
Et
Ethyl
Fmoc
9-Fluorenylmethoxycarbonyl
HOBt
1-Hydroxybenzotriazole
Me
Methyl
MTBE
Methyl tert-buty ether
Mtr
4-Methoxy-2,3,6-trimethylphenylsulfonyl
NMe
N-methylated &agr;-amino group
NMP
N-methylpyrrolidone
OtBu
tert-Butyl ester
OMe
Methyl ester
OEt
Ethyl ester
Pbf
2,2,4,6,7-Pentamethyldihydrobenzofuran-5-
sulfonyl
Pmc
2,2,5,7,8-pentamethylchroman-6-sulfonyl
POA
Phenoxyacetyl
Pr
Propyl
Su
Succinimide
TBTU
2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyl-
uronium tetrafluoroborate
TFA
Trifluoroacetic acid
Z
Benzyloxycarbonyl
It was furthermore surprisingly found that the specific selection of the side-chain protective groups Pbf on Arg and Bzl on Asp even in the synthesis of the linear peptides, which, as mentioned beforehand, are intermediates in the synthesis of cyclo(Arg-Gly-Asp-DPhe-NMeVal), leads to improved yields in the respective synthesis stages. As a result, the yield of cyclo(Arg-Gly-Asp-DPhe-NMeVal) is increased overall and as a result thereof the costs of the synthesis are lowered.
The improvement in yield is achieved in this case both in the solid-phase peptide synthesis and in the synthesis in solution of the linear peptides
H-Arg(Pbf)-Gly-Asp-(OBzl)-DPhe-NMeVal-OH,
H-Gly-Asp(OBzl)-DPhe-NMeVal-Arg(Pbf)-OH,
H-Asp(OBzl)-DPhe-NMeVal-Arg(Pbf)-Gly-OH,
H-DPhe-NMeVal-Arg(Pbf)-Gly-Asp(OBzl)-OH or
H-NMeVal-Arg(Pbf)-Gly-Asp(OBzl)-DPhe-OH, in particular H-Asp(OBzl)-DPhe-NMeVal-Arg(Pbf)-Gly-OH.
The protected amino acids or amino acid fragments used in the two synthesis methods are customarily prepared by methods of amino acid and peptide synthesis, such as described in the standard works Principles of Peptide Synthesis, ed. M. Bodansky, Springer Verlag Berlin 1984; Houben Weyl, Methoden der organischen Chemie [Methods of Organic Chemistry],

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