Liquid phase process for the preparation of GNRH peptides

Details Liquid phase process for the preparation of GNRH peptides Liquid phase process for the preparation of GNRH peptides

1999-07-08

2001-05-22

Moezie, F. T. (Department: 1654)

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

C530S331000, C530S328000, C530S329000

Reexamination Certificate

active

06235876

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to liquid phase processes for the preparation of biologically active peptides. More particularly, it relates to the liquid phase synthesis of peptides having activity as GNRH antagonists which inhibit gonadal function and the release of the steroidal hormones progesterone and testosterone.
BACKGROUND OF THE INVENTION
GnRH is a hypothalmic hormone which triggers the release of the gonadotropic hormones, particularly leutinizing hormone. These hormones regulate the functioning of the gonads to produce testosterone in the testes and estrogen in the ovaries; and they also regulate the production and maturation of gametes. Mammalian GnRH is a well known decapeptide as described in U.S. Pat. No. 4,072,668.
GnRH peptide analogs which are antagonistic to endogeneous GnRH and inhibit the secretion of gonadotropins and the release of steroids by the gonads are known from U.S. Pat. Nos. 5,506,207 and 5,169,932. These compounds are analogs of the decapeptide GnRH which include modified amino acids in the 5 and 6 position. Particularly useful are the peptide analogs which have the general formula:
Ac-&bgr;-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-AA
5
-D-AA
6
-Leu-Lys(isopropyl)-Pro-D-Ala-NH
2
(SEQ ID No:1)
wherein AA
5
and AA
6
are residues of amino-substituted phenylalanine (or its equivalent) where the substituent is preferably in the 4-position and is preferably an acylating agent of 5 carbon atoms or less or one which contains an unsaturated heterocyclic ring containing from 2 to 4 nitrogen atoms. Particularly useful is the GnRH peptide analog known as Azaline B, which contains an “Aph(Atz)” substituent, that is, a 5 membered triazole ring (specifically, a 1,2,4-triazole) as the substituent in the 4 position of the amino substituted phenylalanine of both AA
5
and AA
6
. Also useful are the pepuides known as Azaline C, which has a methyl group on the backbone nitrogen of residue 5, and Acyline, which has an acetamido group as the substituent in the 4 position of the substituted phenylalanine. Further useful GnRH peptide analogues are reported by Rivier et al, J.Med.Chem, 1995, 38, 2649-2662, wherein the amino substituent is acylated with a simple amino acid residue, e.g. Gly, Ser, Ala, etc, followed by deprotection and triazole construction.
Previously, as described in the patents mentioned above, these GnRH analogs have generally been prepared by a classical solid phase peptide synthesis (SPPS) in which the peptide chain is elongated via sequential addition of amino acids to the resin-bound C-terminal amino acid by reaction with an excess of an appropriately activated and protected amino acid. In this method, to maximize coupling efficiencies and reduce side reactions, all couplings are performed with a minimal 3-5 fold excess of activated amino acid, coupling reagents and additives and all reactive side chain functionalities must be protected throughout the entire synthesis. In Azaline B, the key amino-triazoles on the phenylalanine residues of AA
5
and AA
6
are generally constructed only after the entire backbone has been assembled and immediately prior to global deprotection with concomitant resin cleavage. In one exception to this Rivier et al, J.Med.Chem, 1995, 38, 2649-2662, reports the construction of the amino-triazole units simultaneously on the protected resin anchored [5-10] hexapeptide followed by incorporation of the remaining 4 amino acids in the usual solid-phase synthesis manner.
While this SPPS procedure provides the GnRH peptides in acceptable yield and purity, the SPPS procedure exhibits several inherent limitations which make it undesirable for large scale production of the peptides. These include:
capacity of the resin which limits the amount of the final product;
expense attributable to the C-terminal amide cleavable resins and the large excesses of coupling reagents, additives, and amino acids required;
necessity of full protection for all reactive side chains as in the hydroxy group in Ser
4
, the aromatic amino groups in Aph
5
, D-Aph
6
and the &egr;-i-propylamino group in Lys(i-Pr)
8
;
strongly acidic conditions required to affect global deprotection and cleavage of the peptide from the resin.
In addition, because each amino acid is incorporated in a separate reaction, any side reaction or decoupling of the peptide in the later stages requires the process to be repeated from the beginning. Accordingly, there is a need for an improved process for the preparation of GnRH peptide analogs which is amenable to large scale manufacture. Other methods for the bulk scale production of peptides include liquid phase peptide synthesis (LPPS), enzymatic synthesis, or fermentation with genetically manipulated microorganisms. However, none of these methods have been applied to the production of GnRH peptide analogs.
SUMMARY OF THE INVENTION
To address the limitations associated with the solid phase syntheses of GnRH peptide analogs, a liquid (or solution) phase peptide syntheses (LPPS) method is provided in which the GnRH peptide is assembled via condensation of judiciously selected peptide fragments using critical fragment couplings. In this method, the critical fragment couplings are kept to a minimum number to reduce potential side reactions and epimerization of the activated C-terminus. Accordingly, the peptide is synthesized by an LPPS method by first preparing the critical central peptide fragments of AA
5
and AA
6
with the side chains fully elaborated and then assembling the peptide through a “4-2-4”, “3-3-4” or “3-4-3” fragment assembly pattern. In the preferred “4-2-4” assembly pattern embodiment, the Aph(Atz) residues are constructed and incorporated simultaneously. Alternative coupling patterns in accordance with the LPPS method of the present invention, such as a “2-2-2-2-2” pattern, are also contemplated to be within the scope of this invention.
More particularly, in accordance with the invention, a process for preparing GnRH peptide analogs of the formula:
G-AA
1
-(A)D-Phe-AA
3
-AA
4
-(R
2
)AA
5
-AA
6
-AA
7
-AA
8
-Pro-AA
10
-NH
2
(SEQ. ID NO. 3)   Formula 1
or an acid addition salt thereof, wherein G is an acyl group having 7 or less carbon atoms; AA
1
is (A)D-Phe, (B)D-Trp, or &bgr;-D-NAL; A is H, Cl, F, NO
2
, Br, CH
3
, or OCH
3
; B is H, NO2, NH2, OCH
3
, F, Cl, Br or CH
3
; AA
3
is D-PAL, &bgr;-D-NAL or (B)D-Trp; AA
4
is Ser or P
1
-Ser wherein P
1
is a hydroxy protecting group; R
2
is H, N
60
CH
3
or N
&agr;
CH
2
CH
3
; AA
5
is Aph(P
2
), Aph(Ac), Aph(atz), Lys(P
2
), Lys(atz), Aph(Q-Atz) or Lys(Q-Atz); AA
6
is D-Aph(P
2
), D-Aph(ac), D-Aph(atz), D-Lys(P
2
), D-Lys(atz), D-Aph(Q-Atz) or D-Lys(Q-Atz); wherein P
2
is an amino protecting group; Ac is acetyl, atz is 5′-(3′-amino-1H-1′,2′,4′-triazolyl), and Q is the acyl residue of an amino acid; AA
7
is Leu, NML, Nle or Phe; AA
8
is iPr-Lys, (P
2
)iPr-Lys, or Arg; AA
10
is D-Ala, Gly, NH
2
NHCO or NH(R
3
) wherein R
3
is lower alkyl; is provided, which comprises:
(a) reacting a peptide of the formula:
T-(R
2
)AA
5
-AA
6
-X
 where T is (P
2
)AA4 or P
2
and X is AA
7
-OH or is —OH, with a peptide of the formula:
X′-AA
8
-Pro-AA
10
-NH
2
 or acid-addition salt form thereof, where X′ is AA
7
when X is absent and X′ is absent when X is AA
7
-OH;
 in a liquid reaction medium in the presence of a peptide coupling reagent and a stron organic amine base to obtain a product of the formula:
T-(R
2
)AA
5
-AA
6
-AA
7
-AA
8
-Pro-AA
10
-NH
2
(b) removing the P
2
protecting group at the N-terminus, and
(c) reacting the product of step (b) or an acid addition salt thereof, with a peptide of the formula:
G-AA
1
-(R
1
)D-Phe-AA
3
-T′
 or acid-addition salt form thereof, where T′ is AA
4
-OH when T is absent and is absent when T is P
2
-AA
4
, in a liquid reaction medium to obtain a GnRH peptide of the formula:
G-AA
1
-(A)D-Phe-AA
3
-AA
4
-(R
2
)AA
5
-AA
6
-AA
7
-AA
8
-Pro-AA
10
-NH
2
. (SEQ. ID NO. 3)
In this synthetic approach the requisite ce

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