DD295857A5 - PROCESS FOR THE PREPARATION OF GNRH DECAPEPTIDES - Google Patents

Abstract

The invention relates to a process for the preparation of GnRH decapeptides. The object is achieved by the partial fragments Pyr-His-Trp-OR and H-Ser-Tyr-Xaa-Leu-Arg-Pro-Gly-NH 2 prepared in a manner known per se, where R is an aliphatic or aromatic radical and Xaa is a D-amino acid, according to the invention be coupled by means of chymotrypsin. The process ensures a racemization-free production of GnRH Dekapeptide in high yield and purity. The fields of application of the invention lie in the production of medicaments which are used for the treatment of fertility disorders, prostate carcinoma or for ovulation synchronization. {Peptides; enzymatic synthesis; GnRH; chymotrypsin; racemization}

Description

Thus, a GnRH synthesis is described which is based almost exclusively on enzymatic coupling steps (Peptides 1986, Ed, D. Theodoropoulos, W. de Gruyter, Berlin 1987, p 183): The N-term. Tripeptide ester Pyr-His-Tro-OMe is extended by means of chymotrypsin and serine ethyl ester to the tetrapeptide Pyr-His-Trp-Ser-OEt. In further coupling steps carboxypeptidase Y, carboxypeptidase YM, post-proline-specific endoprotease and chlostripain, which, however, are very expensive compared to chymotrypsin, are used. A disadvantage of using this exclusively enzymatic coupling strategy is also the high optimization effort, especially to avoid unwanted peptide cleavage, since all five enzymes used require different reaction conditions for coupling and cleavage.

Object of the invention

Ziol of the invention is to develop a simple and economical process for the industrial production of GnRH Peptlden, formula I.

Explanation of the essence of the invention

The invention is based on the objects, a simple process for the preparation of GnRH peptides, formula I,

Pyr-His-Trp-Ser-Tyr-Xaa-LeU'Arg-Pro-Gly-NH ₂ , I

to develop, after which the peptides can also be produced on an industrial scale in high yield and purity without racemization. The Aufgäbe is achieved in that the prepared in a conventional manner partial fragments of the formula II and III

Pyr-His-Trp-OR II,

H-Ser-Tyr-Xaa-Leu-Arg-Pro-Gly-NH ₂ III,

where R is an aliphatic or aromatic radical, preferably -CH 3, -C ₂ Hb and Xaa is a D-amino acid, preferably D-Phe, D-Ala, D-Trp, D-Leu, D-β-Nal, D-Ser ( tBu) according to the invention are coupled by means of chymotrypsin. The enzyme-catalyzed segment synthesis is advantageously carried out in a mixture of dimethylformamide and water, wherein the reaction in a solvent mixture of 30 to 60% DMF, at a temperature of -10 ⁰ C to 35 ° C and at a pH from 7.8 to 8 9 is performed. The reaction rate is determined by the selected temperature range and the amount of enzyme used, the reaction time being up to 2 days, preferably 4 to 8 hours. The process according to the invention ensures a racemization-free preparation of the GnRH decapeptides in high yield and purity. In the enzymatic peptide synthesis, especially in the coupling of peptide fragments, expected difficulties due to side splits occurring do not take place. Due to the known specificity of chymotrypsin for reactions on carboxyl groups of aromatic amino acids, it is surprising that in the coupling according to the invention the Tyr-Xaa peptide bond is not hydrolytically cleaved by chymotrypsin. The process has a number of other advantages:

The structure of the decapeptides is based on fragments which can be prepared well by classical synthesis methods in solution (fragment II: eg DE 2307010) and by solid-phase synthesis (fragment III, B. B. DD 135078). Thus, the heptapeptide III using N ° -Boc protected amino acids, Arg (NO ₂ ), Ser (Bzl) side chain protection on benzhydrylamine resin by DCC / HOBt easily in the 50-1000g resin scale and after HF cleavage in a Crude peptide purity of 85-90% can be obtained. The acid labile Trp residue is not subject to the critical RF cleavage procedure while maintaining the 3 + 7 strategy. In contrast to other production processes in which the 3 + 7 condensation is carried out by means of DCC or DCC additives, in the process according to the invention the coupling with chymotrypsin is carried out, whereby a racemization of the Trp ³ radical is excluded. In contrast, in the condensations carried out with DCC / additives, a Trp ³ racemization of 5-15% is observed. The racemization can be reduced to <1% when the coupling is carried out via the tripeptide azide. However, the yields in this process are 20-50% (see, for example, DO 2307010), while in the enzymatic linkage according to the invention, when using a tripeptide excess of 1.3 equivalents, a quantitative conversion of the heptapeptide used is achieved. The enzymatic coupling moreover has the advantage that acylations on the unprotected peptide side chains are not possible and the purity of the decapeptide crude product depends exclusively on the purity of the fragments II and III used. The final purification of the crude products obtained is thus possible in a simple manner, especially since the separation of the fragment II and the tripeptide acid formed in the enzymatic synthesis is problem-free both by ion exchange chromatography and by reversed-phase chromatography on hydrophobic polymers

 The following examples are intended to explain in more detail the process according to the invention for the preparation of GnRH peptides:

embodiments

example 1

Preparation of Pyr-His-Trp-Ser-Tyr-DPhe-Leu-Arg-Pro-Gly-NH ₂ :

21 g of Pyr-His-Trp-OEt (43.7 μmol) and 20 g of H-Ser-Tyr-DPhe-Leu-Arg-Pro-Gly-NH ₂ (23.87 mmol) are stirred with stirring at room temperature in 66 ml of DMF and 70 , 5 ml of water and the pH adjusted to 8.0 with 2 N KOH. 450 μl of a solution of 50 mg of α-chymotrypsin (SERVA, 45 U / mg) in 1 ml of 1 × 10 ^-3 N HCl are added, and the enzyme is repeated after 2 hours under pH stat conditions (pH 8, 0 with 2 N KOH), a nearly quantitative conversion of the heptapeptidamide after 6.5 h

reached (HPLC control) and the enzyme reaction stopped by the addition of 10 ml of acetic acid. It is concentrated in vacuo with 3 × 50 ml of methanol and the oily residue is stirred into 3 l of acetone.

The precipitated synthesis product is filtered off with suction, washed with acetone and ether and dried.

Yield: 24.5 g Excessively used Pyr-His-Trp-OEt can be isolated from the acetone supernatant of the product precipitation by concentration and, after washing out of formed Pyr-His-Trp-OH, with Hydrogencaibonatlösung and reused.

Example 2

Like Example 1, but instead of a-chymotrypsin (SERVA, 45U / mg), a-chymotrypsin is used by the VEB Berlin-Chemie (275 U / mg, N-acetyl-L-tyrosine ethyl ester).

Yield; 24,4g

Example 3

As in Example 1, only instead of 23.87 mmol 0.75 mmol Heptapeptidamid be used and instead of synthesized Pyr-His-Trp-OEt recovered Pyr-His-Trp-OEt is used.

Yield: 0.79 g

Example 4

As Example 1, but instead of 450μ1 (22.5 mg) dissolved a-chymotrypsin is 200mg immobiliertes a-chymotrypsin (20U / mg, N-acetyl-L-tyrosine ethyl ester, pH 8.0; 25 ⁰ C) was used.

Yield: 24.2g

Claims (5)

1. Process for the preparation of GnRH decapeptides of the formula I. Pyr-His-Trp-Ser-Tyr-Xaa-Leu-Arg-Pro-Gly-NH ₂ I where Xaa = DPhe or another D-amino acid, characterized in that the structure of the GnRH decapeptides from the fragments of the formula I and II Pyr-His-Trp-OR I ₁ H-Ser-Tyr-Xaa-Leu-Arg-Pro-Gly-NH ₂ II wherein R is alkyl or aryl and Xaa has the abovementioned meaning, carried out by chymotrypsin-catalyzed coupling. 2. The method according to claim 1, characterized in that the reaction is carried out at a temperature between -1O ⁰ C and 35 ° C. 3. The method according to claim 1 and 2, characterized in that is used as the organic solvent dimethylformamide. 4. The method according to claim 1 to 3, characterized in that the fragments are brought to a pH = 8.5 in the water / DMF mixture, the reaction is started by adding the enzyme and added in a period of 2 h further enzyme becomes. 5. The method according to claim 1 to 4, characterized in that bound to a polymeric carrier chymotrypsin is used. Field of application of the invention The invention relates to a novel process for the preparation of decapeptides, especially of the GnRH Pyr-His-Trp-Ser-Tyr-Xaa-Leu-Arg-Pro-Gly-NH ₂ I « wherein Xaa means DPhe or another D-amino acid. The peptides are useful in the preparation of medicines used to treat fertility disorders, prostate cancer or ovulation synchronization. The fields of application of the invention are in human medicine as well as in veterinary medicine. Characteristic of the known state of the art The synthesis of GnRH and GnRH analogues has been described several times. The syntheses are carried out both in a homogeneous phase and by building on polymeric supports. The construction of the peptides in solution takes place via partial sequences, eg Pyr-His-NHNH ₂ , Pyr-His-Trp-OH, Pyr-His-Trp-NHNH ₂ , H-Ser ('Bu) -Tyr-Gly-Leu-OH , H-Trp-Ser ('Bu) -Try-Gly-Leu-OH, H-Arg-Pm-Gly-NH ₂ , H-Arg (NO ₂ ) -Pro-Gly-NH ₂ or H-Arg (TOS ) -Pro-Gly-NH ₂ (DE 2307010, DE 2424287, Tetrahedron Letters 41, 4071 (1973) Coll Check Chem Chem 42,2018 (1976); J. Med. Chem. 15,222 (1972); Chem. Pharm. Bull., 23, 299 (1975); J. Chem. Soc. Perkin Trans., 1.1979, 389; HeIv. Chem. Acta, 57, 231 (1974); J. Med. Chem., 17, 1060 (1974); Med. Chem., 15, 633 (1972); J. Med. Chem., 18, 613 (1975); US 3953416; Chem. Pharm. Bull., 27, 1907 (1979); Peptides, 1972 (Ed H. Hanson, HD Jakubke); North Holland Publ. Amsterdam 1973, p. 93). The couplings of the fragments are realized in part via azide syntheses and partly via DCC condensations, some authors adding racemization-reducing reagents such as HOBt, HOOBt, HONB, HONSu. It is also known to carry out the coupling of partial sequences such as Pyr-His-Trp-OH by means of DCC without additives (HeIv. Chim. Acta., 57, 231 [1974], US Pat. No. 3,953,416). About the extent of racemization at the C-term. however, no information was provided on activated amino acid. In studies on the coupling of Pyr-His-Trp-OH, 5-15% D-Trp peptide was found in the final product in all DCC / Additiva variants. To avoid the Razemisierungsrisikos is therefore chosen by other authors, the azide synthesis despite the dabpKoft unsatisfactory yields or a stepwise construction of the fragment 1-6. In the case of the step-by-step construction of the GnRH peptides by solid-phase synthesis, racemization is in most cases ruled out. Only for histidine derivatives is racemization expected depending on the side chain protection used. Disadvantages of the solid-phase syntheses carried out, especially in syntheses on a production scale, are side reactions, which are mainly due to the high acid lability and alkylability of the tryptophan residue, due to the high racemization tendency of some histidine derivatives, and through N ^{in the} -N ^a migration of N ^lm protective groups as well as incomplete couplings in the N-terminus. This led to the strategy of condensing a tripeptide prepared in solution by classical methods onto a solid-phase synthesized heptapeptide before cleavage from the resin (DD 135078) or after cleavage (Ann et al., Endocrinol. [Paris] 37,215 [1976]) , However, the disadvantage here again is the racemization observed at the C-terminal Trp. Furthermore, it is known that peptide fragments can be linked to racemization-free by enzymatic catalysis (Angew Chem .. Int., Ed., 24.85 [1985]).