CS246162B1 - Peptide with hormone activity releasing luteinizing and follicules stimulating hormone - Google Patents

Abstract

Peptide with luteinizing and follicle-stimulating hormone (LH / RH) releasing hormone of the formula <Glu-Hi-Trp-Se r-Ty-D-p-Et-Phe-Leu-Arg'2 3 4 5 6 7 8 -Pro-Gly-NHg 9 10 and pharmaceutically acceptable organic acid addition salts thereof. This peptide with LH / RH activity is used as a means to synchronize heat in industrial breeding of non-farm animals and also for diagnostic and therapeutic purposes. It is much more effective and longer acting than the original luteinizing and follicle-stimulating hormone, luliberin.

Description

(54) A peptide with luteinizing and follicle-stimulating hormone activity

Peptide with luteinizing and follicle-stimulating hormone (LH / RH) releasing activity of the general formula <Glu-Hi a-Trp-Se-Tyr-D-p-Et-Phe-Leu-Arg'2 3 4 5 6 7 8

-Pro-Gly-NHg

10 and pharmaceutically acceptable organic acid addition salts thereof.

This peptide with LH / RH activity is used as a means for the synchronization of heat in industrial breeding of non-farm animals and for diagnostic and therapeutic purposes. It is much more effective and longer acting than the original luteinizing and follicle-stimulating hormone, luliberin.

The invention relates to peptides with luteinizing hormone releasing and follicle-stimulating hormone (LH / HH) activity of the general formula I-Glu-His-Trp-Ser-Tyr-Dp-Et-Phe-Leu-Arg-Pro-Gly-NHg 12 345 6789 10 and pharmaceutically acceptable organic acid addition salts thereof.

The peptide of formula I is an analogue of the natural luteinizing hormone releasing and follicle-stimulating hormone, called luliberin: it outperforms it by its greater efficacy in the production and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) with extended duration of action. The peptide of the formula I according to the invention is intended for the synchronization of oestrus of farm animals and also for diagnostic and therapeutic purposes in human medicine,

For practical use in these indications, the desired substances are more effective, more stable in the body and thus protracted. Although structural modifications of luliberin have generally resulted in less potent substances, the replacement of glycine at position 6 of the luliberin amino acid sequence by some D-amino acids has succeeded in achieving efficiencies. /AND. Arimura et al .: Endocrinology 95. '* 74 (1974), J. A. Vilchez-Martinez et al .: Biochem. Biophys. Res. Commun. 59, 1226 (1974), DE DOS No. 2,625,843, D. H, Coy, A.V. Schally: Aniraals of Clinical Res. ia, 139 (1978)].

The peptide with LH / RH activity according to the invention of the above formula I meets the requirements for higher activity and longer action than the natural hormone.

The activity and sustained action of the compound of the invention were compared to pharmacological standards / A. Arimura et al., Endocrinology 22, 14 (1974)]. Testing for LH levels was performed using the RIA technique with sheep antibodies, where the reference compound was the standard of the American Institute of Arthritis, Metabolism, and in Disease Diseases - rat LH / RP. Statistical control of the result obtained by the t-test was performed according to Student and Welch, respectively, and the calculation of the activity coefficient according to Borth et al .: Arch. Gynac. 187. 497 (1957).

According to the above-described experimental assays in rats given 50 ng or 250 ng of the compound subcutaneously, a strong effect on LH release with a maximum of between 1 and 2 hours was demonstrated, with significant activity still observed after 6 hours. the LH / RH activity of the invention (50 ng, 250 ng, subcutaneously) achieved a maximum effect after 20 minutes and no activity against the comparator group was registered after 1 hour. For the various groups, it was calculated that the potency ratio of the standard (LH / RH) and the peptides with LH / RH activity of the invention within 6 hours from the time of administration corresponded to an increase of about 31-fold at 50 ng;

The peptide with LH / RH activity of the invention can be synthesized by known methods, for example by condensation on polymeric supports, as described in GDR patent specification 135 078 or in U.S. Pat. AO No. 230 614.

The peptides with LH / RH activity of the invention were prepared by the solid phase method. The tripeptide ζ Glu-His-Trp was prepared by successive construction on a suitable support, cleaved from the resin, and condensed to a resin containing the ser-Tyr-Dp-Et-Phe-Leu-Arg-Pro-Gly heptapeptide or Ser-Tyr-Dp- Et-Phe-Leu-Arg-Pro, also prepared by gradual construction on a suitable carrier. After cleavage of the peptides from the carrier and cleavage of the protecting groups by treatment with liquid hydrogen fluoride, a relatively pure product is obtained in high yield.

Peptides with LH / RH activity are constructed on polymeric supports of known type, the backbone of which consists of cross-linked polystyrene hydrocarbons. By active methods, active centers are introduced into the polymer to which the C-terminal protected amino acid or the corresponding di- or tripeptide is attached, which forms the basis for the further construction of the peptide. Benzhydrylamino or chloromethyl groups are preferably used as reactive centers. The preparation of peptides with LH / RH activity according to the invention consists of several steps.

The amino acid glycine in an appropriately protected form is anchored to the benzhydrylamine resin using known condensation methods, preferably the dicyclohexylcarbodiimide method or symmetric anhydride method, and the appropriately protected amino acids Pro, Arg, Leu, Dp-Et-Phe, Tyr and Ser or the corresponding peptide fragments in an appropriately protected form.

When building a shortened sequence

Ser-Tyr-D-β-Et-Phe-Leu-Arg-Pro is bound appropriately protected. For known methods, the chloromethylated resin and other amino acid derivatives or corresponding peptide fragments are sequentially attached in suitably protected form.

The tripeptide < Glu-His-Trp is exposed to a chloromethylated resin, wherein the Trp is attached in a suitably protected form, e.g., with cesium salt method and the like. The tripeptide is released from the carrier and condensed with a protected heptapeptide bonded to a benzhydrylamine resin or a hexapeptide bonded to a chloromethylated resin. The peptides with LH / RH activity of the invention are cleaved from the carrier while concomitantly cleavage of the protecting groups with liquid hydrogen fluoride or by cleavage of the peptide in a known manner, preferably by ammonolysis and final cleavage of side chain protecting groups, preferably with liquid hydrogen fluoride.

The peptides with LH / RH activity thus obtained already have a content of 70 to 80% by weight. For use in veterinary practice, one purification operation is sufficient, for example precipitation in the form of a soluble acid addition salt and final release by ion exchange. A biologically highly active preparation is obtained.

Further purification operations, such as column chromatography purification, are then performed for diagnostic and therapeutic purposes.

The following scheme illustrates peptide synthesis and LH / RH activity of the invention:

+ Boc-His * Glu

Gly-NH- (?) + Boc-Pro + Boc-ArgdOg) <Qlu-Hi ₃ -Trp- (F) (Glu-His-Trp-OH)

+ Boc-Leu + Boc-D-pEt-Phe-Boc-Tyr (Bzl) + Boc-Ser (Bzl)

Se (Bzl) -Ty (Bzl) -D-pEt-Phe-Leu-Arg (NOg) -Pro-Oly-NH- («)

7. Glu-Hi-T rp-Se r (Bzl) -Ty r (Bzl) -D-pEt-Phe-Leu-Arg (NOg) -Pro-Gly-NH- / Pj + HF

IN

LH / RH analogue

Amino acids containing a side-chain functional group are suitably protected in the synthesis, particularly by groups which can be easily cleaved by hydrolysis, reduction or liquid hydrogen fluoride, using groups known in the synthesis of peptides such as benzyl-, ρ-chlorobenzyl-, benzyl-, ρ-chlorobenzyl-, and the like. 6-dichlorobenzyl-, ρ-nitrobenzyl-, p-methoxybenzyl-, benzhydryl-, tert-butyl-, tosyl-, dinitrophenyl- and nitro.

Aliphatic oxycarbonyl groups such as cyclohexyloxycarbonyl, adamantyloxycarbonyl and especially tert-butyloxycarbonyl (Boc-), or carbobenzoxy substituted in the aromatic ring (e.g., carbobenzoxy, p-bromo- or chlorocarbobenzyl) may be used as amino-protecting groups o-nitrophenylsulfenyl and optionally substituted aralkyl groups (e.g., di- or triphenylmethyl).

Trifluoroacetic acid, trifluoroacetic acid in dichloromethane, hydrogen chloride in glacial acetic acid or dioxane are preferably used to cleave the alpha-amino protecting groups during the synthesis. Other cleavage reagents and conditions for cleavage of specific protecting groups are described in E. Schroeder and K. Lubke, The Peptides I, Academic Press, 965, pp. 72-75.

After synthesis, the peptide may be cleaved from the polymer carrier in a known manner, preferably by glacial acetic acid hydrobromide, by alcoholysis, hydrazinolysis, hydrogenolysis, ammonolysis or liquid hydrogen fluoride with the addition of anisole or mercaptoethanol.

The following examples illustrate the process according to the invention, but do not limit it in any way.

Example 1 g of benzhydrylamine resin (produced according to I. Rivier et al., J. Med. Chem.

545 ('973) or P. Pietta et al., J. Org. Chem. 22 »44 ('977)) was condensed with β 5.3 g of Boc-Gly in methylene chloride using 6.2 g of dicyclohexylcarbodiimide. Obeah Gly did

0.96 mmol / g resin. The following amino acid derivatives were used for the gradual construction of the heptapeptide! Boc-Pro, Boc-Argin (Bzl), Boc-Leu, Boc-Tyr, Boc-Ser (Bzl)

Protected amino acids were condensed in a triple excess relative to the C-terminal Gly by the symmetric anhydride method. The gradual construction of the heptapeptide was performed according to the following program:

1. 3 x 80 ml methylene chloride (for 3 minutes)

2. 3 x 80 ml dioxane (3 minutes)

3. 1 x 80 ml of a 4 N solution of hydrochloric acid in dioxane (10 minutes)

4. 1 x 80 ml of a 4 N solution of hydrochloric acid in dioxane (25 minutes)

5. 3 x 60 ml dioxane (3 minutes)

6. 3 x 80 ml methylene chloride (3 minutes)

7. 3 x 80 ml chloroform: methanol 2: 1 (3 minutes)

8. 3 x 80 ml chloroform (3 minutes)

9. 2 x 80 ml chloroform: triethylamine 9: 1 (5 minutes)

10. 3 x 80 ml chloroform (3 minutes)

11. 3 x 80 ml methylene chloride (3 minutes)

12. 1 x 80 ml symmetrical anhydride of the corresponding protected amino acid (condensation time to negative ninhydrin test)

13. 3 x 80 ml methanol: methylene chloride (3 minutes)

18 g of Boc-SerfBzD-D-pEt-Phe-Leu-ArgOIC-Pro-Gly-NH- were obtained.

Example 2 g Boc-Trp-O- which was prepared by esterification of a chloromethylated polymeric carrier (Cl content 7%) with Boe-Trp cesium salt (Trp content 0.91 mmol Trp / g polymer) were condensed sequentially with BocrHis and Glu by symmetrical anhydride method according to the program of Example 1.

3.45 g <Glu-His-Trp-O-® was obtained. The peptide was cleaved from the resin according to Sakakibara; 1 g of the tripeptide polymer was suspended in 15 ml of hydrogen fluoride and 1.5 ml of anisole at 0 ° C for 30 minutes. After hydrogen fluoride was distilled off, the residue was washed with ether, dissolved in 1 Sfc of acetic acid and converted into acetate using an ion exchanger (Wofatit AD-41 in an acetate cycle). Lyophilization yielded 220 mg of Glu-His-Trp-OH as acetate.

Example 3 luGlu-His-N ₂ Hj (produced by Gillesen et al., Helv. Chim. Acta., 60 (1970)) was condensed by the azide method with Trp (OBzl) according to US Pat. No. 3,888,838 to give < Glu-His-Trp-OBzl. The product was then hydrogenated according to Sievertsson et al., J. Med. Chem. 1, 222 (1972) in the presence of a palladium / barium sulfate catalyst.

Example 4 g of Ser (Bzl) -Tyr-Dp-Et-Phe-Leu-ArgiOg-Pro-Gly-NH-® obtained according to Example 1 was condensed in dimethylformamide with 2.88 mmol of Glu-His-Trp-OH, which was prepared according to Example 2 or 3j, 2.88 mmol of dicyclohexylcarbodiimide was used as condensing agent. After washing with dimethylformamide, chloroform: methanol 2: 1, methylene chloride and methanol, 1.2 g of product were obtained.

1.2 g of the decapeptide polymer were suspended according to Sakakibara in 15 ml of hydrogen fluoride and 1.5 ml of anisole (or mercaptomethanol) for 1 hour at 0 ° C. After hydrogen fluoride was distilled off, the residue was washed with ether and dissolved in 1% acetic acid. The product was converted to decapeptide acetate and lyophilized using an ion exchanger (tfofatit AD 41). 334 mg of the LH / RH analogue of formula I was obtained in the diacetate monohydrate form.

Example 5

250 mg of 4, Glu-Hia-Trp-Ser-Tyr-Dp-Et-Phe-Leu-Arg-Pro-Gly-NH ₂ was dissolved in 10 ml water β 0.25 ml acetic acid and precipitated with 25 ml of a saturated solution of stific acid (1,3-dihydroxy-2,4,6-trinitrobenzene). The precipitate was washed with a solution of stific acid, dissolved in 1: 1 acetone-water and converted by ion exchanger (Wofatit AD 41, in acetate form) to the LH / RH analogue in diacetate form. After lyophilization, 188 mg of pure product of the above formula were obtained.

Example 6

1.0 g of the LH / RH analogue obtained according to Example 5 for diagnostic and therapeutic purposes was chromatographed by gradient elution with triethylamine: acetic acid buffer (0.01 / 0.4 m pH 4.5) to carboxymethylcellulose (column size 2 χ 30) cm). Detection was performed by UV measurement at 278 nm and purity was monitored by thin layer chromatography (System I: chloroform: methanol; 30% acetic acid 60: 45: 20; System II: n-butanol: acetic acid: water 4: 1: 5). The product was obtained in the di-acetate form.

Yield: 0.638 g of the LH / RH analogue, _mp ⁼ -60.3 ° -0.5 (c = 1.1, acetic acid),

Value Rj. by thin-layer chromatography: System I: 0.49

System II: 0.13.

Amino acid analysis:

Olu-Hi s-Trp-Ser-Tyr-Dp-Et-Phe-Leu-Arg-Pro-Gly Theory: 11111 11111 found: / 1.0 / 1.02 0.97 0.85 0.98 1, 01 1.0 1.04 1.0 1.02,

Explanation of abbreviations used in the text:

<Glu pyroglutamic acid His histidine Trp tryptophan Ser serine Tyr tyrosine Et ethyl Phe phenylalanine Leu leůcin Arg arginine For proline Gly glycine Boc target, butyloxycarbonyl Bzl benzyl HF hydrogen fluoride

RIA radioimmunoanalysis

Claims (1)

SUBJECT OF THE INVENTION A peptide having luteinizing and follicle-stimulating hormone activity of Formula I 4Glu-His-Trp-Ser-Tyr-D-β-Et-Phe-Leu-Arg-Pro-Gly-NHg (I) and pharmaceutically acceptable organic acid addition salts thereof.