CS246162B1 - A peptide with luteinizing and follicle-stimulating hormone activity - Google Patents

Abstract

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

Description

(54) Peptide with luteinizing hormone-releasing hormone and follicle-stimulating hormone activity

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

-Pro-Gly-NHg

10 and its pharmaceutically usable organic acid addition salts.

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

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

The peptide of formula I is an analogue of the natural hormone releasing luteinizing and follicle-stimulating hormone, the so-called luliberin: it surpasses it in its higher efficiency in the formation and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) with a prolonged duration of action. The peptide of general formula I according to the invention is intended for the synchronization of oestrus (estrus) in farm animals and also for diagnostic and therapeutic purposes in human medicine,

For practical use in the indicated indications, the desired substances are more effective, more stable in the organism and therefore with prolonged action. Structural changes in luliberin usually led to less effective substances, however, it was possible to achieve an increase in effectiveness by replacing glycine in position 6 of the amino acid sequence of luliberin with some D-amino acids. /A. Arimura et al.: Endocrinology 95. ’ *74 (1974), J. A. Vilchez - Martinez et al.: Biochem. Biophys. Res. Commun. 59. 1 226 (1974), DE DOS No. 2 625 843 j D. H, Coy, A. V. Schally: Aniraals of Clinical Res. ia, 139 (1978)/.

The requirements for higher activity and longer action than the natural hormone are met by the peptide with LH/RH activity according to the invention, of the above-mentioned general formula I.

The activity and prolonged effect of the substance according to the invention were compared with pharmacological standards /A. Arimura et al.: Endocrinology 22» ' '14 (1974)/. Testing of the LH level was carried out using the RIA technique with sheep antibodies, where the standard of the American institute National Institute of Arthritis, Metabolism and vDigestion Diseases - rat LH/RP, served as a reference compound. Statistical control of the result achieved by means of the t-test was carried out according to Student, or according to Welch and calculation of the activity coefficient according to Borth et al.: Arch. Gynac. 188. 497 (1957).

According to the above-described experimental determinations on rats, to which 50 ng or 250 ng of the compound were administered subcutaneously, a strong effect on the release of LH was demonstrated with a maximum between 1 and 2 hours, with significant activity still being observable after 6 hours. In contrast, the same dose of peptides with LH/RH activity according to the invention (50 ng, 250 ng, subcutaneously) reached a maximum effect after 20 minutes and after 1 hour no activity was registered against the comparison group. For the different groups, it was calculated that the ratio of the effectiveness of the standard (LH/RH) and the peptides with LH/RH activity according to the invention within 6 hours from the time of administration corresponds to an increase of about 31 times at a dose of 50 ng, and to an increase of about 43 times at a dose of 250 ng.

The peptide with LH/RH activity according to the invention can be synthesized by known methods, for example by condensation on polymer carriers, as described in the NDR patent document No. 135,078 or in the Czechoslovak Patent Application No. 230,614.

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

The construction of peptides with LH/RH activity is carried out on polymer carriers of a known type, the basic skeleton of which consists of sessile polystyrene hydrocarbons. Active centers are introduced into the polymer by suitable methods, to which the C-terminal protected amino acid or the corresponding di- or tripeptide is bound, which represents the basis for 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 partial stages.

The amino acid glycine in a suitably protected form is anchored to the benzhydrylamine resin using known condensation methods, preferably the dicyclohexylcarbodiimide method or the symmetrical anhydride method, and the suitably protected amino acids Pro, Arg, Leu, D-p-Et-Phe, Tyr and Ser or corresponding peptide fragments in a suitably protected form are successively attached using the same method.

When constructing a shortened sequence

Ser-Tyr-D-p-Et-Phe-Leu-Arg-Pro is attached in a suitably protected form to a chloromethylated resin by known methods and the other amino acid derivatives or corresponding peptide fragments are successively attached in a suitably protected form.

The tripeptide < Glu-His-Trp is exposed to a chloromethylated resin, with Trp being attached in a suitably protected form, e.g. by the cesium salt method, etc. The other amino acids His and < Glu are successively attached in a protected form using the above-mentioned condensation methods. The tripeptide is released from the carrier and condensed with the protected heptapeptide bound to the benzhydrylamine resin, or with the hexapeptide bound to the chloromethylated resin. The peptides with LH/RH activity according to the invention are cleaved from the carrier with simultaneous cleavage of the protecting groups using liquid hydrogen fluoride or by cleavage of the peptide in a known manner, preferably by ammonolysis and final cleavage of the groups protecting the side chains, 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 using an ion exchanger. This results in a biologically highly active preparation.

For diagnostic and therapeutic purposes, further purification operations are then performed, for example purification by column chromatography.

The following scheme further explains the synthesis of peptides and the activity of LH/RH according to the invention:

+ Boc-His * Glu

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

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

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

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

IN

LH/RH analog

Amino acids containing a functional group in the side chain are suitably protected during synthesis, in particular by groups that can be easily cleaved by hydrolysis, reduction or liquid hydrogen fluoride, using groups known in peptide synthesis, such as benzyl, ρ-chlorobenzyl, 2,6-dichlorobenzyl, ρ-nitrobenzyl, p-methoxybenzyl, benzhydryl, tert-butyl, tosyl, dinitrophenyl and nitro groups.

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

Trifluoroacetic acid, trifluoroacetic acid in dichloromethane, hydrogen chloride in glacial acetic acid or dioxane are preferably used to remove alpha-amino protecting groups during synthesis. Other cleaving agents and conditions for the removal 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 can be cleaved from the polymeric support in a known manner, preferably with hydrogen bromide in glacial acetic acid, alcoholysis, hydrazinolysis, hydrogenolysis, ammonolysis or liquid hydrogen fluoride with the addition of anisole or mercaptoethanol.

The following examples of embodiments only illustrate the method according to the invention, but are not limiting in any way.

Example 1 g of benzhydrylamine resin (prepared according to I. Rivier et al., J. Med. Chem. £6,

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

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

The protected amino acids were condensed in a three-fold excess relative to the C-terminal Gly by the symmetrical anhydride method. The stepwise construction of the heptapeptide was carried out according to the following program:

1. 3 x 80 ml of methylene chloride (3 minutes each)

2. 3 x 80 ml dioxane (3 minutes)

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

4. 1 x 80 ml of 4 N hydrochloric acid solution 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 of a 2:1 chloroform:methanol mixture (3 minutes)

8. 3 x 80 ml chloroform (3 minutes)

9. 2 x 80 ml of a mixture of 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 of symmetrical anhydride of the corresponding protected amino acid (condensation time to negative ninhydrin test)

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

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

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

3.45 g of <Glu-His-Trp-O-® were obtained. Cleavage of the peptide from the resin was carried out 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 distilling off the hydrogen fluoride, the residue was taken up in ether, dissolved in 1 Sfc of acetic acid and converted to acetate using an ion exchanger (Wofatit AD-41 in the acetate cycle). Lyophilization yielded 220 mg of Glu-His-Trp-OH in the acetate form.

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

Example 4 g of Ser(Bzl)-Tyr-D-p-Et-Phe-Leu-Argd1OgJ-Pro-Gly-NH-® obtained according to Example 1 was condensed in dimethylformamide with 2.88 mmol of ζ Glu-His-Trp-OH, which was prepared according to Example 2 or 3j using 2.88 mmol of dicyclohexylcarbodiimide as the condensing agent. After washing with dimethylformamide, a 2:1 chloroform:methanol mixture, methylene chloride and methanol, 1.2 g of product was obtained.

1.2 g of decapeptide polymer was 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 distilling off the hydrogen fluoride, the residue was washed with ether and dissolved in 1% acetic acid. The product was converted to decapeptide acetate using an ion exchanger (thiophatite AD 41) and lyophilized. 334 mg of the LH/RH analog of the 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 of water β 0.25 ml of acetic acid and precipitated with 25 ml of saturated solution of sthifnic acid (1,3-dihydroxy-2,4,6_trinitrobenzene). The precipitate was washed with a solution of sthifnic acid, dissolved in a mixture of acetone and water 1:' and converted with an ion exchanger (Wofatit AD 41, in acetate form) to the LH/RH analogue in diacetate form. After lyophilization, 188 mg of the pure product of the above formula was 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) on carboxymethylcellulose (column size 2 x 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 diavetate form.

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

Rj. value in thin layer chromatography: System I: 0.49

System II: 0.13.

Amino acid analysis:

Olu-Hi s-Trp-Ser-Tyr-D-p-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,

Explanations of abbreviations used in the text:

< Glu pyroglutamic acid His histidine Suffering tryptophan Shit serine Tyr tyrosine Et ethyl Phe phenylalanine Leo leucine Arg arginine For proline Gly glycine Sorry tert-butyloxycarbonyl Bzl benzyl HF hydrogen fluoride

RIA radioimmunoassay

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.