CZ290347B6 - Cyclopeptides stimulating growth hormone release and process of their preparation - Google Patents

Abstract

The present invention relates to novel cyclic peptides stimulating release of growth hormone of the general formula I, in which A represents radicals of histidine or cysteine in L or D configuration or the radical 3-mercaptopropionic acid, B and D represent the radicals of tryptophan, tert-leucin or neopentylglycine in either L or D configuration, C represents the alanine radical in either L or D configuration, E represents the residues of lysine, phenylalanine or tetrahydroisoquinoline carboxylic acid in either L or D configuration, F represents the residues of lysine or cysteine in either L or D configuration or the radical of 3-mercaptopropionic acid; or the cycle can be formed by a disulfide optionally thiomethylene bond between side chains of the cysteine radicals in either L or D configuration, optionally in combination with the residue of the 3-mercaptopropionic acid. Process for preparing the above described cyclopeptides, forming also a part of the present invention, is characterized in that Ne{alpha}-Fmoc-Ne{epsilon}-Boc-lysine or Ne{alpha}-Fmoc-S-Trt-cysteine, optionally S-Trt-3-mercaptopropionic acid being all attached to a polymer carrier are successively condensed with active hydroxybenzotriazole esters of Ne{alpha}-Fmoc-amino acids in the presence of diisopropyl ethylamine, whereby the condensation reaction is carried out in a synthetic cycle under addition of dimethyl sulfoxide, Ne{alpha}-Fmoc protecting group is split off in each synthesis step by the action of piperidine in dimethyl formamide and in the final stage the hexapeptide, after being split off from a resin by the action of a weak alkali agent with subsequent acidification or by one percent TFA acid, is recycled by the action of diphenyl phosphorylazide, while the hexapeptides with residues of amino acid in either L or D configuration thereof, optionally of the 3-mercaptopropionic acid are split off from the resin under simultaneous closing the disulfide bond by the action of iodine in the mixture of AcOH, TFE and DCM solvents.

Description

The present invention relates to cyclopeptides that stimulate growth hormone release and a process for their preparation

BACKGROUND OF THE INVENTION

Cyclic peptides are potent and specific stimulators of growth hormone secretion (GH) in a variety of animal species having a certain amino acid sequence.

The mechanism of biological role of GH is to regulate the function of various growth factors (GFs, Rothe M., Falanda V., Arch. Dermatol. 125, 1390 (1989); Cohen, S., Carpenter, G., Proc. Natl. Acad. Sci. USA, 72, 1317 (1975): Lynch SE, Nixon JC, Colvin RB, Anthonianes N., Proc., Natl. Acad. Sci. USA 84, 7696 (1987); Baxter RC: Adv. 49 (1986), Gospodarowicz D., Neufeld G., Schweigerer L .: Mol. Cell Endocrinol., 16, 187 (1986), which are of complex importance for cell growth and synthesis. They are considered to be important mediators in wound healing, fibrosis and tissue replacement. Insufficient GH secretion results in impaired function of GFs and consequently limits growth and regulation of the tissue system in various organisms. On the other hand, increased GH intake stimulates GFs function in metabolism and improves growth, condition or healing of tissues and organs of humans and animals (Rothe M., Falanga V., Arch. Dermatol. 125, 1390 (1989)).

The neuroendocrine system, where GH and GFs play such an important function, has also been implicated in the aging process of the organism (Meites, J., Goya, R., Takahasni, S .: Exp. Gerontol. 22, 1 (1987); Fabris, N .: Intem. J. Neuroscience, 51, 373 (1990), Doubal S .: Mech. Ageing Dev. In press). Recently, effects of low doses of GH on the immune system and aging of mice have been described (Khansari D. N., Gustad T .: Mech. Ageing Dev. 56, 87 (1991)).

In addition to the so-called releasing factor (GRF) from the hypothalamus containing 44 amino acid residues, endogenous GH secretion can also be stimulated by the so-called releasing peptide: His-D-TrpAla-Trp-D-Phe-Lys-HN2 (GHRP). 6 amino acid residues and exerting GH secretion both in vitro and in vivo (Momany FA, Bowers CY, Reynolds GA, Chang D., Hong A., Newlander K .: Endocrinology 108, 31 (1981); Bowers CY, Momany FA, Reynolds GA , Hong A., Endocrinology, 114, 1537 (1984).

This hexapeptide amide does not interact with the same receptor site in the pituitary gland as GRF and appears to be involved in another mechanism of GH secretion (Sartor, O., Bowers CY, Chang D .: Endocrinology, 116, 952 (1985); Cheng K., WWS Khan, Barrets , Jr.A., Convey DM, Smith RG: Endocrinology 124, 2791 (1989)). In humans, its potency is even higher than that of the GRF (Yellin TO, Huffman WF in "Peptides"; Chemistry, Structure and Biology. Proc. 11 ^th APS (Rivier JE, Marshall GR, Eds.), P. 214. ESCOM (Leiden, 1990). Because of its short amino acid sequence, GHRP or its analogues are, in practical terms, more interesting than GRF containing tens of amino acid residues and could therefore be used in human and veterinary medicine.

Recently, we discovered that GHRP also significantly slowed the aging process of mice in an experiment lasting 10 months (Hlavacek J., Smekal O., Barth T., Doubal S., Robinson I .: Peptides 1992 (Schneider CH and Eberle AN, Eds. , p. 741, ESCOM, 1993).

Based on calculations (Momany F.A., Bowers C.Y., Reynolds G.A., Hong A., Newlander K .: Endocrinology, 114, 1531 (1984)), a pseudocyclic conformation was proposed for GHRP.

Enabled by flexible alanine residues in the central part of the molecule and stabilized by the interaction between aromatic amino acid residues of opposite chirality in amino- (His, D-Trp) and carboxy- (Trp,

D-Phe) terminal portions. The proposed conformation is associated with the effect of GHRP on stimulating GH release.

The above findings led us to the idea of covalently fixing the aforementioned pseudocyclic conformation. Therefore, we synthesized a series of cyclopeptides (I) according to the head-to-tail scheme and observed their effect on the release of GH into the plasma. Some of the analogs exhibit the agonist effect of the linear hexapeptide amide, confirming our notion that the cyclic alignment is relevant to the interaction of cyclopeptides with the corresponding receptor and does not prevent the following processes that lead to GH secretion. In addition, in a long-term experiment in mice (10 months), one of the cyclic analogs was even more effective than GHRP in slowing the aging process in experimental animals.

These peptides and combinations thereof act directly on the pituitary anterior lobe under specific GH triggering and are potential GHRP analogues with protracted action. Some of them also slow down the processes leading to the aging of experimental animals in the long-term test.

SUMMARY OF THE INVENTION

Cyclic peptides stimulating growth hormone release of formula I

A - B - C (I),

F - E - D where A represents the amino acid residues of histidine or cysteine in their L or D configuration or the residue of 3-mercaptopropionic acid,

B and D are the amino acid residues of tryptophan, tert.leucine or neopentylglycine in their L or D configuration,

C is the amino acid residue of alanine in its L or D configuration,

E represents the amino acid residues of lysine, phenylalanine or tetrahydroisoquinolinecarboxylic acid in their L or D configuration,

F represents the amino acid residues of lysine or cysteine in their L or D configuration or the residue of 3-mercaptopropionic acid;

or the cycle consists of a disulfide or thiomethylene bond between the side chains of the amino acid residues of the cysteine in its L or D configuration, optionally in combination with a 3-mercaptopropionic acid residue.

Process for preparing cyclopeptides according to the invention consists in a N "-Fmoc-N ^e -Boc-lysine or N" -Fmoc-S-Trt-cysteine or S-Trt-mercaptopropionic acid, 3-linked to a polymeric carrier is gradually condensed with an activated with hydroxybenztriazole esters of N ' -Fmocamino acids in the presence of diisopropylethylamine, where the condensation reaction is allowed to proceed in a synthetic cycle with the addition of dimethylsulfoxide, the N ' -Fmoc protecting group is cleaved at each stage of the a weakly alkaline reagent followed by acidification or 1% TFA acid cyclizes with diphenylphosphoryl azide, while

Hexapeptides with the amino acid residues of the cysteine in its L or D configuration, respectively

The 3-mercaptopropionic acids are cleaved from the resin while the disulfide bond is closed by iodine in a mixture of AcOH, TFE and DCM solvents.

The peptides are synthesized according to the general procedure by modifying standard methodologies on a polymeric carrier or in solution. In the first case, the carboxyterminal amino acid attached to the polymeric carrier, preferably the chloromethyl Merrifield resin or its acid labile hydroxymethyl or chlorotrityl modifications, are sequentially attached in triple excess with N ^and -Fmoc-amino acids. Their carboxyl group is activated with diisopropylcarbodiimide (DIC) and N-hydroxybenztriazole (HOBt) as the HOBt ester and condensation is carried out in the presence of dimethylformamide (DMF), respectively. N-methylpyrrolidone (NMP). The synthetic cycle for the desired amino acid attachment includes cleavage of the N ' -Fmoc protecting group by treatment with 20% piperidine in DMF (5 + 20 min), 5 times washing of the peptidyl resin with DMF, 2-propanol, DMF, respectively. NMP. The acylation reaction generally requires a cycle of 2 hours, after which dimethyl sulfoxide (15% in the reaction mixture) is added after 40 min. Completion of the reaction is controlled by a ninhydrin test (KaiserE., Colescott RL, Bossinger CD, Cook PI: Anal. Biochem. 34, 595 (1970)) and a change in bromophenol blue color (Krchňák V., Vagner J., Safar P., Lebl M Chem. Commun., 53, 2542 (1988). In the case of incomplete condensation (below 99%), the BOP reagent is added in a 3 molar excess with DIEA and the reaction is prolonged by 1 hour. Incomplete condensation after this operation is eliminated by acylation reaction with 25% acetic anhydride in DMF for 5-10 min.

The peptide is cleaved from the resin by a slightly alkaline aqueous solution of 0.1 M NaOH in a mixture of solvents MeOH and dioxane, over 3 min, cleaving a total of three times. Then the solution is acidified with acetic acid to regenerate the carboxylic function. In the case of an acid labile resin, the peptide is cleaved with 1% TFA. The crude GHRP analog product was desalted on a Sephadex G-10 biogel column using 0.2M acetic acid. After lyophilization, the peptide is further purified by gel filtration on Sephadex G-25 in 0.2M acetic acid, preparative continuous flow electrophoresis (Prusik Z., Kasicka V., Mudra P., Stepanek J., Smekal O., Hlavacek J .: Electrophoresis 11, 932 (1990)) in 0.5 M acetic acid and finally preparative high performance liquid chromatography (HPLC). Preparative HPLC was performed on a Spectra Physics apparatus with a Vydac C18 column at a gradient of 0 to 30% MeOH, 10 min and 30 to 80% MeOH, 40 min in 0.05% trifluoroacetic acid, flow rate 3 mL / min with detection at 222 resp. 254 nm. Analytical purity of the products was performed by TLC in S1 (2-butanol-98% HCOOH-water, 75: 13.5: 11.5), S2 (2-butanol-25% aqueous ammonia-water, 85: 7, 5: 7.5), S3 (1-butanol-acetic acid-water, 4: 1: 1) and S4 (1-butanol-pyridineacetic acid-water, 15: 10: 3: 6) followed by electrophoresis on Whatman 3MM paper at a potential gradient of 20V / cm, 1 h in 1M acetic acid (pH 2.4) and in pyridine acetate buffer (pH 5.7) and by analytical HPLC (Vydac Cl8 column, 20 to 80% MeCN / 0.5% trifluoroacetic acid) acid, 60 min, flow rate 1 ml / min, detection 222 and 280 nm), amino acid analysis on Durrum Instrum. Corp. USA, after hydrolysis of the peptide in 6M-HCl, 20 h at 110 ° C and mass spectrometry on a VG Analytical, England.

In the following, the invention and the general experimental procedure are explained in more detail in the examples without being limited thereto.

- j CZ 290347 B6

DETAILED DESCRIPTION OF THE INVENTION

Example 1

N ^and -Fmoc-N ^E -Boc-lysyl-4-oxomethylenophenyl resin

The Merrifield resin (0.7 mmol / g substitution, 2% divinylbenzene copolymer, Fluka, 2.8 g) was washed with DMF, water, dried in vacuo and washed with benzene. To the resin was added a solution of the precision salt prepared from N '-Fmco-N ^e -Boc-Lys-OH (3.42 g) and Cs ₂ CO ₃ (3.1 g) by mixing both components in DMF-water for 3 hours. and the whole mixture was stirred at 40 ° C for 48 hours. After removal of the solvent, the polymeric amino acid-bound material was washed on a frit of EtOH and DCM, and after drying, 1.67 g was used to gradually build the peptide chain.

Example 2 cyclo (D-His-T rp-Ala-D-T rp-Pre-Lys)

The N ^and -Fmoc protecting group of the bound Lys (Boc) of Example 1 was cleaved with 20% piperidine in DMF (5 + 20 min) and after washing the polymeric material with DMF, 2-propanol and again DMF the product was acylated sequentially with N'-Fmoc derivatives. Phe (0.75 g, 2 h), D-Trp (0.46 g, 3 h), Ala (0.57 g, 2 h), Trp (0.38 g, 3 h) and D-His ( Boc) (0.42 g, 2 h). The Fmoc group was cleaved with 20% piperidine in DMF and all condensations were treated with DIC in the presence of HOBt in DMF. The partially protected hexapeptide was then cleaved from the resin in aqueous 0.1 M NaOH in MeOH-dioxane (3 x 50 mL) at room temperature and separated from the polymer material by filtration and washing with 50 mL DMF, DCM and MeOH. 0.52 g of crude product was obtained, which was cyclized by treatment with diphenylphosphoryl azide in the presence of a 1M solution of K ₂ HPO ₄ . After cyclization, the product was purified by gel filtration on Sephadex G-10 and G-25 in 0.2M acetic acid and by HPLC. Amino Acid Analysis: His 0.93, Ala 0.98, Trp 1.72, Phe 1.05, Lys 1.0. For C56H69N11O10 (1056.2) found Μ + ΗΛ 1057.1. In the final step, the cyclopeptide was dissolved in TFA (90) -anisole (5) -ethanedithiol (5) with the addition of 100 mg indole and the solution was evaporated to dryness in vacuo after 1 h, extracted with ether, dissolved in 3M acetic acid and washed. 3x ether and lyophilized after dilution with water. The crude product was purified by semi-preparative HPLC in a mobile phase gradient of 50-90% MeOH in 0.05% TFA, 30 min (retention time 5.21 min).

For C ₄₆ H 45 N ₁₁ O 6 (856.02) the assay was found to be Μ + ΗΛ = 857. Amino acid analysis: Lys 1, Phe 1.03, Trp 1.84, Ala 1.04, His 0.96; E and _{G y} ^{2 '4} 1.18 E _His ^{5' 7} 0.82.

Example 3 Cycle (His-D-Trp-Ala-Trp-D-Tic-Lys)

Fmoc-Lys (Boc) -oxymethylenophenyl-resin (0.62 g) was acylated successively after cleavage of the Fmoc-protecting group with 20% piperidine in DMF with HOBt esters of Fmoc derivatives of D-Tic (0.6 g, 3 h), Trp (0 7 g, 4 h) Ala (0.3 g, 2 h), D-Trp (0.7 g, 3 h) and His (Boc) (0.73 g, 2 h). The partially protected hexapeptide was cleaved from the resin as in Example 2 and also cyclized with diphenylphosphoryl azide in the presence of K ₂ HPO ₄ . After cleavage of the Boc protecting groups as in Example 2, the cyclohexapeptide was purified by preparative HPLC using a mobile phase gradient of 50 to 90% MeOH in 0.05% TFA, 30 min, with a retention time of 5.64. Amino Acid Analysis: His 0.94, Trp 1.56, Ala 1.08, Tic0.93, Lys 1.0. E _Gly ^2.4 1.16, EHis ^5.7 0.82. For Ο47Η ₅₃ Ν] ΐθ6 (868.0) is determined / M + H + = 868.4.

-4GB 290347 B6

Example 4 cyclo (His-D-Neo-Ala-Trp-D-Phe-Lys)

The N '-Fmoc protecting group from bound Lys (Boc) was cleaved with 20% piperidine in DMF (5 + 20 min) and after washing the polymeric material with DMF, 2-propanol and again DMF, the product was acylated sequentially with N -Fmoc derivatives D-Phe (0.75 g, 2 h), Trp (0.46 g, 3 h), Ala (0.57 g, 2 h), D-Neo (0.31 g, 2 h) and His (Boc) ( 0.42 g, 3 h). The Fmoc group was cleaved as before. All condensations were treated with DIC in the presence of HOBt in DMF. The partially protected hexapeptide was released from the resin as in Example 2 and cyclized also with diphenylphosphoryl azide in the presence of K 2 HPO 4. After cleavage of the Boc protecting groups as in Example 2, the cyclohexapeptide had the amino acid composition: His 0.94, Neo 0.97, Trp 0.82, Ala 1.08, Phe 0.93, Lys 1.0. E ² _Gly ⁴ · 1.12 _H i E ^{₅ '7} 0.79.

For C 42 H 56 N 10 O ⁶ (796.97) found (M + H) + = 797. Preparative HPLC gave a mobile phase gradient of 50-90% MeOH in 0.05% TFA, 30 min pure product III, retention time 6.68.

Example 5

N '-Fmoc-N ^E -Boc-Lys-2-methoxy-4-alkoxybenzyl alcohol-resin

To the Sasrin resin (substitution 0.9 mmol / g, Bachem, 1 g) in DMF was added the active ester formed by reaction of Fmoc-Lys (Boc) -OH (1.15 g) with HOBt (0.76 g) with stirring, and Pentafluorophenol / DCC "complex" (3.74 g) at 0 ° C, 30 min and dimethylaminopyridine (30 mg). The pH of the mixture was adjusted to 7 by addition of DIEA (1.2 mL) and after 4 days of stirring at room temperature, the amino acid-bound resin was aspirated on a frit and washed with 5x 10 mL DMF, EtOH, iPrOH and MeOH and dried in a desiccator to constant weight. Weighing (1.24 g) and determining the Fmoc group resulted in a substitution of 0.57 mmol amino acid per g resin. Unreacted OH-CH ₂ linker groups were acetylated with acetic anhydride (1.3 mL) -DIEA (2 mL) -DCM (8 mL) and after 1 h the resin was aspirated on a frit and washed 3 x 10 mL with DCM, iPrOH and DMF. After cleavage of the Fmoc group with 20% piperidine in DMF and washing with 5x10 ml DMF, the material was prepared for the sequential construction of the peptide chain as in Example 1.

Example 6

H-Cys-D-Trp-Ala-Trp-D-Phe-Cys-OH

To a solution of Fmoc-Cys (Trt) -OH (0.96 g) in DCM (20 mL) and DMF (1 mL) was added chlorotrityl resin (2 g, substrate 1.3 mmol Cl / g) with stirring at room temperature. , NovaBiochem, Switzerland) and after 5 min 0.26 ml and after another 5 min 0.52 ml DIEA. After stirring for 60 min, the reaction was quenched by the addition of MeOH (2 mL) and after 10 min the solvent was filtered off and washed with 3x20 mL DCM, DMF, iPrOH, DMF, iPrOH, MeOH, ether and dried over KOH in a desiccator. The substitution of 0.54 mmol / g resin was found by weighing and determining the Fmoc group. The Fmoc protecting group was then cleaved off with 5% and 5% respectively. 20% piperidine in DCM / DMF 1: 1 (10 + 15 min) and after washing with DMF 4 times, the product was acylated sequentially with N-Fmoc derivatives of D-Phe (0.75 g, 2 h), Trp (0.46 g, 3 h), Ala (0.57 g, 2 h), D-Trp (0.38 g, 3 h) and Cys (Trt) (0.72 g, 2 h). The Fmoc group was cleaved with 20% piperidine in DMF and all condensations were treated with DIC in the presence of HOBt in DMF. The partially protected hexapeptide was then released from the resin and at the same time the disulfide bond between the two Cys residues was closed by treatment with iodine (0.76 g) in a mixture of solvents: AcOH (1.2 mL), TFE (2.4 mL),

-5 CZ 290347 B6

DCM (8.4 mL), 10 min at room temperature. The solution was extracted with 10 mL of 0.1% Na ₂ S ₂ O ₃ until the solution was decolorized, which was then evaporated and the residue dissolved in 3M AcOH and lyophilized.

The crude product was purified by semi-preparative HPLC in a mobile phase gradient of 5% to 90% ACN in 0.05% TFA, 30 min (retention time 24.3 min) and found / M + for C ₄ H ₄ N ₈ O ₇ S ₂ (813.0) Hf = 813.3. Amino Acid Analysis: Ala 1, Trp 1.64, Phe 1.03, Cys 2.02.

Abbreviations

Amino acid abbreviations correspond to the nomenclature proposed by IUPAC-IUB (Nomenclature and Symbolism for Amino Acids and Peptides, Recommendations 1983: Eur. J. Biochem. 138, 9 (1984)). In addition, the following abbreviations are used for amino acid protecting groups: AcOH, acetic acid; Boc; tert-butyloxycarbonyl; DCC; Ν, Ν-dicyclohexylcarbodiimide; DCM, dichloromethane; DIC, Ν, Ν-diisopropylcarbodiimide; DIEA, diisopropylethylamine; DMF, dimethylformamide; EtOH, ethanol; Fmoc, fluorenylmethyloxycarbonyl; HOBt, 1-hydroxybenztriazole, 15 MeOH, methanol; iPrOH, 2-propanol; TFA, trifluoroacetic acid; Trt, triphenylmethyl.

Industrial applicability

The compounds of the invention can be used in human and veterinary medicine to affect the immune system in connection with the regulation of the function of certain growth factors in order to slow down the processes involved in the aging process.

Claims (2)

30 1. Cyclic peptides stimulating growth hormone release of Formula I A - B - C (I), F - E - D where A represents the amino acid residues of histidine or cysteine in their L or D configuration or the residue of 3-mercaptopropionic acid, 35 B and D are the amino acid residues of tryptophan, tert.leucine or neopentylglycine in their L or D configuration, C is the amino acid residue of alanine in its L or D configuration, 40 E means the amino acid residues of lysine, phenylalanine or tetrahydroisoquinolinecarboxylic acid in their L or D configuration, F represents the amino acid residues of lysine or cysteine in their L or D configuration or the residue of 3-mercaptopropionic acid; or the cycle consists of a disulfide or thiomethylene linkage between the side chains of the cysteine amino acid residues in its L or D configuration, optionally in combination with a 3-mercaptopropionic acid residue. -6GB 290347 B6 A process for the preparation of cyclopeptides according to claim 1, characterized in that N'-Fmoc-N ^E -Boc-lysine or N'-Fmoc-S-Trt-cysteine or S-Trt-3-mercaptopropionic acid attached to the the polymeric carrier is sequentially condensed with the active hydroxybenzotriazole esters of bT-Fmoc-amino acids in the presence of diisopropylethylamine, 5 wherein the condensation reaction is allowed to proceed in a synthetic cycle with the addition of dimethylsulfoxide, N'-Fmoc protecting group is cleaved at each synthesis step step, the hexapeptide is cleaved from the polymeric carrier resin by weakly alkaline reagent followed by acidification or 1% TFA acid by cyclization with diphenylphosphoryl azide, while the hexapeptides with amino acid cysteine residues in its L or D configuration or 3-mercaptopropionic acid are cleaved from the resin. closed dis ulide bonds by iodine in a mixture of solvents AcOH, TFE and DCM.