DD276483B5 - Process for the preparation of new cholecystokinin pentapeptides - Google Patents

Description

Field of application of the invention

The invention relates to a process for the preparation of novel acylated cholecystokinin sequences of general formula I.

ACyI-TYr (SE) -X-Gly-TrP-Y-NH-CH ₂ -CH ₂ CH ₆ H ₆ ,

in the acyl is an amino acid residue, preferably aspartic acid, or a carboxylic acid residue, preferably succinic acid, or a protecting group commonly used in peptide chemistry, preferably butyloxycarbonyl (BOC) and benzyloxycarbonyl (Z), and SE is a titer residue for a sulfate ester moiety. X is an amino acid residue, preferably Thr, Nie, Leu or methionine and Y is methionine, norleucine or leucine. For better enzymatic stability, instead of Trp, D-Trp can also be used purely.

Such peptides represent novel important antagonists of cholecystokinin and are valuable tools for pharmacological studies. Fields of application of the invention are medical research and the pharmaceutical industry.

Characteristic of the known technical solutions

The gastrointestinal peptide hormone cholecystokinin (CCK) exists in various molecular forms including CCK-39, CCK-33, CCK-12 and CCK-8 (by Mutt and E. Jorpes, Biochem., Journal 125, 57, 1971).

It shows a number of important effects in the gastrointestinal, pancreatic and hepatobiliary system. Thus it stimulates the gallbladder contraction and causes the relaxation of the sphincter oddi (for reviews see JE Morley, Life Sci 30,479,1982 and Nieber, K., Henklein, P. Ott, T. and P.Oehme Z. Gesam inn. Med. 42 [1987] 501).

After finding CCK in the CNS, further effects were discovered. For example, CCK plays a role in appetite regulation, analgesia, ptosis or catalepsy. Antagonists are important tools for the pharmacological investigation of all these effects. The presently known antagonists of cholecystokinin are derivatives of glutamic acid - proglumide - (A.L Rovati, Minerva Medica 1967, 58, 36551, Macovec et al., Eur. J. Med. Chem.-chim. Ther. 21 [1986] 9 78 (1987) 6304) or truncated or cyclic sequences of cholecystokinin (Spanarkel et al., J. Org. J. Biol. Chem. 258, 6746 [1983];

M. Ch. Galas Am. J. Physiol. 254, G176 [1988] and R.M. Freidinger et al., Proc. 19th. Europ. Peptide Symp. 1986 Porto Carras Greece, EP-PS 0132919).

Recently, it has been found that 3-substituted benzodiazepines also have high peripheral antagonist activity (Chang

R.U. Science 230 [1985] 177). Variations of the substituents resulted in further enhancement of action (Evans, B.E., Proc. Natl.

Acad. Be. USA83, [1986] 4918; U.S. Patent 4,554,272, U.S. Patent 4,563,451, U.S. Patent 4,559,338, European Patent 166,353, European Patent 166,354, European Patent 167920, European Patent 170024).

Recently, new effective antagonists have been developed based on truncated sequences (DD 272,652 and US Pat

DD 272 653). All peptide-based antagonists found so far had too little antagonistic activity

on (IC50 values 10 "" ⁶ to 10 " ⁷ moles).

Derivatives of benzodiazepine are only peripheral.

Object of the invention

The aim of the invention is to produce novel antagonists of cholecystokinin peptide-based, which have a higher antagonist activity than the comparable known antagonists.

Explanation of the essence of the invention

The invention has for its object to develop methods by which novel sequences of cholecystokinin (CCK) can be produced with antagonistic properties. The object was achieved by synthesizing C-terminally truncated sequences lacking the two C-terminal amino acids aspartic acid and phenylalanine. The amino acid Y, which now stands as the C-terminal amino acid, has been converted to the β-phenethylamide (PEA). The thus obtained truncated CCK sequences surprisingly show no CCK properties more, but are capable of CCK in concentrations to inhibit 10 ^-8 to 10 ^-9 mol / l. The compounds were prepared by peptide chemistry in a conventional manner using known protecting groups. For this purpose, Boc-Trp-X-ß-phenethylamide was prepared, freed from the N-protecting group with 2 N HCL / glacial acetic acid and reacted with Boc-Tyr-X-Gly-ONB to the pentapeptide. The Boc-Tyr-X-Gly-Trp-Y-PEA pentapeptide is deprotected, acylated and then sulfated with pyridinium acetyl sulfate. Final purification is isocratic by RP-18 medium pressure chromatography on 28% acetonitrile / 0.05N ammonium acetate pH 6.5. The invention will be explained in more detail with reference to embodiments.

example 1

Suc-Tyr (SE) -Met-Gly-Trp-Met-NH-CH ₂ -CH ₂ C ₆ H ₅

Boc-Trp-Met-PEA

The dipeptide was obtained by reaction of Boc-Trp-ONB with H-MeI-NH-CH ₂ -CH ₂ C ₆ H ₅ .

Mp 161-163 DC: CHCl ₃ / MeOH 9: 1, R _F = 0.72

[a] 2o ° = _14 ₍ 37 с = 1

SUC-Tyr-Met-Gly-Trp-Met-PEA

5 mmol of Boc-Tyr-Met-Gly-ONB (prepared according to Henklein et al. Z. Pharmazie 42, 414 [1987]) are reacted with 5 mol of H-Trp-Met-PEA in 30 ml of DHF. The solvent is concentrated to ¹ A of volume and the remaining oil is stirred into 10% KHSO ₄ .

The precipitate is dried and removed from the protective group by treatment with 10 ml of 2 N HCl / glacial acetic acid with the addition of 0.5 ml of mercaptoethanol.

The crude hydrochloride is acylated as usual with succinic anhydride in pyridine / DMF.

The solution is concentrated, triturated with 0.1 η HcI.

The resulting residue is filtered off with suction and washed with water.

SUC-Tyr-Met-Gly-Trp-Met-PEA MW = 1016

Mp 144-50 (dec.), R _F ethyl acetate Schwyzer's basic solution (Pyr: HOAc: H ₂ O = 20: 6: 11) = 70:30 suc-Tyr (SE) -Met-Gly-Trp-Met-PEA

MG 1097 Fp. 144-52X (under softening)

HPLC: Column ODS Hypersil 5μιη 2.1 x 100mm

Eluent A: 0.01 H ₃ PO ₄ pH 2.2 / CH ₃ CN

Gradient 28-42% CH ₃ CN

Flusso, 7ml; R _T = 1.18min Eluent B: 0.01m ammonium acetate pH 4.13 / 25% CH ₃ CN; R ₁ = 4.13 min.

Example 2

On the isolated guinea pig ileum SUC-Tyr- antagonized (SE) -Met-Gly-Trp-Met-NH-CH ₂ -CH ₂ -C _e H5 contraction inducing effect of CCK-8 competitive. The substance showed up to a concentration of 10 ~ ⁵ mol no contracting effect. The pA ₂ value for SUC-Tyr (SE) -Met-Gly-Trp-Met-NH-CH ₂ -CH ₂ -C ₆ H ₅ is 8.04 ± 0.19 (n = 6).

Claims (3)

1. Process for the preparation of novel cholecystokinin pentapeptides of the general formula I, ACyI-TYr (SE) -X-Gly-TrP-Y-NH-CH ₂ -CH ₂ -C ₆ H ₅ I, in which acyl is an amino acid residue A or a carboxylic acid residue or a protecting group, SE is a sulfate ester residue on tyrosine, X is an amino acid residue B and Y is Met, Nie or Leu, characterized in that the β-phenethylamide (PEA) derivatives Boc Trp-X-PEA, deprotected from the N-protecting group, reacted with Boc-Tyr-X-Gly-ONB to give the pentapeptides Boc-Tyr-X-Gly-Trp-Y-PEA which deprotected then acylated and then sulfated. 2. The method according to claim 1, characterized in that the amino acid residue A preferably from aspartic acid, the carboxylic acid residue preferably from succinic acid, the protective group z. Example of t-butyloxycarbonyl (Boc) or benzyloxycarbonyl (Z) and the amino acid residue B preferably Thr, Nie, Leu or Met means. 3. The method according to claim 1 and 2, characterized in that in the compounds of formula I L-Trp can be replaced by D-Trp.