DD276482B5 - Process for the preparation of new cholecystokinin sequences - Google Patents

Description

Field of application of the invention

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

Acyl-Tyr (SE) -X-Gly-Trp-Y-NH-Ad,

in which 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, such as t-butyloxycarbonyl (Boc) or benzyloxycarbonyl (Z), and SE is a sulfate ester residue on tyrosine. X is an amino acid residue, preferably Thr, No, Leu or Met and Y is Met, No, Leu, Met-Asp, Nle-Asp or Leu-Asp. For better enzymatic stability, L-Trp can be replaced by D-Trp. Such peptides are novel, highly effective antagonists of cholecystokinin and are valuable tools for pharmacological studies. Areas 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 еіпѣ series 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. OEZZ., Total 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 (AL Rovati, Minerva Medica 1967, 58, 6551, Macovec et al., Eur. J. Med. Chem.-chim. Th., 21 [1986] 9), des Tryptophan Benzotript- (Hahne, WF Jensen, T; LempG.F.

and J.D. Gardner 1983, Proc. Natl. Acad. Be. 78 [1987] 6304) or truncated or cyclic sequences of cholecystokinin (Spanarkel et al., J. Biol. Chem. 258, 6746 [1983]; M. Ch. Galas Am. J. Physiol. 254, G 176 [1988] and RM 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

RU Science 230 [1985] 177). Variations of the substituents led to further enhancement of activity (Evans, BE, Proc. Natl.

Acad. Be. USA 83, [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 potent antagonists have been developed based on truncated sequences. (DD 272 652 and DD 272 653). All previously found antagonist peptide-based had a too low antagonistic activity at IC ₅₀ values {10 ^-6 to 10 ^-7 mol).

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 has been achieved by synthesizing C-terminally truncated sequences lacking the C-terminal amino acids phenylalanine or phenylalanine and aspartic acid. The terminal amino acid Y was converted to the 1-adamantylamide. The thus obtained truncated CCK sequences surprisingly show no agonist properties more but are capable of cholecystokinin in concentrations of 10 ^-7 to 10 ^-8 mol / Izu inhibit. The novel compounds were constructed by peptide chemistry in usually either stepwise or by fragment condensation using known protecting groups starting from BOC-Y-NH-adamantyl. Subsequently, the protected peptide is deprotected, acylated and then sulfated with pyridinium acetyl sulfate. The final purification is carried out by preparative HPLC on RP-18isocratic with 25% acetonitrile / 0.05 N ammonium acetate solution pH 6.5

 The invention will be explained in more detail with reference to exemplary embodiments.

embodiments

Example 1 Suc-TyrlSEl-Met-Gly-Trp-Met-Asp-NH-adamantyl

Boc-Trp-Met-OH is reacted with H-Asp (OtBu) -NH-Ad to form Boc-Trp-Met-Asp (OtBu) -NH-Ad.

After cleavage of the Boc and OtBu ester protecting group, the tripeptide H-Trp-Met-Asp-NH-Ad is extended to hexapeptide with Boc-Tyr-Met-Gly-ONB. Renegotiation of the Boc-protecting group and acylation with succinic anhydride lead to the Suc-Tyr-Met-Gly-Trp-Met-Asp-NH-Ad.

Provide Suc-TyrtSEl-Met-Gly-Trp-Met-Asp-i-adamantyl amide sulfation with Pyridiniumacetylsulfat and purification by preparative HPLC 0.05 N ammonium acetate / 25% _CH3 CN pH 6.5.

Mp. 205-211 ⁰ C [a] = -42.34 g ° (C = 1.022, DMF)

HPLC: Column ODS-Hypersil 5pm 2.1 x 100mm

0.01 moles of H 2 PCa-acetonitrile 28 ° / o-A2% (6 min)

Rt = 2.218 uniform

Example 2

On isolated guinea pig ileum, suc-TyriSEl-Met-Gly-Trp-Met-Asp-NH-adamantyl competitively antagonizes the contraction-inducing effect of CCK-8. Up to a concentration of 10 ~ ⁵ mol / l, the substance showed no contracting intrinsic activity

The pA ₂ value of Suc-Tyr (SE) -Met-Gly-Trp-Met-Asp-NH-adamantyl is 7.20 ± 0.15 (n = 8).

Claims (4)

1. A process for the preparation of new cholecystokinin sequences of general formula I, Acyl-Tyr (SE) -X-Gly-Trp-Y-NH-Ad I, in which acyl represents an amino acid residue A or a carboxylic acid residue or a protecting group, SE stands for a sulfate ester moiety on tyrosine, X stands for an amino acid residue B and Y stands for an amino acid residue C, characterized in that the compounds of formula I are built up stepwise or by fragment condensation and after Cleavage of the protecting groups are acylated and sulfated. 2. The method according to claim 1, characterized in that the synthesis is carried out using protective groups and starting from BOC-Y-NH-adamantyl. 3. The method according to claim 1 and 2, characterized in that the amino acid residue A preferably from aspartic acid, the carboxylic acid residue preferably from succinic acid, the protective groups z. B. from t-butyloxycarbonyl (BOC) or benzyloxycarbonyl (Z), the amino acid residue B preferably for Thr, Nie, Leu or Met and the amino acid residue C for Met, No, Leu, Met-Asp, Nle-Asp or Leu-Asp stand. 4. The method according to claim 1 to 3, characterized in that in the compounds of formula I L-Trp can be replaced by D-Trp.