DE2727048C2 - 8-norleucyl-ceruletide - Google Patents

Description

The invention relates to decapeptides referred to as 8-norleucyl-ceruletide and the salts according to the preceding claims.

The decapeptide according to the invention according to claim 2 is pyroglutamyl-glutaminyl-aspartyl-tyrosyl-threonyl-glycyl-tryptophyl-norleucyl-aspartyl-phenylalaninamide, in which the phenol group of the tyrosyl residue is substituted with a sulfuric acid residue and in which all amino acids (with the exception of glycine) are L-configuration exhibit. The invention also includes the non-toxic, pharmaceutically acceptable salts of this decapeptide with organic and inorganic bases. The decapeptide according to the invention shows a strong biological activity and can be used in the diagnosis of various diseases in humans and animals.

In US-PS 34 72 832 the synthesis of a very similar decapeptide (Ceruletid: generic name) is described. However, because of the presence of a methionyl radical in the molecule, this decapeptide is associated with many disadvantages, in particular with regard to the purification of the intermediate products and the end product of the synthesis and with regard to the stability of the finished dosage form.

These disadvantages also occur due to the slight oxidation of the methyl mercapto group in the methionine residue to the corresponding sulfoxide by atmospheric oxygen.

The oxidized ceruletide is almost completely devoid of biological activity. In order to remedy this deficiency - and this is the aim of the present invention - methionyl was substituted by norleucyl in the "ceruletide" molecule.

The new decapeptide obtained in this way is very stable to oxidizing agents and surprisingly shows increased biological effectiveness in comparison with the "ceruletide".

The synthesis of the new decapeptide according to the present invention consists essentially in successive condensations of the protected amino acids or polypeptides. In the amino acids and polypeptides that are condensed according to methods known per se in polypeptide chemistry, the amino and carboxyl groups that are not involved in the formation of the peptide bond are protected by a protective group that can be removed by acidolysis, saponification or hydrogenolysis . The following protective groups can be used to protect the amino groups: carbobenzoxy (benzyloxycarbonyl), tert-butyloxycarbonyl, trityl (triphenylmethyl), formyl or trifluoroacetyl. The following protective groups, for example, can be used to protect the carboxyl groups: methyl, ethyl, tert-butyl, benzyl or p-nitrophenyl.

The condensation between an amino group of one molecule and a carboxyl group of another molecule to form a peptide bond can be effected by an activated acyl derivative such as a mixed anhydride, an azide, a p-nitrophenyl ester or a 2,4,5-trichlorophenyl ester and the like, or by direct condensation between a free amino group and a free carboxyl group in the presence of a condensing agent such as dicyclohexylcarbodiimide or 1-cyclohexyl-3-morpholinylcarbodiimide. The condensation can be carried out in a solvent such as N, N-dialkylformamide, a lower aliphatic nitrile, a pyridine, tetrahydrofuran, dimethylformamide, acetonitrile or pyridine. The reaction temperatures are - 20 ° C to room temperature. The reaction time is generally 6 to 120 hours.

The synthesis scheme is chosen so that the risk of racemization is avoided; accordingly, the two pentapeptides I and II are coupled by the azide method. The azide pentapeptide (I A) is obtained from the hydrazide (I), which was obtained by the stepwise construction as described in Example 1. The pentapeptide pyroglutamyl-glutaminyl-aspartyl-tyrosyl-O-acetyl-threonine-azide (IA) is prepared in a solvent such as dimethylformamide with the pentapeptide glycyl-tryptophyl-norleucyl-aspartyl-phenylalanine amide (II), as in Example 2 described, condensed, the decapeptide pyroglutamyl-glutaminyl-aspartyl-tyrosyl-O-acetyl-threonyl-glycyl-tryptophyl-norleucyl-aspartyl-phenylalanine amide (III) is obtained. The decapeptide (III) is treated at low temperature with an anhydrous pyridine-sulfuric anhydride complex, the decapeptide being pyroglutamyl-glutaminyl-aspartyl-tyrosyl (O-sulfate) -O-acetyl-threonyl-glycyl-tryptophyl-norleucyl-aspartyl-phenylalaninamide (IV ) is obtained in which the phenol group of the tyrosyl radical is sulfated and the hydroxyl group of the threonyl radical is protected by an acetyl group. By mild alkaline hydrolysis of the decapeptide (IV) the decapeptide pyroglutamyl-glutaminyl-aspartyl- tyrosyl (O-sulfate) -threonyl-glycyl-tryptophyl-norleucyl-aspartyl-phenylalanine amide (V) in which the hydroxyl group of the threonyl radical is free and the phenol group of the tyrosyl radical is sulfated. The condensation can be represented by the following scheme in which the symbols correspond to the symbols commonly used in polypeptide chemistry.

(IA) (II)

(III)

(IV)

The examples are intended to explain the present invention in more detail.

example 1

Preparation of the compound (I)

(I)

8.04 ml of N-methylmorpholine and 7.01 ml of ethyl chloroformate were added successively at a temperature of -15 ° C. to a solution of 16.05 g of Boc-Thr-OH in 170 ml of anhydrous tetrahydrofuran. After stirring at this temperature for 2 minutes, 12.16 g of NH [deep 2] -NH-Z (Z = benzyloxycarbonyl) in 50 ml of anhydrous tetrahydrofuran were added. The reaction mixture was left with stirring for 2 hours at -10 ° C and then for 16 hours at 0 to 5 ° C. The solvent was removed in vacuo and the residue was taken up in ethyl acetate and washed successively with 1N hydrochloric acid, a 5% strength aqueous sodium bicarbonate solution and a saturated sodium chloride solution to neutrality. After removal of the solvent, 26.5 g of Boc-Thr-NH-NH-Z (Boc = tert-butyloxycarbonyl) (VI) were obtained as a white foam, yield 98%. The analytical sample was obtained by crystallization from diethyl ether, melting point 83 to 84 ° C .;

(c = 1 in dimethylformamide).

15 ml of 6N HCl in 15 ml of glacial acetic acid were added to a solution of 26.5 g of Boc-Thr-NH-NH-Z (VI) in 50 ml of diethyl ether. After the diethyl ether had evaporated, 150 ml of acetyl chloride were added at a temperature of 0 ° C. over the course of 2 hours. The mixture was kept at 0 ° C. for a further 10 minutes, after which 1500 ml of diethyl ether were added. The precipitate thus formed was filtered and crystallized from methanol-isopropanol, whereby (VII)

Mp. 120 ° C (dec.),

(c = 1 in demethylformamide), yield 84%, were obtained,

(VII)

were condensed by ethoxyformic anhydride with 7.52 g of Boc-Tyr-OH, as described for the preparation of the compound (VI). The crude synthetic material thus obtained was crystallized from diethyl ether, whereby

(VIII)

Mp. 133 to 134 ° C,

(c = 1 in dimethylformamide), 78% yield.

(VIII)

were dissolved in 130 ml of 1.33 N hydrogen chloride in acetic acid. After 25 minutes at room temperature, the solution was evaporated to dryness in vacuo and the residue was triturated with diethyl ether. The solid obtained in this way was filtered and crystallized from anhydrous ethanol-diethyl ether, with a quantitative yield

(IX)

Mp. 150 to 160 ° C,

(c = 1 in AcOH), 95% yield.

(IX)

were by using ethoxyformic anhydride

(prepared as described in J. Am. Chem. Soc. 81, p. 620 [1965]). After crystallization from dimethylformamide-ethyl acetate were

(X)

obtained, m.p. 185 to 186 ° C,

(c = 1 in dimethylformamide), yield 90%.

By acidolysis of

(X)

with a solution of 1.33 N hydrogen chloride in acetic acid

(XI)

obtain. After crystallization from methanol diethyl ether, the compound melted at 173 to 176 ° C,

(c = 1 in AcOH 95%), yield 98%.

(XI)

were condensed by ethoxyformic anhydride with 3.79 g of Boc - Gln - OH, after crystallization from methanol-ethyl acetate

(XII)

Mp. 205 to 206 ° C,

(c = 1 in dimethylformamide), yield 83%. By acidolysis of

(XII) with a solution of 1.33 N hydrogen chloride in acetic acid

10.76 g (quantitative yield)

(XIII)

obtained, m.p. 120-140 ° C. After crystallization from anhydrous ethanol-diethyl ether, the product melted at 125 to 135 ° C (decomp.),

(c = 1 in dimethylformamide).

(XIII)

were condensed by ethoxyformic anhydride with 2.89 g of Z - Pyr - OH (prepared according to Liebigs Ann. 640, p. 145 [1961]), after crystallization from dimethylformamide-methanol

(XIV)

were obtained, m.p. 216 to 220 ° C,

(c = 1 in dimethylformamide), yield 74%.

(XIV)

were dissolved in 150 ml of dimethylformamide containing 1.1 equivalents of hydrochloric acid and hydrogenated at room temperature in the presence of 10% palladium on carbon for 5 hours. The catalyst was filtered off and washed thoroughly with dimethylformamide. The filtrate was then evaporated to dryness in vacuo.

The solid residue was crystallized from absolute ethanol, whereby

(I)

were obtained, m.p. 189 to 191 ° C,

(c = 1 in dimethylformamide), yield 80%.

Example 2

Preparation of the compound (II)

H - Gly - Trp - Nle - Asp - Phe - NH [deep 2] (II)

To a solution of 6.8 ml of N-methylmorpholine and 5.9 ml of ethyl chloroformate were added successively at a temperature of -15 ° C. in 120 ml of anhydrous tetrahydrofuran. After stirring at this temperature for 2 minutes, 12.4 g of Phe - NH [deep 2] HCl in 100 ml of anhydrous dimethylformamide containing 6.8 ml of N-methylmorpholine were added.

The reaction mixture was allowed to stand with stirring for 2 hours at -10 ° C and then for 16 hours at 0 to 5 ° C. The solvent was removed in vacuo and the residue was crystallized from ethyl acetate-petroleum ether, whereby

(XV)

Mp. 138 ° C,

(c = 1 in dimethylformamide), yield 70%.

(XV) were dissolved in 200 ml of 1.33 N hydrogen chloride in acetic acid. After 25 minutes at room temperature, the solution was evaporated in vacuo and the residue was triturated with diethyl ether. The solid product thus obtained was filtered and crystallized from ethyl acetate-petroleum ether, whereby

(XVI)

were obtained, m.p. 183 to 184 ° C,

(c = 1.2 in MeOH), 99% yield.

(XVI)

were condensed by ethoxyformic anhydride, as described for the compound (XV), with 8.4 g of Boc - Nle - OH, with 192 g of protected tripeptide

(XVII)

were obtained, m.p. 156 ° C,

(c = 1.3 in MeOH), yield 91%.

By acidolysis of

(XVII)

with 1.33 N hydrogen chloride in acetic acid as described for compound (XVI)

(XVIII)

obtained, m.p. 224 to 225 ° C,

(c = 1.1 in MeOH), 97% yield.

(XVIII)

were condensed by ethoxyformic anhydride with 10.76 g of Z - Trp - OH, as described for the preparation of the compound (XV), with 24.7 g of the protected tetrapeptide

(XIX)

were obtained, m.p. 220 to 222 ° C,

(c = 1.2 in dimethylformamide), 97% yield.

(XIX)

were dissolved in 200 ml of a mixture of dimethylformamide and acetic acid (1: 1 V / V) and reduced in a stream of hydrogen in the presence of 10% palladium on carbon at room temperature. After 1 equivalent of hydrochloric acid had been added, the catalyst was filtered off, washed with dimethylformamide and the solvent was evaporated off in vacuo, with

15.75 g of H - Trp - Nle - Asp - PhNH [deep 2]. HCl (XX)

were obtained, m.p. 222 ° C,

(c = 1 in MeOH), yield 83%.

To a solution of the tetrapeptide

H - Trp - Nle - Asp - PheNH [deep 2]. HCl (XX)

in 100 ml of dimethylformamide, 12.7 g of Boc-Gly-OCP (OCP = 2,4,5-trichlorophenyl ester) and 2.86 ml of N-methylmorpholine were added at 0 ° C. in succession. After stirring for 60 hours at 0 to 5 ° C, the solvent was removed in vacuo and the residue was triturated with diethyl ether, filtered and first from di- methylformamide-ethyl acetate and then crystallized from methanol, whereby

8.55 g Boc - Gly - Trp - Nle - Asp - Ph - NH [deep 2], (XXI)

were obtained, m.p. 218 ° C

(c = 1 in dimethylformamide), yield 45%.

By acidolysis of 8.55 g of the protected pentapeptide

Boc - Gly - Trp - Nle - Asp - Phe - NH [deep 2] (XXI)

in 170 ml of formic acid for 4 hours at room temperature and in the presence of 0.85 ml of 2-mercaptoethanol

5.98 g H - Gly - Trp - Nle - Asp - Phe - NH [deep 2] (II)

obtained, m.p. 212 ° C,

(c = 0.44 in dimethylformamide-HMPA 1: 1), yield 81%.

Example 3

Preparation of the compound (III)

(III)

To a solution of

(I)

in 10 ml of anhydrous dimethylformamide, 1.22 ml of 1.84 N hydrogen chloride in anhydrous tetrahydrofuran and 0.176 ml of n-butyl nitrite were added successively at a temperature of -25 ° C. After stirring at this temperature for 15 minutes, 0.58 ml of N-methylmorpholine and a solution of

0.953 g H - Gly - Trp - Nle - Asp - Phe - NH [deep 2] (II)

in 20 ml of a mixture of dimethylformamide and hexamethylphosphoramide (1: 1 V / V) containing 0.165 ml of N-methylmorpholine, the reaction mixture was kept at -10 ° C for 3 days and at 5 ° C for 2 days. After evaporation of the volatile solvents in vacuo, the product was precipitated by dilution with a cold aqueous citric acid solution. The raw material yielded after two crystallizations from dimethylformamide-methanol

(III)

Mp. 225 ° C,

(c = 1 in dimethylformamide), yield 40%.

Example 4

Preparation of the compound (IV)

(IV)

0.320 g of the compound (III) dissolved in 10 ml of anhydrous dimethylformamide was added at -10 ° C. to 5 g of a pyridine-sulfuric anhydride complex suspended in 25 ml of anhydrous pyridine. The mixture was allowed to stand at room temperature for 18 hours and then cooled with a 1 M aqueous solution of potassium bicarbonate. After evaporation to dryness in vacuo, the residue was triturated with dimethylformamide and filtered to remove the insoluble inorganic salts. The filtrate was again evaporated to dryness in vacuo to give 0.320 g of the crude sulfated decapeptide of formula (IV).

Example 5

Production of the decapeptide (V)

(V)

0.320 g of the raw material (IV) obtained in Example 4 was dissolved in 10 ml of water. 2.5 equivalents of 1N sodium hydroxide was added and the solution was kept at room temperature for 3 hours. After neutralizing the alkaline solution with solid CO [deep 2], the mixture was evaporated to dryness in vacuo. The crude residue obtained in this way was taken up in dimethylformamide and filtered from inorganic salts. The yellow filtrate was again evaporated to dryness in vacuo and the residue was dissolved in a little water and treated at a temperature of 0 ° C. with Dowex® 50W (H (+)) with stirring for 5 minutes. The mixture was filtered and the filtrate was extracted three times with n-butanol. The combined organic extracts were washed with a little water and made alkaline with diethylamine. The solvent was evaporated off in vacuo and diethyl ether was added to the concentrated solution, whereby

(V)

were precipitated as diethylammonium salt. The product was then purified by dissolving it in 2 ml of dimethylformamide and applying the resulting solution on a small column (27 x 1.5 cm) Amberlite® XA-D2 using water and then a mixture of water and methanol as the eluent increasing amount of methanol was eluted to a concentration of 50%. 0.150 g of end product were obtained,

(c = 1 in dimethylformamide).

The biological activities of the [Nle [high 8]] ceruletide prepared according to Example 5 were determined in comparison with ceruletide. Ceruletide is a decapeptide developed by Erspamer et al. (Nature 212, 204, 1966) was found in the skin of Australian frogs (Hyla caerulea) and which shows physiological activities similar to those of cholecystokinin-pancreozymin (G. Bertaccini et al., Br. J. Pharmacol. 34, 291, 1968 ; G. Bertaccini et al., Ibidem 37, 185, 1969).

1) The effect of [Nle [power 8]] ceruletide on gallbladder motility was examined in situ in guinea pigs by intravenous injection. If the strength of the ceruletide is set equal to 100, the relative strength of the [Nle [to the power of 8]] ceruletide turns out to be 130 to 160.

2) The effect on pancreatic secretion was determined in anesthetized rats as described by G.J. Dockray (J. Physiol. 225, 679, 1972). Two 10 minute fractions of the pancreatic juice were collected before and seven fractions after intravenous administration of the peptides. The ratio of dose to volume and protein output within 70 minutes after treatment are shown in the table.

If the strength of the ceruletide is set equal to 100, the strength of the [Nle [to the power of 8]] ceruletide appears to be around 120 to 130.

Based on the above results, the [Nle [power 8]] ceruletide can be advantageously used as a diagnostic agent for the same clinical indications as ceruletide in both biliary and pancreatic diseases.

Tabel

Effect of intravenous injections of [Nle [power 8]] ceruletide and ceruletide on pancreatic secretion in rats

Dose volume output protein output

ng / kg IV µl / 70 min mg / 70 min

[Nle [to the power of 8]] - Cerul. Cerul. [Nle [to the power of 8]] - Cerul. Ceruletide

25 59 57 7 5

50 67 66 11 9

100 79 70 18 16

200 96 93 28 21

Mean value from 6 to 7 rats.

Claims (2)

1. 8-Norleucyl-ceruletide of the sequence H-Pyr-Gln-Asp-Tyr-Thr-Gly-Trp-Nle-Asp-Phe-NH [deep 2], in which the tyrosyl radical with free hydroxyl group or as sulfuric acid monoester and the threonyl radical with free hydroxyl group or as acetate, and their non-toxic salts with bases. 2. 8-norleucyl-ceruletide of the formula and its sodium, potassium, magnesium, calcium, diethylammonium and dicyclohexylammonium salts.