JPS6115080B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel therapeutically useful decapeptide, a salt thereof, and a method for producing the same. The novel decapeptide according to the invention is pyroglutamyl-glutaminyl-aspartyl-tyrosyl-
Threonyl-glycyl-tryptopyl-norleucyl-aspartyl-phenylalanine amide, in which the phenyl group of the tyrosyl group is blocked by a sulfate group, and all amino acids except glycine have the L configuration. The present invention also includes non-toxic, pharmaceutically acceptable salts of the decapeptides with organic and inorganic bases. The novel decapeptides according to the invention have a high degree of biological activity and can be usefully used in the treatment of various unpleasant conditions both in the human and veterinary fields. U.S. Patent No. 3,472,832 (October 14, 1969)
The production of a similar decapeptide (species name: serretide) has been described and claimed.
However, the decapeptide has many drawbacks due to the presence of a methionyl group in the molecule. Thus, there are difficulties with respect to the purification steps of the intermediate and final products, and especially with respect to the stability of the "finished dosage form". These drawbacks arise because the mercapto group in the methionine group is easily oxidized by, for example, oxygen in the air and converted into the corresponding sulfoxide. The oxidized ceruletide so produced is almost completely devoid of biological activity. The object of the invention is to eliminate the above-mentioned drawbacks,
For this purpose, the amino acid methionine is replaced with n-leucine in the molecule of "ceruletide". The novel decapeptide thus obtained is very stable towards oxidizing agents and also exhibits surprisingly significant biological activity compared to the known "ceruletide". The preparation of the novel decapeptides according to the invention essentially consists of a suitable sequential condensation of protected amino acids or polypeptides. The condensation is carried out in such a way that the resulting decapeptide has the desired sequence of amino acids. Amino acids and polypeptides condensed by methods known to polypeptide chemistry contain amino groups and carboxyl groups that do not participate in the formation of the peptide chain, which are blocked by protecting groups that can be removed by hydrolysis, saponification or hydrogenolysis. It has a group. For the protection of amino groups, the following protecting groups can be used, for example: carbobenzoxy (benzyloxycarbonyl), t-butyloxycarbonyl, trityl (triphenylmethyl),
Formyl or trifluoroacetyl. For the protection of carboxyl groups, the following protecting groups can be used, for example: methyl, ethyl, tert-butyl, benzyl or p-nitro-phenyl. The reaction that condenses the amino group of one molecule with the carboxyl group of another molecule to form a peptide chain is performed using active acyl derivatives such as mixed anhydrides, azides, p-nitro-phenyl-esters or 2,4,5-trichlorophenyl. This can be done using ester or the like. Alternatively, it can also be carried out by directly condensing free amino groups and free carboxyl groups in the presence of a condensing agent such as dicyclohexyl-carbodiimide or 1-cyclohexyl-3-morpholinyl-carbodiimide. The condensation can be carried out in solvents such as N.N-di-lower-alkyl-formamides such as dimethylformamide, lower aliphatic nitriles such as acetonitrile, pyridine or tetrahydrofuran. Reaction temperature is -20℃
and room temperature. Reaction times are generally 6 to 120 hours. The manufacturing method is selected in such a way that the risk of racemization is avoided. Therefore, the coupling of two pentapeptides ()+() is performed by the azide method. The azide pentapeptide (A) is obtained from the hydrazide () obtained by stepwise elongation as described in Example 1.
The pentapeptide, glycyl-tryptopyl-norleucyl-aspartyl- prepared as described in Example 2 from the pentapeptide, pyroglutamyl-glutaminyl-aspartyl-tyrosyl-O-acetyl-threonine-azide (A) in a solvent such as dimethylformamide. Condensation with phenylalaninamide () gives the decapeptide, pyroglutamyl-glutaminyl-aspartyl-tyrosyl-O
-acetyl-threonyl-glycyl-tryptopyl-norleucyl-aspartyl-phenylalaninamide () is obtained. By treating the same decapeptide () with an anhydrous pyridine-sulfuric anhydride complex at low temperature, a decapeptide with a sulfated phenol group in the tyrosyl group and a hydroxyl group in the threonyl group protected by an acetyl group is produced. Glutamyl-glutaminyl-aspartyl-tyrosyl (O-sulfate)-O-acetyl-threonyl-glycyl-tryptopyl-norleucyl-aspartyl-phenylalaninamide () is obtained. Finally, the same decapeptide ()
By mild alkaline hydrolysis of , the hydroxyl group of the threonyl group is liberated and the phenol group of the tyrosyl group is sulfated. Glycyl-tryptopyl-norleucyl-aspartyl-phenylalaninamide () is obtained. The condensation described above can be expressed as follows using symbols commonly used in polypeptide chemistry: The following examples illustrate the invention without limiting it. Example 1 Intermediate product (): Preparation of Boc−Thr− in 170 ml of anhydrous tetrahydrofuran
8.04 N-methyl-morpholine in a solution of 16.05 g OH
ml and 7.01 ml of ethyl chloroformate are added sequentially at -15°C. After stirring for 2 minutes at the same temperature, 12.16 g of _NH2 -NH-Z (Z=benzyloxycarbonyl) in 50 ml of anhydrous tetrahydrofuran are added. The reaction mixture was heated at -10°C for 2 hours, followed by 0
Keep under stirring for 16 hours at ~5°C. The solvent was removed in vacuo and the residue was taken up in ethyl acetate and diluted with 1 N hydrochloric acid, 5 ml of sodium bicarbonate, and
% aqueous solution and a saturated solution of sodium chloride until neutral. Boc− after removing the solvent
26.5 g of Thr-NH-NH-Z (Boc=t-butyloxycarbonyl) () are obtained as a white foam. Yield = 98%. Analytical grade samples are obtained by crystallization from diethyl ether: melting point 83-84°C, [α] ²⁵ _D -14
゜(c=1, DMF). Boc−Thr−NH− in 50 ml of diethyl ether
A solution of 26.5 g of NH-Z() in 15 ml of glacial acetic acid
Add 15 ml of specified HCl. After evaporating the diethyl ether, 150 ml of acetyl chloride are added over a period of 2 hours at 0°C. The mixture was allowed to stand at 0°C for 10 minutes and then diluted with diethyl ether.
Add 1500ml. The precipitate formed at that time was filtered and crystallized from methanol-isopropanol.

[Formula] Obtain 21g. Melting point: 120°C (decomposed), [α] ²⁰ _D +10° (c=1, DMF), yield = 84%. 15.19 g of Boc-
Condenses with 7.52g of TyrOH. The crude product so obtained was crystallized from diethyl ether. Obtain 15.19g. Melting point 133-134°C, [α] ²⁰ _D −1.6° (c=1, DMF), yield 78%. 12.8g to 130ml of a 1.33N solution of hydrogen chloride in acetic acid
Dissolve inside. After 25 minutes at room temperature, the solution is evaporated to dryness in vacuo and the residue is slimed with diethyl ether. The solid thus obtained was filtered and crystallized from anhydrous ethanol-diethyl ether in quantitative yield. Obtain 11.4g. Melting point 150-160°C, [α] ²⁰ _D +29° (c=1, AcOH), yield = 95%. 11.4 g using ethoxy formic anhydride,

[Formula] (Bzl=benzyl) (prepared as described in Journal of the American Chemical Society (J. Am. Chem. Soc., 1965, Vol. 81, p. 620)) is condensed with 7.28 g. By crystallization from dimethylformamide-ethyl acetate, Obtain 15.66g. Melting point 185-186℃, [α] ^20D _-15.7
°(c=,DMF), yield=90%. In a 1.33N solution of hydrogen chloride in acetic acid By hydrolyzing 12.2g, Get 11g. After crystallization from methanol-diethyl ether, the compound has a melting point of 173-176°C. [α] ²⁰ _D +16.3° (c=1, AcOH95%), yield = 98%. 11 g of Boc-Gln-
By condensing with 3.79g of OH and crystallizing from methanol-ethyl acetate Obtain 11.6g. Melting point: 205-206°C, [α] ²⁰ _D -19.9° (c = 1, DMF), yield = 83%. By acidolyzing 11.58 g with a 1.33N solution of hydrogen chloride in acetic acid, 10.76 g (quantitative yield) are obtained. Melting point 120-140
℃. The melting point of the same product after crystallization from anhydrous ethanol-diethyl ether is 125-135°C (decomposed). [α] ²⁰ _D −8.7° (c=1, DMF). 9.25g of Z-Pyr-
OH (Liebig´s Ann.)
1961, Vol. 640, p. 145) and crystallization from dimethylformamide-methanol. Obtain 8.55g. Melting point 216-220°C, [α] ²⁰ _D -24° (c=1, DMF), yield = 74%. 8.55 g are dissolved in 150 ml of dimethylformamide containing 1.1 equivalents of hydrochloric acid and hydrogenated for 5 hours at room temperature in the presence of 10% palladium on charcoal. Separate the catalyst and wash thoroughly with dimethylformamide. The liquid is then evaporated to dryness in a vacuum. The solid residue was crystallized from absolute ethanol and Obtain 4.75g. Melting point 189-191°C, [α] ²⁰ _D −12° (c=1, DMF), yield = 80%. Example 2 Preparation of intermediate product (): H-Gly-Trp-Nle-Asp-Phe- _NH2 () in 120 ml of anhydrous tetrahydrofuran

[Formula] To 20 g of solution, add 6.8 ml of N-methyl-morpholine and 5.9 ml of ethyl chloroformate to -15
Add sequentially at ℃. After stirring for 2 minutes at the same temperature, in 100 ml of anhydrous dimethylformamide containing 6.8 ml of N-methyl-morpholine.
Add 12.4 g of Phe-NH ₂ .HCl. The reaction mixture is kept with stirring at -10°C for 2 hours and then at 0-5°C for 16 hours. The solvent was removed in vacuo and the residue was dissolved in ethyl acetate.
Crystallized from petroleum ether,

[Formula] Obtain 20.5g. Melting point 138℃, [α] ^20D _-28.3゜(c
=
1, DMF), yield = 70%.

[Formula] 20.2g to 200ml of a 1.33N solution of hydrogen chloride in acetic acid
Dissolve inside. After keeping at room temperature for 25 minutes, the solution is evaporated in vacuo and the residue is slimed with diethyl ether. The solid product so obtained was filtered and crystallized from ethyl acetate-petroleum ether,

[Formula] Obtain 17.3g. Melting point 183-184°C, [α] ²⁰ _D +11.9° (c = 1.2, MeOH), yield = 99%.

[Formula] 14.77g of Boc-Nle
- condensed with 8.4 g of OH, protected tripeptide, Obtain 19.2g. Melting point 156℃, [α] ^20D -30゜( _c =
1.3, MeOH), yield = 91%. 19.2 g by hydrolysis with a 1.33N solution of hydrogen chloride in acetic acid as described for the preparation of compound (). Obtain 16.6g. Melting point: 224-225°C, [α] ²⁰ _D -2.4° (c = 1.1, MeOH), yield = 97%. 16.5 g of Z-Trp-
Condensed with 10.76g of OH, protected tetrapeptide, Obtain 24.7g. Melting point: 220-222°C, [α] ²⁰ _D -33.7° (c = 1.2, DMF), yield = 97%. 24.7 g are dissolved in 200 ml of a mixture of dimethylformamide and acetic acid (1:1 v/v) and reduced at room temperature in a stream of hydrogen in the presence of palladium on charcoal (10%). 15.75 g of H-Trp-Nle-Asp-Phe- _NH2.HCl () are obtained by adding 1 equivalent of hydrochloric acid, separating off the catalyst, washing with DMF and evaporating the solvent in vacuo. Melting point 222℃, [α] ^20D _-21.1゜(c
= 1, MeOH), yield = 83%. Boc-Gly-OCP ( _OCP =2,4,5
-trichlorophenyl ester) 12.7g and N-
2.86 ml of methyl-morpholine are added sequentially at 0°C. After stirring for 60 hours at 0°-5°C, the solvent is removed in vacuo and the residue is slimed with diethyl ether, filtered and crystallized first from dimethylformamide-ethyl acetate and then from methanol. 8.55 g of Boc-Gly-Trp-Nle-Asp-Phe- _NH2 (XI) is obtained. Melting point 218℃, [α] ^25D _-18.3゜(c
=
1, DMF), yield = 45%. _H -Gly-Trp-Nle-Asp-Phe- _NH2 () 5.98 g are obtained. Melting point 212℃, [α] ^20D -35゜( _c =
0.44, DMF:HMPA 1:1), yield = 81%. Example 3 Intermediate product (): Preparation of in 10 ml of anhydrous dimethylformamide To 1.095 g of the solution at -25 DEG C. 1.22 ml of a 1.84N solution of hydrogen chloride in anhydrous tetrahydrofuran and 0.176 ml of n-butyl nitrite are added sequentially. After stirring for 15 minutes at the same temperature, 0.58 ml of N-methyl-morpholine and
A mixture of dimethylformamide and hexamethylphosphoramide (1:1 v/v) containing 0.165 ml20
A solution of 0.953 g of H-Gly-Trp-Nle-Asp-Phe- _NH2 () in ml is added. The reaction mixture is kept at -10°C for 3 days, followed by 2 days at 5°C. After evaporating the volatile solvent in vacuo, the product is precipitated by dilution with a cold aqueous solution of citric acid. The same crude product was purified from dimethylformamide and methanol.
By crystallizing the decapeptide, Obtain 0.780g. Melting point 225℃, [α] ^20D -15゜( _c =
1, DMF), yield = 40%. Example 4 Intermediate product (): Preparation 0.320 g of intermediate product () dissolved in 10 ml of anhydrous dimethylformamide are added at -10 DEG C. to 5 g of the pyridine-sulfuric anhydride complex suspended in 25 ml of anhydrous pyridine. The same mixture was prepared at room temperature for 18
Allow to stand for an hour, followed by quenching with a 1 molar aqueous solution of potassium bicarbonate. After evaporation to dryness in vacuo, the residue is slimed with dimethylformamide and filtered to remove insoluble inorganic salts. The solution was again evaporated to dryness in vacuo to obtain the crude sulfated decapeptide: Obtain 0.320g. Example 5 Decapeptide (): Preparation 0.320 g of the crude product () obtained as described in Example 4 above is dissolved in 10 ml of water. 2.5 equivalents of 1N sodium hydroxide are added and the solution is kept at room temperature for 3 hours. solid alkaline solution
After neutralization with _CO2 , the mixture is evaporated to dryness in vacuo. The crude residue so obtained is taken up in dimethylformamide and the inorganic salts are separated off. The yellow liquid was again evaporated to dryness in vacuo and the residue was dissolved in a small amount of water and dissolved in Dowex 50W (H ⁺ ) (registered trademark) with stirring at 0°C.
Process for 5 minutes. The same mixture was filtered and the liquid was
- Extract three times with butanol. Wash the combined organic extracts with a small amount of water;
Make alkaline with diethylamine. Evaporate the solvent in vacuo and add diethyl ether to the same concentrated solution to obtain a crude solution. 0.320 g of diethylammonium salt of The same product was then dissolved in 2 ml of dimethylformamide and the solution so obtained was purified using Amberlite ( Purify by chromatography on a small column (27 x 1.5 cm) of Amberlite) XA-D2®. Yield is 0.150
It is g. [α] ²⁰ _D −23° (c=1, DMF). Biological Activity [Nle ⁸ ]-Celluletide The biological activity of the novel [Nle ⁸ ]-Cellletide according to the present invention was determined in comparison with that of known celluletide. Ceruletide is a decapeptide discovered in the skin of the Australian frog, Hyla caerulea, by Erspamer and others (Nature).
1966, Vol. 212, p. 204), and has a physiological activity similar to that of cholecystokinin-pancreothymine (G. Bertaccini et al., Journal of Pharmacology, 1968, Vol. 34, p. 291; G. Bertaccini et al., 1969, Volume 37, 185
page). (1) The relative effects of [Nle ⁸ ]-celretide on gallbladder activity were evaluated in situ by intravenous injection into guinea pigs. The efficacy of celretide
100, the relative potency of [ ^Nle8 ]-cereletide was 130-160. (2) The relative effects on pancreatic secretions were evaluated in anesthetized mice as described by GJ Dockray (J. Physiol., 1972, Vol. 225, p. 679). did. Pancreatic secretions were collected twice at 10 min intervals before each peptide intravenous injection and seven times after the injection. The dose-response relationship for volume and protein excretion within 70 minutes after treatment is shown in the table below.

Table: The average values shown in this table are from 6-7 mice. From the table above, the potency of known celretide is 100
Therefore, it is clear that the potency of the new [Nle ⁸ ]-cereletide according to the present invention is about 120-130. Based on the above results, [Nle ⁸ ] according to the present invention
- Ceruletide can be advantageously proposed as a therapeutic agent for both cholecystitis and pancreatitis, in the same direction as the known ceruletide.

Claims (1)

[Claims] 1. The following formula using symbols used in polypeptide chemistry: or a non-toxic, pharmaceutically acceptable salt thereof with an organic or inorganic base. 2 The following formula using symbols used in polypeptide chemistry: The pentapeptide, pyroglutamyl-
Glutaminyl-aspartyl-tyrosyl-O-acetyl-threonine-azide is condensed with the pentapeptide, glycyl-tryptopyl-norleucyl-aspartyl-phenylalanine amide, in a solvent to yield the decapeptide, pyroglutamyl-glutaminyl-aspartyl-tyrosyl-O- Acetyl-threonyl-glycyl-tryptopyl-norleucyl-aspartyl-phenylalanine amide was formed, and the same decapeptide was converted to anhydrous pyridine-
The decapeptide, pyroglutamyl-glutaminyl-aspartyl-tyrosyl (O-sulfate)-O-acetyl-threonyl-glycyl-tryptopyl-norleucyl-aspartyl-phenylalaninamide, is produced by treatment with an anhydrous sulfuric acid complex, and the decapeptide The decapeptide, pyroglutamyl-glutaminyl-aspartyl-tyrosyl (O-sulfate)-threonyl-glycyl-tryptopyl-norleucyl-aspartyl-phenylalanine amide was produced by mild alkaline hydrolysis of A method for producing a novel decapeptide, which comprises converting the decapeptide into a nontoxic and pharmaceutically acceptable salt thereof by optionally reacting with an organic or inorganic base. 3 The following formula using symbols used in polypeptide chemistry: A pancreatic juice and bile secretion promoting agent containing a decapeptide having the formula or a non-toxic, pharmaceutically acceptable salt thereof with an organic or inorganic base.