HU189204B - Process for producing cholecistokinin-octapeptide-sulfate-ester and salts thereof - Google Patents

Abstract

The present invention relates to a novel process for the preparation of compounds of general formula (I) which can be used in medical practice L-Asp-L-Tyr-L-Met-Gly-L-Trp-L-Met-L-Asp-L-Phe-NH2 (I) SO3X - a wherein X is hydrogen or an alkali metal ion. These compounds are prepared from the hexapeptide of formula (II) by starting with L-Met-Gly-L-Trp-L-Met-L-Asp-L-Phe-NH2 (II) such that: a) the hexapeptide is of formula III; protected from dipeptide hydrazide YL-Asp-L-Tyr-Ν2Η, (III) I SO2X - wherein X is as defined above and can be cleaved with Y, but it is resistant to known acid-protected azide, or b) hexapeptide optionally present with YO-sulfate-L-Tyr or an alkali metal salt of an activated carboxyl group, wherein Y is as defined above, cleaves the Y-protecting group of the resulting product and then reacts the heptapeptide formed with Y-protected aspartic acid, and finally according to method a) or b). the protected octapeptide Y is deprotected. -1-

Description

The present invention relates to a novel process for the preparation of the cholecystokinin octapeptide sulfate ester of formula (I) and its salts.

L-Asp-L-Tyr-L-Met-Gly-L-Trp-L-Met-L-Asp-L-Phe-NH ₂ (I) SO ₃ X

- where X = hydrogen or an alkali metal ion.

Cholecystokinin octapeptide (I) is the active center of the 33 amino acid cholecystokinin pancreasozyme. It occurs independently in the central nervous system and small intestine, and can be isolated in large quantities from the cortex. According to Ondetti's study, the compound of formula I, on a molar basis, is one order of magnitude more potent than the 33-amino acid molecule. Of its many biological activities, it is the first known as the so-called gall bladder contraction. cholecystokinetic effect and so-called pancreatic enzyme secretion enhancers. the effect of pancreozyme became known [Amer. J. Physiol. 86,599 (1928); J. Physiol. 102, 115 (1943)]. Cholecystokinin octapeptide has a strong relaxing effect on sphincter Oddi (J, Amer. Digestive Diseases, 1970, 15, 149) and enhances intestinal motility. In addition to the gastrointestinal effects of cholecystokinin octapeptide, a number of central nervous system effects have recently been reported: it affects the functioning of the hypothalamus's hunger center and mobilizes various neurotransmitters in certain regions of the brain. It is also well-known in the literature for the treatment of schizophrenia.

Therefore, beyond the diagnostic value of the formulation (gall bladder test), it may be of therapeutic significance.

The preparation of cholecystokinin octapeptide is known from several articles and patents (Ondetti et al., J. Am. Chem. Soc. 92, 195 (1970);

No. 922,185 Disclosure in the Federal Republic of Germany; Penke et al., No. 174,500; Hungarian Patent Specification). A common feature of the known processes is that the cholecystokininoctapeptide sulfate ester is prepared from a protected octapeptide by sulfate esterification followed by deprotection. The product thus obtained showed uniformity in analytical methods previously known (electrophoresis, TLC), but according to recent high performance chromatographic tests, it contains significant amounts of impurities (mainly sulfonated tyrosine derivatives). The formation of contaminating by-products is due to the over-reactivity of sulfate ester-forming reagents (H ₂ SO ₄ , pyridine sulfur trioxide, chlorosulfonic acid) and sulfonation and even oxidation of amino acids. It has now been found that the use of a new, more gentle sulfating reagent, acetyl sulfuric acid pyridinium salt, can prevent the sulfonation or oxidation of tyrosine and peptides containing tyrosine by using ch ₃ -co-oso ₃ oc ₆ h ₆ n ©. By the sulfate esterification of this reagent with the appropriately protected L-tyrosine or L-asparaginyl-L-tyrosine, very pure derivatives directly suitable for further synthesis can be prepared.

Thus, starting from the C-terminal hexapeptide, the cholecystokinin octapeptide sulfate ester of formula (I) is constructed in two steps with the appropriate protected L-tyrosine sulfate ester and L-aspartic acid, or in one step with a suitable derivative of L-asparaginyl L-tyrosine sulfate.

(The preparation of the C-terminal hexapeptide is described in Ondetti, supra. The preparation of the related component is described by Guttmann et al., Helv. Chim. Acta 44, 721 (1961).)

The present invention thus relates to a novel process for the preparation of cholecystokinin octapeptide sulfate esters of the formula I wherein X is hydrogen or an alkali metal ion. According to the invention, the process is carried out by:

a) the hexapeptide of formula II

With the protected dipeptide hydrazide of L-Met-Gly-L-Trp-L-Met-L-Asp-L-Phe-NH ₂ (II) YL-Asp-L-Tyr-N ₂ H ₃ (III )

SO ₃ X

wherein X is as defined above and Y is capable of being cleaved by alkali, but is acid-resistant, known protecting group, preferably fluorenylmethyloxycarbonyl or o-hydroxy-phenyloxycarbonyl, is reacted via an azide compound, or

b) reacting the hexapeptide of formula II with Y-O-sulfate-LTyr or an alkali metal salt thereof, optionally having a carboxyl group, deprotecting the resulting Y product and then forming the resulting heptapeptide of formula V

L-Tyr-L-Met-Gly-L-Trp-L-Met-L-Asp-L-Phe-NH ₂ (V)

SO ₃ X

wherein X is as defined above, by reaction with an alkaline but acid-resistant aspartic acid, preferably a fluorenylmethyloxycarbonyl protecting group, and finally the protected octapeptide derivative of formula IV obtained according to process aza or b)

YL-Asp-LTyr-L-Met-Gly-L-Trp-L-Met-L-Asp-L-Phe-NH ₂ (IV)

SO ₃ X

wherein X and Y are as defined above, and the resulting cholecystokinin octapeptide sulfate ester of formula (I) is isolated as an acid (X = H) or a salt (X = alkali metal ion).

The protected protecting group Y of the protected octapeptide of formula IV may be cleaved by alkaline treatment. 1N NaOH / dioxane or 10% piperidine in dimethylformamide are suitable for deprotection.

The hexapeptide of formula (II) used as starting material can be used according to methods known in the art of peptide chemistry, e.g. dipeptides are prepared by fragment condensation using the mixed anhydride, azide and active ester techniques.

189,204

The compounds of formula (III) wherein Y is fluorenylmethyloxycarbonyl, which are new substances, are conveniently prepared as follows: The benzyloxycarbonyl-L-tyrosyl-tert-butoxycarbonylhydrazide known from the literature is acetyl sulfuric acid pyridinium and the resulting O-sulfato-L-tyrosyl-tert-butoxycarbonylhydrazide with fluorenylmethyloxycarbonyl-L-aspartic acid β-tert-butyl ester. acylated. The acylation can be carried out using either the active ester or the dicyclohexylcarbodiimide technique. By gentle acidolysis of the resulting protected peptide in a single step with a novel solution of 85% trifluoroacetic acid, 10% water, 2% thioglycolic acid and 3% dithiothreitol, the dipeptide hydrazide of formula (III) is not cleaved under these conditions. juice. The dipeptide of formula (III) can be converted in situ to an acid azide which is capable of synthesizing the protected octapeptide sulfate ester of formula (IV) without protecting the side functional groups of the hexapeptide of formula (II). The absence of side chain protection and the use of an alkali-sensitive N-terminal protecting group optionally imply the need for an acidolytic cleavage, thereby avoiding many of the side effects during acidolysis (sulfate ester cleavage, Trp tert-butylation, indolease damage, Side reactions of Met moieties, etc.). These side reactions are mainly explained by the acidolysis of the tert-butyl and tert-butyloxycarbonyl groups by the reaction of the ₄ -tert-butyl cation. The indole ring of tryptophan is e.g. it can be tert-butylated at multiple sites (2-, 5-, and 7-positions) and a sulfonium ionic structure is formed on the sulfur atom of methionine, which can be decomposed to homocysteine. All these transformations result in the complete loss of the biological activity of the molecule, so it is extremely important to replace the final acidolytic step with alkaline hydrolysis. Thus, the only Y protecting group of the compound of formula (IV), preferably FMOC, under very mild reaction conditions

It can be removed by treatment with piperidines for 5 to 10 minutes and this reaction does not involve any side reaction.

According to a preferred embodiment of the synthesis, N-fluorenylmethyloxycarbonate is first prepared from fluorenylmethyloxycarbonyl-L-tyrosine in chloroform solution with isobutyl ester of chloroformate in the presence of morpholine with tert-butyloxycarbonylhydrazide. Removal of the tyrosyl tert-butyloxycarbonyl hydrazide followed by the tert-butyloxycarbonyl group in trifluoroacetic acid to convert the resulting hydrazide to the azide thereby acylating the hexapeptide of formula II to afford the protected compound of formula VI heptapeptide sulfate ester is obtained: FMOC-L-Tyr-L-Met-Gly-L-Trp-L-Met-L-Asp-L-Phe-NH ₂

SO ₃ H (VI)

In the next step, the fluorenylmethyloxycarbonyl of formula (VI) is cleaved with piperidine to give the free heptapeptide sulfate ester of formula (VII)

L-Tyr-L-Met-Gly-L-Trp-L-Met-L-Asp-L-Phe-NH ₂ (VII)

Acylation of SO ₃ H with fluorenylmethyloxycarbonyl-L-aspartic acid azide yields the protected octapeptide sulfate ester of formula IV.

The synthesis of the sulfate ester of benzyloxycarbonyl-lithyrosyl-tert-butyloxycarbonylhydrazide and fluorenylmethyloxycarbonyl-L-tyrosyl-tert-butyloxycarbonylhydrazide is advantageously accomplished by: the corresponding protected amino acid derivatives are sulfated with acetyl sulfuric acid in a suitable solvent (pyridine, acetonitrile, dimethylformamide). The major advantage of the process over the previously known tyrosine sulfate ester formation reactions is that it does not cause either sulfonation, oxidation or other side reactions, so that the starting compound of formula III can be prepared purely and the alkali metal salts of the products can be readily isolated. In a preferred embodiment of the process, the two most problematic reactions (sulfate ester formation, acidolysis of protecting groups) are carried out with amino acid or dipeptide derivatives which do not contain the sensitive amino acids of the octapeptide, so that the side reactions mentioned above can be eliminated. By using this method, a single purification step after purification of the final protecting group by silica gel column chromatography affords the pure cholecystokinin octapeptide sulfate ester in substantially better yields than the methods previously described.

The octapeptide sulfate ester of formula (I) prepared by the above method, when administered at 2 to 5 pg / kg in animal studies, exhibits gastrointestinal effects characteristic of cholecystokinin, 60-80% more potent than Squibb cholecystokinin octapeptide, as measured on isolated gallbladder strip. The preparation also has appropriate CNS activity and is suitable for both diagnostic and therapeutic purposes.

The melting point values given in the examples were determined on a Koffler block. For thin layer and column chromatography a silica gel ("Kieselgel 60") adsorbent or layer ("Merck DC Fertigplatten, Kieselgel 60F254") and the following solvent systems were used:

1. 95 ml ethyl acetate: 5 ml (: n-butanol: acetic acid: water 75: 10: 24).

2. 90 ml ethyl acetate: 10 ml (: n-butanol: acetic acid: water 75: 10: 24).

3. 85 ml of ethyl acetate: 15 ml (: n-butaryol: acetic acid: water 75: 10: 24).

4. 70 ml ethyl acetate: 30 ml (: n-butanol: acetic acid: water 75: 10: 24).

5. 60 mL ethyl acetate: 40 mL (: pyridine: acetic acid: water 20: 6: 11).

6. 50 mL ethyl acetate: 50 mL (: pyridine: acetic acid: water 20: 6: 11).

7. n-butanol: acetic acid: water 8: 5: 4.

8. n-butanol: acetic acid: water 4: 1: 1.

A practical embodiment of the process of the invention is illustrated by the following examples.

_ 189,204

Example 1 _t g (23.3 mmol) of benzyloxycarbonyl-L-tyrosyl tert-butyloxycarbonyl hydrazide was dissolved in 100 ml of dry pyridine at 0 ° C with stirring, then ⁵ µl of 0 g (50 mmol) acetyl sulfuric acid pyridinium salt is added. The reaction mixture was stirred at room temperature for two days, concentrated and the residue taken up in water to pH 7.5 with 2N sodium hydroxide solution.

The solution is extracted with ether and the aqueous phase is evaporated to dryness. The amorphous residue was dissolved in acetone, the insoluble matter was filtered off, the acetone was evaporated to dryness in vacuo and the oily material was triturated with ether. The obtained product (9.2 g, 67%) of ^{benzyl-benzyloxycarbonyl-O-sulfato-L-tyrosine-15-tert-butyloxy-carbonyl-hydrazide,} sodium salt, very hygroscopic, R / = 0.35 (retention chromatographic value).

9.2 g of benzyloxy-carbonyl-O-sulfato-L-tyrosyl ₂₀ tert-butyl-oxycarbonyl-hydrazide sodium salt are dissolved in 100 ml of ethanol and hydrogenated over 1 g of 10% Pd-C catalyst in 10 to 20 hours After filtration of the catalyst, the filtrate is concentrated in vacuo and the residue is triturated with ether. The product is 6.2 g (90%) of O-sulfate-L-tyrosine-tert-butyloxycarbonylhydrazide hygroscopic crystal R _f ⁷ = 0.71 .

400 mg (1 mmol) of sodium salt of O-sulfate-L-tyrosyl-tert-butyloxycarbonylhydrazide and 4.13 mg of ₃₀ (1 mmol) fluorenylmethyloxycarbonyl-L-aspartic acid P-tert-butyl- The ester is dissolved in 5 ml of dimethylformamide and the solution is cooled to 0 DEG C. and 210 mg (1 mmol) of dicyclohexylcarbodiimide are added with stirring. The reaction mixture was allowed to stand overnight for _3g and then 100 μΐ of acetic acid was added. The precipitated dicyclohexylurea was filtered off and the filtrate was evaporated. The resulting oily residue was triturated with ether to give crystals. The product is a hygroscopic crystalline sodium salt of fluorenylmethyloxycarbonyl-L-asparaginyl- _4θ β-tert-butyl-O-sulfate-L-tyrosine-tert-butyloxycarbonylhydrazide (536 mg, 66%). R1 = 0.2; R f = 0.5.

1.4 g of the protected dipeptide sulfate ester sodium salt are dissolved in ml of trifluoroacetic acid at 0 ° C with stirring. After standing at room temperature for 40 minutes, the dipeptide trifluoroacetate was precipitated with dry ether, filtered, washed with ether and dried in a desiccator. Sodium salt of fluorenylmethyloxycarbonyl-L-asparaginyl-O-sulfato-L-tyrosyl- _50- hydrazide trifluoroacetate (hygroscopic crystal) (1.3 g, 95%) is obtained. R1 = 0.55.

1.5 g (2 mmol) of the sodium salt of fluorenylmethyloxycarbonyl-Lasparaginyl-O-sulfato-L-tyrosylhydrazide trifluoroacetate are dissolved in 7.5 ml of dimethylformamide. The solution was cooled to -15 'C with stirring, followed by 0.5 mL of 5 M sodium nitrite and 0.4 mL of cc. hydrochloric acid is added. The reaction mixture

- Stir at 15 ° C for 15 minutes, then neutralize with 0.8 mL of triethylamine and 1.2 g (1.3 mmol) of θθ L-methionylglycyl-L-tryptophyl-L-methionyl-L-asparaginyl-L -phenylalanine trifluoroacetate dissolved in ml DMF. The pH was adjusted with triethylamine to 7.0, and the temperature 'C is maintained, and the reaction to rt _g5' 24 + 4 hours

leave it at room temperature for another night. The solution was concentrated in vacuo to pH 6.0 and the residue was treated with 1N hydrochloric acid (30 mL). The resulting amorphous powder was filtered, washed with water, ether and then dissolved in a mixture of 30 ml of dimethylformamide and 5 ml of water. The pH was adjusted to 7.0 with 5% sodium bicarbonate solution and evaporated. The product (3.7 g) was dissolved in aqueous pyridine acetate (pyridine: acetic acid: water 20: 6: 11) (10 ml), the octapeptide sodium salt was precipitated with ethyl acetate (90 ml), filtered and washed with ethyl acetate. Dissolve 2 g (1.2 mmol) of the protected octapeptide sodium salt of formula IV in 15 ml of dimethylformamide and add 300 μΐ (3 mmol) of piperidine. After 10 minutes, the solution is neutralized with 250 μΐ of acetic acid, evaporated, the free octapeptide sulfate ester is precipitated with 10-15 ml of 1N hydrochloric acid at 0 ° C, filtered and washed with water. The resulting amorphous powder was dissolved in a mixture of dimethylformamide (15 mL) and methanol (5 mL), adjusted to pH 7.0 with 5% sodium bicarbonate and evaporated to dryness. Trituration with ethyl acetate gave 1.4 g of an amorphous material which was chromatographed on a silica gel column in solvent system 5. After combining and evaporating the appropriate fractions, 860 mg of the pure sodium salt of L-asparaginyl-O-sulfato-L-tyrosyl-L-methionylglycyl-L-tryptopyl-L-methionyl-L-asparaginyl-L-phenylalanine amide (I) are obtained. . Yield 57%, Rf = 0.33 [α] D 20 = -23.0 ° (c = 1, DMF).

Example 2

Example 1 is the preparation of the cholecystokininoctapeptide sulfate ester protected at the N-terminus with a fluorenylmethyloxycarbonyl group and the fluorenylmethyloxycarbonyl group cleaved with piperidine. The free octapeptide sulfate ester (2 g, 1.3 mmol) is precipitated with 1N hydrochloric acid at 0 ° C, filtered and washed with water. The resulting amorphous solid was dissolved in a mixture of dimethylformamide (15 mL) and methanol (5 mL), adjusted to pH 7.0 with 5% potassium bicarbonate, and evaporated to dryness. The octapeptide was purified as described in Example 1. 900 mg (59%) of the pure L-asparaginyl-O-sulfate-L-tyrosyl-L-methionylglycyl-L-tryptophyl-L-methionyl-L-asparaginyl-L-phenylalanine amide tripotassium salt are obtained. R1 = 0.33. [α] D ₂₀ = -22.6 '(c = 1, DMF).

Example 3 Fluorenylmethyloxycarbonyl-L-tyrosine (5 mmol) was dissolved in chloroform (5 ml), cooled to -15 ° C, added 550 µl N-methylmorpholine and 650 µl chloroformic acid isobutyl ester. After stirring for 10 minutes, t-butyloxycarbonyl hydrazide (0.65 g, 5 mmol) was added and the solution was left to stand in the refrigerator overnight. The precipitated crystals are filtered off, the filtrate is evaporated, dissolved in ethyl acetate and extracted with potassium bisulfate and potassium bicarbonate. The ethyl acetate solution was dried over anhydrous sodium sulfate, evaporated, dissolved in 10 ml of pyridine to form the mole-41.189204-donated sulfate ester from Example 1, and then reacted with sodium hydroxide to recover the sodium salt. Yield 1.5 g (50%) of fluorenyl-methyloxy-carbonyl-L-tyrosyl-sulphatoethyl-tert-butyloxycarbonyl-hydration _zid-5 sodium salt.

1.0 g of the resulting material is dissolved in 4 ml of trifluoroacetic acid at 0 'C and allowed to stand at room temperature for 30 minutes. The final product is precipitated with dry ether, filtered and washed with ether. Yield: 0.9 g (90%) of the fluorenylmethyloxycarbonyl-O-sulfate-L-tyrosine hydrazide trifluoroacetate sodium salt.

Dissolve 620 mg (1 mmol) of the resulting product in 2 ml of dimethylformamide, cool to -15 ° C and stir with 250 μϊ of 5 M sodium nitrite solution and 200 μϊ of cc. hydrochloric acid is added. After fifteen minutes, neutralize with 400 μϊ of triethylamine and add 540 mg (0.6 mmol) of L-methionylglycyl-ltrripophyl-L-methionyl-L-asparaginyl-L-phenylalaninamide trifluoroacetate in 2 mL of dimethyl _2θ formamide. The reaction mixture was allowed to stand overnight and the pH was maintained at 7.0 by the addition of triethylamine. The solution was concentrated at pH 6.0 and the residue was triturated with 20 ml of 1N hydrochloric acid. The resulting amorphous powder was filtered, washed with water and ether, followed by dissolution of the pH of 20 ml of dimethylformamide was adjusted to 7.0 with ²⁵ 5% sodium bicarbonate solution and evaporated. Further purification of 600 mg (80%) of the protected fluorenylmethyloxycarbonyl-O-sulfato-L-tyrosyl-L-methionylglycyl-L-tryptoyl-L-asparaginyl-L-phenylalanine amide sodium salt ³ without application.

500 mg (0.4 mmol) of the protected heptapeptide sulfate ester sodium salt are dissolved in 3 mL of dimethylformamide. Add 400 μΐ of piperidine, solution 10. Allow to stand for a minute, then neutralize with acetic acid and evaporate. The residue was triturated with 5 ml of n hydrochloric acid solution at 0 ° C to an amorphous powder. The resulting material was dissolved in dimethylformamide (5 mL) and the solution was adjusted to pH 7.5 with sodium hydroxide and evaporated. The final product was triturated with ethyl acetate ^{to 40} and is filtered off. 380 mg (92%) of the sodium salt of O-sulfate-L-tyrosyl-L-methionylglycyl-L-tryptophyl-L-methionyl-L-asparaginyl-L-phenylalanine amide are obtained.

Dissolve 2.1 g (5 mmol) of fluorenylmethyloxycarbonyl-L-aspartic acid β-tert-butyl ester in 5 ml of chloroform, cool to -15 ° C, add 550 µl of N-methylmorpholine and add 650 μϊ chlorobutyl ester of formic acid. After stirring for 10 minutes, t-butyloxycarbonyl hydrazide (0.65 g, 5 mmol) in chloroform (5 mL) was added. After stirring at 50 ° C for one hour, allow to stand overnight and evaporate. The residue was dissolved in ethyl acetate, extracted with potassium bisulfate and potassium bicarbonate solution and dried over anhydrous sodium sulfate. The resulting material was dissolved in 5 mL of trifluoroacetic acid at 0 C, allowed to stand at room temperature for 45 minutes after dissolution, and precipitated with ether. The precipitated crystals are filtered off and washed with ether. The product was 1.2 g (50%) of fluorenylmethyloxycarbonyl-L-aspartic acid α-hydrazide trifluoroacetate.

Dissolve 240 mg (0.5 mmol) of fluorenylmethyloxycarbonyl L-aspartic acid α-hydrazide trifluoroacetate in 2 ml of dimethylformamide, cool to 15 ° C and 125 μϊ of 5 molar sodium nitrite solution and 100 ml cc. hydrochloric acid is added. After stirring for 15 min, neutralize with 200 μϊ triethylamine and add 300 mg (0.3 mmol) of O-sulfate-L-tyrosyl-L-methionylglycyl L-tryptophyl-L-methionyl-L-asparaginyl-L-phenylalaninamide sodium salt dissolved in 2 ml of dimethylformamide. The solution was maintained at pH 7.0 with triethylamine, stirred at room temperature for one day, and worked up in a manner analogous to Example 1 to give 250 mg (71%) of L-asparaginyl-O-sulfate-L-tyrosyl-L-methionyl- yields the octapeptide sulfate ester of glycyl-L-tryptophyl-L-methionyl-Lasparaginyl-L-phenylalanine amide (I).

Claims (1)

Claims Process for the preparation of L-Asp-L-Tyr-L-Met-Gly-L-Trp-L-Met-L-Asp-L-Phe-NH ₂ (I) SO ₃ X for the preparation of cholecystokinin octapeptide sulfate esters, wherein X is hydrogen or an alkali metal ion, characterized in that: a) the hexapeptide of formula II From the protected dipeptide hydrazide of L-Met-Gly-L-Trp-L-Met-L-Asp-L-Phe-NH ₂ (II) YL Asp-L-Tyr-N ₂ H ₃ (III) SO ₃ X - wherein X is as described above and Y is capable of being cleaved by alkali, but is an acid-resistant, known protecting group, preferably fluorenylmethyloxycarbonyl, - with an azide, or b) reacting the hexapeptide of formula II with Y-O-sulfate-LTyr or an alkali metal salt thereof, optionally having a carboxyl group, deprotecting the resulting Y product and then forming the resulting heptapeptide of formula V L-Tyr-L-Met-Gly-L-Trp-L-Met-L-Asp-L-Phc-NH ₂ (V) SO ₃ X - wherein X is as defined above, by reaction with an alkaline but acid-resistant aspartic acid protected by an acid-protecting group, preferably a fluorenylmethyloxycarbonyl group, and finally the protected octapeptide derivative of formula IV obtained according to a) or b) -ί-Αδρ- ^ Γ-υΜε (-ΟΝ-υΤΓρ-ΕΜο1-υΑ5ρ-υΡΚε NH ₂ SO ₃ X (IV) wherein X and Y are as defined above, and the resulting cholecystokinin octapeptide sulfate ester of formula (I) is isolated as an acid (X = H) or a salt (X = alkali metal ion).